MXPA00009629A - Non-maltogenic exoamylases and their use in retarding retrogradation of starch - Google Patents

Abstract

The present invention relates to the use of non-maltogenic exoamylases of retarding the detrimental retrogradation of starch. Furthermore, the invention relates to a novel non-maltogenic exoamylase from Bacillus Clausii.

Description

NON-MALTOGENIC EXOAMYLASES AND THEIR USE TO DELAY STARCH RETRODEGRADATION FIELD OF THE PRESENT INVENTION The present invention relates to proteins, especially proteins that are capable of degrading starch. In particular, the present invention relates to the use of proteins that are capable of retarding the damaging retrogradation of starch. Harmful retroregradation procedures, such as rancidity, typically occur after heating or cooling starch media, in particular aqueous suspensions of starch, and are due to the transformation of gelatinized starch into an increasingly orderly state. More particularly, the present invention relates to the use of proteins that are capable of retarding the detrimental retrogradation of amylopectin. More particularly, the present invention relates to the use of proteins to prepare baked bread products, as well as the baked bread products themselves. More particularly, the present invention relates to the retardation of rancidity in baked mealy bread products. More specifically, the present invention relates to a method for making a baked bread product having delayed or reduced rancidity, which comprises adding a non-maltogenic exoamylase to the bread dough. The present invention also relates to an improving composition for baked dough and bread products comprising a non-maltogenic exoamylase.

BACKGROUND OF THE PRESENT INVENTION The starch comprises amylopectin and amylose. Amylopectin is a highly branched carbohydrate polymer with short a- (1? 4) -D-glucan chains that are linked together at branching points through a- (1- »6) bonds forming a branched structure and type brush. On average, there is a branching point for every 20-25 glucose residues linked with a- (1? 4). In contrast, amylose is a linear structure consisting mainly of unbranched a- (1? 4) -D-glucan units. Typically, the starches contain about 75% amylopectin molecules and about 25% amylose molecules. More specifically, the linear malto-oligosaccharides are composed of 2-10 a-D-glucopyranose units linked by an a- (1 → 4) bond. Thanks to their properties such as low sweetness, high water retention capacity and prevention of the crystallization of sucrose [1] these compounds have potential applications in the food industry. The preparation of malto-oligosaccharides with a degree of polymerization (DP) of more than 3 (ie, DP> 3) in larger amounts is nevertheless tedious and expensive. As previous information, DP1 = glucose, DP2 = maltose, DP3 = maltotriose, DP4 = maltotetraose, DP5 = maltopentaose, DP6 = maltohexaose, DP7 = maltoheptaose, DP8 = maltooctase, DP9 = maltononae and DP10 = maltodecaose. The discovery of microbial enzymes, which produce malto-oligosaccharides of a specific length, could allow the production of larger quantities of these oligosaccharides [2]. Amylases are starch degrading enzymes, classified as hydrolases, which cut a-D- (1-4) O-glycosidic bonds in starch. Generally, a-amylases (EC 3.2.1.1, aD- (1? 4) -glucan glucanohydrolase) are defined as endo-action enzymes that cut aD- (1? 4) O-glycosidic bonds within the starch molecule in a random way [3]. In contrast, exo-acting amylolytic enzymes such as β-amylases (EC 3.2.1.2, aD- (1 ?4) -glucan maltohydrolase), and some product-specific amylases cut the starch molecule from the non-reducing end of the substrate [4]. The ß-amylases, a-glucosidases (EC 3.2.1.20, aD-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3, aD- (1-4) -glucan glucohydrolase) and product-specific amylases can produce malto-oligosaccharides from a specific length from starch.

Several amylases that produce malto-oligosaccharides of a specific DP have been previously identified, including maltose-producing amylases from Klebsiella pneumonia [5, 6], Bacillus subtilis [7], B. circulans G-6 [8], ß. circulans F-2 [2, 10] and ß. caldovelox [11, 12]. The amylases that produce maltopentase have been detected in ß. licheniformis 584 [13] and Pseudomonas spp. [14, 15]. In addition, maltotetraose-producing amylases from Pseudomonas stutzeri NRRL B-3389 [16, 17], Bacillus sp. MG-4 [18] and Pseudomonas sp. IMD353 [19] and maltotriose-producing amylases from Streptomyces gríseus NA-468 [20] and B. subtilis [21]. EP-B1-298,645 describes a process for preparing exo-maltotetraohydrolase from Pseudomonas stutzeri or P. saccharophila using genetic engineering techniques. US-5,204,254 discloses a native and a genetically modified exo-maltopentaohydrolase of an alkalophilic bacterium (DSM 5853). Very few product-specific amylases active at high pH have been identified. Examples of those that have been identified include Bacillus sp. Amylases. H-167 producing maltohexaose [22, 23], from a bacterial isolate (163-26, DSM 5853) that produce maltopentaose [24], from Bacillus sp. IMD370 that produce maltotetraose and smaller malto-oligosaccharides [25], and from Bacillus sp. GM 8901 that initially produced maltohexaose from starch that became maltotetraose during periods of extended hydrolysis [26].

The starch granules heated in the presence of water undergo a transition of order-disorder phases called gelatinization, in which the liquid is assimilated by the granules in swelling. The gelatinization temperatures vary for different starches and depend for the native and unmodified starches of their biological source. The cooling converts the gelatinized phase into a viscoelastic paste or elastic gel, depending on the concentration of starch. During this process, the amylose and amylopectin chains anneal to form a more ordered structure. With the passage of time, more associations are formed and become more orderly. It is believed that the 15-15 amylopectin DP chain associations lead to a structure and quasi-crystalline and thermoreversible. As a consequence of the damaging retrodegradation, the water retention capacity of the paste or gel system is changed with important implications on gel texture and dietary properties. It is known that the quality of baked bread products deteriorates gradually during storage. The crumb loses softness and elasticity and becomes firm and crumbly. This so-called rancidity is mainly due to the damaging retrodegradation of the starch, which is understood to be a transition from the gelatinized starch during cooking from an amorphous state to a quasi-crystalline state. The increase in firmness of the crumb is commonly used as a measure of the process of bread rancidity. After cooling the freshly baked bread the amylose fraction, in hours, is retrograde to develop a network. This process is beneficial because it creates a desirable crumb structure with a low degree of firmness and improved slicing properties. More gradually the crystallization of amylopectin takes place within the gelatinized starch granules during the days after cooking. In this process it is believed that amylopectin reinforces the amylose network in which the starch granules are embedded. This reinforcement leads to an increased firmness of the bread crumb. This reinforcement is one of the main causes of bread rancidity. The rate of retrodegradation or damaging crystallization of amylopectin depends on the length of the amylopectin side chains. Accordingly, the cereal amylopectin is retrograde at a lower rate than the amylopectin of pea or potato, which has an average chain length longer than the cereal amylopectin. This is supported by observations of amylopectin gel systems that amylopectin with average DP chain length, ie, degree of polymerization, <; 11 does not crystallize at all. In addition, the presence of very short chains of DP 6-9 seems to inhibit the crystallization of surrounding longer side chains probably due to steric hindrance. Therefore, these short chains seem to have a strong anti-damaging retrodegradation effect. According to this, retrograde degradation of aminopectin is directly proportional to the molar fraction of side chains with DP 14-24 and inversely proportional to the molar fraction of side chains with DP 6-9. In wheat and other cereals the external side chains in amylopectin are on the DP 12-19 scale. In this way, the enzymatic hydrolysis of the amylopectin side chains can markedly reduce their crystallization tendencies. The retardation of bread rancidity is known in the art using glycogenic and maltogenic exo-amylases - such as amyloglucosidases that hydrolyze starch releasing glucose - and exoamylases or maltogenic β-amylases - which hydrolyze starch releasing maltose from non-reducing chain ends . In this regard, Jakubczyk et al, (Zesz. Nauk, Sck. GI.Gospod Wiejsk, Warzawie, Technol. Kingdom-Spozyw, 1973 223-235) reported that amyloglucosidase can retard the rancidity of bread baked in wheat flour. JP-62-79745 and JP-62-79746 indicate that the use of β-amylase produced by Bacillus stearothermophilus and Bacillus megaterium, respectively, can be effective in retarding the rancidity of starchy foods, including bread. EP-A-412,607 discloses a process for the production of a bread product having delayed rancidity properties by adding to the dough a thermostable exoamylase, which is not inactivated prior to gelatinization. Only amyloglucosidases and β-amylases are listed as suitable exoamylases to be used. The exoamylase is in an amount that is capable of selectively modifying the crystallization properties of the amylopectin component during baking by separating glucose or maltose from the non-reducing ends of amylose and amylopectin. According to EP-A-412,607, exoamylase selectively reduces the crystallization properties of amylopectin, without substantially affecting the crystallization properties of amylose. EP-A-494,233 discloses the use of a maltogenic exoamylase to release maltose in the a configuration and which is not inactivated prior to gelatinization in a process for the production of a baked product having delayed rancidity properties. Only one maltogenic α-amylase of Bacillus strain NCIB 11837 is specifically described. Apparently, maltogenic exoamylase hydrolyzes (1-4) -a-glucosidic bonds in starch (and related polysaccharides) by removing a-maltose units from the non-reducing ends of the polysaccharide chains in steps. Thus, the prior art teaches that certain glycogenic exoamylases and maltogenic exoamylases can provide an anti-flushing effect by selectively reducing the detrimental degradation tendencies of amylopectin through the shortening of the amylopectin side chains. However, there is still a need to provide different and effective, preferably more effective, means for delaying harmful retrogradation, such as retarding the rancidity of starch products, in particular baked goods, more particularly bread products.

BRIEF DESCRIPTION OF THE PRESENT INVENTION The present invention provides a method for making a starch product that has a delayed detrimental retrogradation property. The present invention also provides enzymes that are useful in the process of the present invention. The enzymes of the present invention are amylase enzymes. More particularly, the enzymes of the present invention are non-maltogenic exoamylase enzymes. It should be noted that non-maltogenic exoamylases have not been used to date to retard the damaging retrogradation of starch products, much less to retard rancidity in baked goods.

Thus, according to a first aspect of the present invention there is provided a process for making a starch product comprising adding to a starch medium a non-maltogenic exoamylase which is capable of hydrolyzing starch by cutting the linear maltooligosaccharides, which consist predominantly of in four to eight D-glucopyranosyl units, from the non-reducing ends of the amylopectin side chains. The addition of the non-maltogenic exoamylase to the starch medium can occur during and / or after the heating of the starch product. Thus, according to a second aspect of the present invention, a baked product obtained by the process according to the present invention is provided. Thus, according to a third aspect of the present invention, an improving composition for a dough is provided; wherein the composition comprises a non-maltogenic exoamylase, and at least one additional dough or additive ingredient. Thus, according to a fourth aspect of the present invention the use of a non-maltogenic exoamylase in a starch product is provided to retard the damaging retrogradation of the starch product. Thus, according to a fifth aspect of the present invention, a novel non-maltogenic exoamylase is provided. These and other aspects of the present invention are presented in the appended claims. In addition, these and other aspects of the present invention, as well as preferred aspects thereof, are presented and described below.

General Definitions In this manner, the present invention relates to the use of proteins that are capable of retarding the damaging retrogradation of starch media, in particular starch gels. In a preferred aspect, the present invention relates to the use of proteins that are capable of retarding rancidity of starch. In another aspect, the present invention relates to the use of proteins that are capable of retarding the damaging retrogradation of starch media, such as starch gels. According to the present invention, the term "starch" means starch per se or a component thereof, especially amylopectin. According to the present invention, the term "starch medium" means any suitable medium comprising starch. The term "starch product" means any product that contains, is based on, or is derived from, starch. Preferably, the starch product contains, is based on, or is derived from, starch obtained from wheat flour. The term "wheat flour" as used herein, is a synonym for finely ground wheat flour or other grain. However, preferably, the term means flour obtained from wheat per se and not from another grain. Also, and unless otherwise indicated, references to "wheat flour" as used herein preferably mean references to wheat flour per se, as well as to wheat flour when present in a medium, such as a dough. A flour that is preferred is wheat flour or rye flour or mixtures of wheat and rye flour. However, masses comprising flour derived from other types of cereals such as for example rice are also contemplated, corn, barley and durra. Preferably, the starch product is a confectionery product. Most preferably, the starch product is a bread product. Still more preferably, the starch product is a baked bread product. The term "baked bread product" is understood to refer to any baked product based on ground cereals and baked in a dough that can be obtained by mixing flour, water and a fermentation agent under dough forming conditions. However, it is within the scope of the present invention that additional components can be added to the dough mixture. The term "amylase" is used in its normal sense-for example, an enzyme that is inter alia capable of catalyzing the degradation of starch.

In particular there are three hydrolases that are capable of cutting a-D- (1? 4) or o-glycosidic bonds in starch. The term "non-maltogenic exoamylase enzyme" means that the enzyme does not initially degrade starch to substantial amounts of maltose. In a highly preferred aspect, the term also means that the enzyme does not initially degrade starch to substantial amounts of maltose and glucose. Prior to the present invention, non-maltogenic exoamylase enzymes had not been suggested to retard the damaging retrogradation of starch media, in particular starch gels. A suitable test for determining the amylase activity according to the present invention is presented below. For reasons of convenience, this test is called the "amylase test protocol." Thus, preferably, the term "non-maltogenic exoamylase enzyme" means that the enzyme does not initially degrade starch to substantial amounts of maltose as analyzed in accordance with the process of product determination as described in the "amylase test protocol" presented herein. In a further aspect, the non-maltogenic exoamylase can be further characterized in that if an amount of 0.7 units of said non-maltogenic exoamylase were to be incubated for 15 minutes at a temperature of 50 ° C at pH 6.0 in 4 ml of an aqueous solution of 10. mg of pre-warmed waxy corn starch per ml of pH-regulated solution containing 50 mM of 2- (N-morpholino) ethanesulfonic acid and 2 mM of calcium chloride, then the enzyme would generate hydrolysis products that would consist of one or more maltooligosaccharides linear of two to ten units D-glucopyranosyl and optionally glucose; whereby at least 60%, preferably at least 70%, most preferably at least 80% and more preferably at least 85% by weight of the aforementioned hydrolysis products would consist of linear maltooligosaccharides of three to ten D units -glucopyranosyl, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units. For ease of reference, and for the present purposes, the characteristic of incubating an amount of 0.7 units of the non-maltogenic exoamylase for 15 minutes at a temperature of 50 ° C at pH 6.0 in 4 ml of an aqueous solution of 10 mg of pre-warmed waxy corn starch per ml of pH-regulated solution containing 50 mM of 2- (N-morpholino) ethanesulfonic acid and 2 mM of calcium chloride, could be referred to as the "waxy corn starch incubation test". Thus, alternatively said, a preferred non-maltogenic exoamylase is characterized in that it has the ability in the incubation test of waxy maize starch to generate hydrolysis products which may consist of one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and optionally glucose; whereby at least 60%, preferably at least 70%, most preferably at least 80% and more preferably at least 85% by weight of said hydrolysis products would consist of linear maltooligosaccharides of three to ten D units. glucopyranosyl, preferably linear maltooligosaccharides consisting of about four to eight D-glucopyranosyl units. The hydrolysis products in the incubation test of waxy corn starch include one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and optionally glucose. The hydrolysis products in the waxy maize starch incubation test may also include other hydrolytic products. However, the weight percent amounts of linear maltooligosaccharides of three to ten D-glucopyranosyl units are based on the amount of the hydrolysis product consisting of one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and optionally glucose. In other words, the weight percent amounts of linear maltooligosaccharides of three to ten D-glucopyranosyl units are not based on the amount of hydrolysis products other than one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and glucose. The hydrolysis products can be analyzed by any suitable means. For example, the hydrolysis products can be analyzed by anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with, for example, known linear maltooligosaccharides of glucose to maltoheptase as standards. For ease of reference, and for the present purposes, the characteristic of analyzing the hydrolysis products using anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and with known linear maltooligosaccharides of glucose to maltoheptaose used as standards, can be referred to as "Analysis by anion exchange". Of course, and as just indicated, other analytical techniques would be sufficient, as well as other specific anion exchange techniques. Thus, in other words, a preferred non-maltogenic exoamylase is characterized in that it has the ability in an incubation test of waxy maize starch to generate hydrolysis products which could consist of one or more linear maltooligosaccharides of two to ten D units. -glucopyranosyl and optionally glucose, said hydrolysis products being able to be analyzed by anion exchange; whereby at least 60%, preferably at least 70%, most preferably at least 80% and more preferably at least 85% by weight of said hydrolysis products would consist of linear maltooligosaccharides of three to ten D units. glucopyranosyl, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units. As used herein with respect to the present invention, the term "linear maltooligosaccharide" is used in the normal sense as meaning 2-10 units of a-D-glucopyranose linked by an a- (1 → 4) bond.

The term "can be obtained from P. saccharophila" means that the enzyme does not necessarily have to be obtained from P. saccharophila. Instead, the enzyme could be prepared by using recombinant DNA techniques. The term "functional equivalent thereof" in relation to the enzyme that can be obtained from P. saccharophila means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme may have a different chemical structure and / or formula but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme does not necessarily have to have exactly the same non-maltogenic exoamylase activity as the non-maltogenic exoamylase enzyme obtained from P. saccharophila. For some applications, preferably, the functionally equivalent enzyme has at least the same activity profile as the enzyme obtained from P. saccharophila. The term "can be obtained from Bacillus clausii" means that the enzyme does not necessarily have to be obtained from Bacillus clausii. Instead, the enzyme could be prepared by using recombinant DNA techniques. The term "functional equivalent thereof" in relation to the enzyme obtainable from Bacillus clausii means that the functional equivalent could be obtained from other sources. The functionally equivalent enzyme may have a different amino acid sequence but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme may have a different chemical structure and / or formula but will have non-maltogenic exoamylase activity. The functionally equivalent enzyme does not necessarily have to have exactly the non-maltogenic exoamylase activity as the non-maltogenic exoamylase enzyme obtained from Bacillus clausii. For some applications, preferably, the functionally equivalent enzyme has at least the same activity profile as the enzyme obtained from Bacillus clausii (such as the reactivity profile shown in Figure 7).

General Comments The present invention is based on the surprising discovery that non-maltogenic exoamylases are highly effective in retarding or reducing damaging retrogradation, such as rancidity, in starch products, in particular baked goods. The present inventors have found that non-maltogenic exoamylases according to the present invention can be more effective in delaying damaging retrogradation, such as rancidity, in bread than glucogenic and maltogenic exoamylases. The reduction of detrimental retrogradation can be measured by standard techniques known in the art. As an example, some techniques are presented later in the section entitled "Test for the measurement of retrodegradation" In the present studies, it has been found that by incorporating a sufficient amount of activity of a non-maltogenic exoamylase, such as an exoamylase. -maltotetraohydrolase (EC 3.2.1.60), which has sufficient thermostability, baked goods are provided in a dough with reduced harmful degradation and in some cases significantly reduced, compared with that of a control bread, such as under storage conditions . In contrast, the reducing effect on the damaging retrogradation of incorporating the same amount of activity of a maltogenic exoamylase with a thermostability comparable to that of non-maltogenic exoamylase is significantly lower. In this way, the anti-retrodegradation effect of non-maltogenic exoamylase is more efficient than that of a maltogenic exoamylase. It is believed that this difference may be due, in part, to the degree to which the amylopectin side chains shorten. It is also believed that the anti-retrodegradation effect may be even more pronounced when a non-maltogenic exoamylase according to the invention is used which releases maltoheptaose and / or maltoctate and / or maltohexose. In the present studies, a specific amylase of active product at high pH that produces maltohexaose has also been purified and characterized. This amylase was isolated from an alkaline-tolerant strain of Bacillus clausii BT-21.

In addition, it has been found that the retardation of the detrimental retrogradation that can be obtained using non-maltogenic exoamylases according to the present invention responds to doses over a very wide range. This contrasts with the effect of maltogenic exoamylases, which is quite limited and has a strongly descending dose response.

Amylases In one aspect, the present invention provides the use of certain amylases to prepare starch products, such as confectionery products. In this regard, amylases - which are non-maltogenic exoamylases - retard or reduce the rancidity properties (i.e., decrease the rate of rancidity) of the starch product, in particular a baked bread product. Preferably, the amylase is in an isolated form and / or in a substantially pure form. Here, the term "isolated" means that the enzyme is not in its natural environment. As indicated above, the non-maltogenic exoamylase enzyme of the present invention does not initially degrade starch to substantial amounts of maltose. In accordance with the present invention, the non-maltogenic exoamylase is capable of cutting linear maltooligosaccharides, consisting predominantly of four to eight D-glucopyranosyl units, from the non-reducing ends of the amylopectin side chains. Non-maltogenic exoamylases having this characteristic and which are suitable for use in the present invention are identified by their ability to hydrolyze gelatinized waxy maize starch in the model system presented in the Amylase Test Protocol (below). When inced 15 minutes under the conditions described in the Amylase Test Protocol, non-maltogenic exoamylases that are suitable for use in accordance with the present invention could generate hydrolysis products that would consist of one or more linear maltooligosaccharides of two to ten units D-glucopyranosyl and optionally glucose, whereby the product standard of that hydrolysis product would consist of at least 60%, in particular at least 70%, most preferably at least 80% and more preferably at least 90% by weight of starch hydrolysis degradation products that were not maltose and glucose. For a preferred aspect of the present invention, non-maltogenic exoamylases which are suitable for use in accordance with the present invention could provide, when inced 15 minutes under the conditions described for the waxy maize starch incion test, the aforementioned hydrolysis product. , whereby the hydrolysis product would have a product standard of at least 60%, in particular at least 70%, most preferably at least 80% and more preferably at least 90% by weight of linear maltooligosaccharides of three to ten D-glucopyranosyl units, in particular linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units. In a more preferred aspect of the present invention, said hydrolysis product in said test would have a product standard of at least 60%, in particular at least 70%, most preferably at least 80% and more preferably at least 85% by weight of linear maltooligosaccharides of 4 or 6 D-glucopyranosyl units. In a more preferred aspect of the present invention, said hydrolysis product in said test would have a product standard of at least 60%, in particular at least 70%, most preferably at least 80% and more preferably at least 85% by weight of linear maltooligosaccharides of 4 D-glucopyranosyl units. In a more preferred aspect of the present invention, said hydrolysis product in said test would have a product standard of at least 60%, in particular at least 70%, most preferably at least 80% and more preferably at least 85% by weight of linear maltooligosaccharides of 6 D-glucopyranosyl units. Preferably, the non-maltogenic exoamylase does not substantially hydrolyze its primary products to convert them into glucose, maltose and maltotriose. If that were the case, the primary products would compete as substrates with non-reducing amylopectin chain ends for the enzyme; so its anti-retrodegradation efficiency could be reduced.

Thus, preferentially, non-maltogenic exoamylase when incubated for 300 minutes under conditions similar to those of the waxy maize starch incubation test but where the 15 minute period extends to 300 minutes - as an aspect apart and for convenience for the present purposes, this incubation test of modified waxy corn starch could be called the "incubation test of extended waxy corn starch" -there would still be said hydrolysis product in which the hydrolysis product would have a standard of product of at least 50%, in particular at least 60%, most preferably at least 70% and more preferably at least 80% by weight of four to eight D-glucopyranosyl units. By way of example, a non-maltogenic exoamylase useful in the method of the present invention can be characterized in that it has the ability in an incubation test of waxy maize starch to generate hydrolysis products which could consist of one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and optionally glucose; whereby at least 60%, preferably at least 70%, most preferably at least 80% and more preferably at least 85% by weight of the aforementioned hydrolysis products would consist of linear maltooligosaccharides of three to ten D units -glucopyranosyl, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units; and wherein the enzyme can be obtained from P. saccharophila or is a functional equivalent thereof.

By way of further example, another non-maltogenic exoamylase useful in the process of the present invention can be characterized in that it has the ability in an incubation test of waxy maize starch to generate hydrolysis products which would consist of one or more linear maltooligosaccharides of two. to ten D-glucopyranosyl units and optionally glucose; whereby at least 60%, preferably at least 70%, most preferably at least 80% and more preferably at least 85% by weight of said hydrolysis products would consist of linear maltooligosaccharides of three to ten D units glucopyranosyl, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units; wherein the enzyme can be obtained from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101,000 Da (calculated by electrophoresis with sodium dodecylsulfate polyacrylamide) and / or the enzyme has an activity optimum at pH 9.5 and 55 ° C. Preferably, non-maltogenic exoamylases which are suitable for use in accordance with the present invention are active during baking and hydrolyze starch during and after the gelatinization of the starch granules which starts at temperatures of about 55 ° C. The more thermostable the non-maltogenic exoamylase, the longer the time it can be active and thus the greater the anti-rancidity effect it will provide. However, during baking at temperatures of about 85 ° C the non-maltogenic exoamylase is preferably inactivated gradually so that there is substantially no activity after the baking process in the final bread. Therefore, preferably non-maltogenic exoamylases suitable for use in accordance with the present invention have an optimum temperature of more than 45 ° C and less than 98 ° C when they are incubated for 15 min. at 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 ° C in a test tube with 4 ml of 10 mg / ml waxy corn starch in 50 mM of MES, 2 mM calcium chloride, pH 6.0 prepared as described above and tested for the release of hydrolysis products as described above. Preferably, the optimum temperature of the non-maltogenic exoamylase is more than 55 ° C and less than 95 ° C, and still more preferably more than 60 ° C and less than 90 ° C. Non-maltogenic exoamylases that can be found less thermostable can be improved using protein engineering so that they become more thermostable and thus better adapted for use in accordance with the present invention. In this way, the use of non-maltogenic exoamylases modified to become more thermostable by protein engineering is encompassed by the present invention. It is known that some non-maltogenic exoamylases may have some degree of endoamylase activity. In some cases, this type of activity may need to be reduced or eliminated since the endoamylase activity could possibly adversely affect the quality of the final bread product by producing a sticky or rubbery crumb due to the accumulation of branched dextrins. Thus, in a preferred aspect, the non-maltogenic exoamylase of the present invention will have less than 0.5 endoamylase units (EAU) per unit of exoamylase activity. Preferably, the non-maltogenic exoamylases that are suitable for use in accordance with the present invention have less than 0.05 EAU per unit of exoamylase activity and most preferably less than 0.01 EAU per unit of exoamylase activity. The endoamylase units can be determined by using the endoamylase test protocol presented below. Examples of non-maltogenic exoamylases suitable for use in accordance with the present invention include exo-maltotetraohydrolase (EC3.2.1.60), exo-maltopentaohydrolase and exo-maltohexaohydrolase (EC3.2.1.98) which hydrolyze 1,4-a-glucosidic bonds in amylaceous polysaccharides to remove successive residues of maltotetraose, maltopentaose or maltohexaose, respectively, from non-reducing chain ends. Examples are exo-maltotetraohydrolases from Pseudomonas saccharophila and P. stutzeri (EP-0 298 645 B1), exo-maltopentaohydrolases from a gram-positive alkalophilic bacterium (US 5,204,254) and from Pseudomonas sp. (Shida et al., Biosci, Biotechnol, Biochem., 1992, 56, 76-80) and exo-maltohexaohydrolases from Bacillus sp. # 707 (Tsukamoto et al., Biochem. Biophys., Res. Commun., 1988, 151, 25-31), ß. circulans F2 (Taniguchi, ACS Symp., 1991, Ser. 458, 111-124) and Aerobacter aerogenes (Kainuma et al., Biochim, Biophys.Acta, 1975, 410, 333-346). Another example of a non-maltogenic exoamylase suitable for use in accordance with the invention is the exoamylase of an alkalophilic Bacillus strain, GM8901 (28). This is a non-maltogenic exoamylase that produces maltotetraose as well as maltopentase and starch maltohexaose. In addition, non-maltogenic exoamylases suitable for use in accordance with the present invention also include exomaltoheptaohydrolase or exomaltooctaohydrolase that hydrolyze 1,4-a-glycosidic linkages in amylaceous polysaccharides to remove maltoheptaose or maltooctase residues, respectively, from the non-reducing chain ends. Exo-maltoheptaohydrolase and exo-maltooctaohydrolase can be found either by selecting wild-type strains or can be developed from other amylolitic enzymes by protein engineering. In this manner, non-maltogenic exoamylases developed by engineering proteins from other amylolytic enzymes to become non-maltogenic exoamylases are also suitable for use in the present invention.

NEW AMYLASE In one aspect, the present invention also provides a novel amylase which is suitable for preparing starch products according to the present invention, such as confectionery products. The novel amylase of the present invention is a non-maltogenic exoamylase. In studies carried out by the present inventors, this new amylase has been characterized which is suitable for the preparation of foods, in particular doughs for use in the preparation of confectionery products. Thus, the present invention also provides a non-maltogenic exoamylase, wherein the non-maltogenic exoamylase is further characterized because it has the ability in an incubation test of waxy maize starch to generate hydrolysis products that would consist of one or more linear maltooligosaccharides. from two to ten D-glucopyranosyl units and optionally glucose; whereby at least 60%, preferably at least 70%, most preferably 80% and more preferably at least 85% by weight of said hydrolysis products could consist of linear maltooligosaccharides of three to ten D-glucopyranosyl units, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units; wherein the enzyme can be obtained from Bacillus clausii or is a functional equivalent thereof; and wherein the enzyme has a molecular weight of about 101,000 Da (calculated by electrophoresis with sodium dodecylsulfate polyacrylamide) and / or the enzyme has an activity optimum at pH 9.5 and 55 ° C. Preferably, the amylase is in an isolated form and / or in a substantially pure form. Here, the term "isolated" means that the enzyme is not in its natural environment.

Antibodies Enzymes of the present invention can also be used to generate antibodies-such as by the use of standard techniques. In this manner, antibodies can be developed for each enzyme according to the present invention. The or each antibody can be used to select other suitable amylase enzymes according to the present invention. In addition, the or each antibody can be used to isolate amounts of the enzyme of the present invention. For the production of antibodies, several hosts including goats, rabbits, rats, mice, etc., can be immunized by injection with the inhibitor or any portion, variant, homologue, fragment or derivative thereof or oligopeptide that retains immunogenic properties. Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin., pluronic polyols, polyanions, peptides, oil emulsions, limpet hemocyanin and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants that can be used. Monoclonal antibodies to the enzyme can be prepared even using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256: 495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote ef al (1983) Proc Nati Acad Sci 80: 2026-2030) and the EBV hybridoma technique (Colé et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes can be used to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al (1984) Proc Nati). Acad Sci 81: 6851-6855; Neuberger et al (1984) Nature 312: 604-608; Takeda et al (1985) Nature 314: 452-454). Alternatively, the techniques described for the production of single chain antibodies (US-A-4946779) can be adapted to produce individual chain antibodies specific for inhibitor. Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening from recombinant immunoglobulin libraries or highly specific binding reagent panels such as those described in Orlandi et al (1989, Proc Nati Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349: 293-299).

IMPROVING COMPOSITION As indicated, one aspect of the present invention relates to an improving composition for a starch product, in particular a dough and / or a baked flour bread product made from the dough.

The enhancer composition comprises a non-maltogenic exoamylase according to the present invention and at least one dough or additive ingredient for additional dough. According to the present invention, the dough ingredient or additive for additional dough can be any of the dough ingredients and dough additives described above. In a simplified manner, the improving composition is a dry pulverulent composition comprising the non-maltogenic exoamylase according to the invention mixed with at least one additional ingredient or additive. However, the improving composition may also be a liquid preparation comprising the non-maltogenic exoamylase according to the invention and at least one additional ingredient or additive dissolved or dispersed in water or another liquid. It should be understood that the amount of enzyme activity in the improving composition will depend on the amounts and types of additional ingredients and additives that are part of the improving composition. Optionally, the improving composition may be in the form of a complete mixture, a so-called premix, which contains all the ingredients and dry additives to make a particular baked product.

Preparation of starch products In accordance with one aspect of the present invention, the process comprises forming the starch product by adding a suitable non-maltogenic exoamylase enzyme, such as one of the novel non-maltogenic exoamylase enzymes presented herein, to a medium of starch. If the starch medium is a dough, then the dough is prepared by mixing flour, water, the non-maltogenic exoamylase according to the invention, and other possible ingredients and additives. By way of further example, if the starch product is a baked bread product (which is a highly preferred embodiment), then the process comprises mixing - in any suitable order - flour, water and a yeast agent under the conditions of mass formation, and also adding a suitable non-maltogenic exoamylase enzyme. The yeast agent can be a chemical yeast agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's yeast). Non-maltogenic exoamylase can be added together with any dough ingredient including water or dough mix or with any additive or admixture of additives. The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry that makes dough based products. The baking of floury bread products such as for example white bread, bread made from rye flour and wheat flour, rolls and the like is typically achieved by baking the bread dough at oven temperatures in the range of 180 to 250 ° C during approximately 15 to 60 minutes. During the baking process, a pronounced temperature gradient (200? 120 ° C) remains in the outer layers of dough, where the characteristic crust of the baked product develops. However, due to the consumption of heat by the generation of steam, the temperature in the crumb is only close to 100 ° C at the end of the baking process. The non-maltogenic exoamylase can be added as a liquid preparation or as a dry powdery composition, whether comprising the enzyme as the sole active ingredient or mixed with one or more ingredients for dough or additional dough additives. To further improve the properties of the baked product and impart distinctive qualities to the baked product, dough ingredients and / or additional dough additives can be incorporated into the dough. Typically, said added additional components may include dough ingredients such as salt, grains, fats and oils, sugar, dietary fiber substances, milk powder, gluten and dough additives such as emulsifiers, other enzymes, hydrocolloids, flavoring agents, oxidizing agents , minerals and vitamins. The emulsifiers are useful as dough softeners and crumb softeners. As dough reinforcers, the emulsifiers can provide tolerance with respect to rest time and shock tolerance during force measurement. Furthermore, dough reinforcers will improve the tolerance of certain dough to variations in fermentation time. Most dough reinforcers also improve in the oven which means the increase in volume of the reinforced items to the baked goods. Finally, the dough reinforcers will emulsify any fat present in the recipe mix. The softening of the crumb, which is mainly a characteristic of monoglycerides, is attributed to an interaction between the emulsifier and the amylose fraction of the starch, leading to the formation of insoluble inclusion complexes with the milose that is not otherwise they will recrystallize after cooling and therefore will not contribute to the firmness of the bread crumb. Suitable emulsifiers that can be used as additives for additional dough include lecithin, polyoxyethylene stearate, mono- and diglycerides of edible fatty acids, acetic acid esters of mono- and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, citric acid esters of mono- and diglycerides of edible fatty acids, esters of diacetyltartaric acid of mono- and diglycerides of edible fatty acids, sucrose esters of edible fatty acids, sodium stearoyl-2-lactylate and stearoyl -2-calcium lactylate. Other enzymes that are useful as additives for additional dough include as examples oxide reductases, such as glucose oxidase, hexose oxidase and ascorbiate oxidase, hydrolases, such as lipases and esterases as well as glucosidases such as α-amylase, pullulanase and xylanase. Oxidase reductases such as for example glucose oxidase and hexose oxidase, can be used to strengthen the dough and control the volume of the baked goods, and xylanases and other hemicellulases can be added to improve the handling properties of the dough, the softness of the crumb and the volume of bread. Lipases are useful as dough softeners and crumb softeners, and a-amylases and other amylolytic enzymes can be incorporated into the dough to control bread volume and further reduce crumb firmness. The amount of the non-maltogenic exoamylase according to the present invention which is added is usually an amount which results in the presence in the finished dough of 50 to 100,000 units per kilo of flour, preferably 100 to 50,000 units per kilo of flour. In useful embodiments of the present invention, the amount is on the scale of 200 to 20,000 per kilo of flour. In the present context, 1 unit of non-maltogenic exoamylase is defined as the amount of enzyme that releases hydrolysis products equivalent to 1 μmole of reducing sugar per minute when incubated at 50 ° C in a test tube with 4 ml of 10 mg / ml of waxy corn starch in 50 mM of MES, 2 mM of calcium chloride, pH 6.0 as described hereinafter.

Foods prepared with amylases The present invention provides suitable amylases for use in the manufacture of a food. Typical foods, which also include animal feed, include dairy products, meat products, poultry products, fish products and baked goods. Preferably, the food is a confectionery product, such as the confectionery products described above. Typical baking products incorporated within the scope of the present invention include bread-such as pancakes, rolls, biscuits, pizza bases, etc.-pretzels, tortillas, cakes, biscuits, biscuits, etc.

Amylase Test Protocol The following system is used to characterize non-maltogenic exoamylases which are suitable for use in accordance with the present invention. As an initial background information, waxy corn amylopectin (obtainable as WAXILYS 200 from Roquette, France) is a starch with a very high amylopectin content (more than 90%). 20 mg / ml of waxy corn starch are boiled for 3 minutes in a pH regulator of 50 mM of MES ((2-N-morpholino) ethanesulfonic acid), 2 mM of calcium chloride, pH 6.0 and then incubated at 50 ° C and are used within one hour.

One unit of non-maltogenic exoamylase is defined as the amount of enzyme that releases hydrolysis products equivalent to 1 μmole of reducing sugar per minute when incubated at 50 ° C in a test tube with 4 ml of 10 mg / ml of starch of waxy corn in 50 mM of MES, 2 mM of calcium chloride, pH 6.0 prepared as described above. Reductive sugars are measured using maltose as standard and using the dinitrosalicylic acid method of Bernfeld, Methods Enzymol. (1954), 1, 149-158 or other method known in the art for quantifying reducing sugars. The pattern of hydrolysis product of the non-maltogenic exoamylase is determined by incubating 0.7 units of non-maltogenic exoamylase for 15 or 300 minutes at 50 ° C in a test tube with 4 ml of 10 mg / ml waxy corn starch in the regulator of pH prepared as described above. The reaction is stopped by immersing the test tube for 3 minutes in a boiling water bath. The hydrolysis products are analyzed and quantified by anion exchange HPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxide and water as eluents, with pulsed amperometric detection and with known linear mantooligosaccharides of glucose to maltoheptase as standards. The response factor used for maltoctane to maltodecase is the response factor found for maltoheptaose.

Endoamylase test protocol 0.75 ml of enzyme solution is incubated with 6.75 ml of 0.5% (w / v) of AZCL-amylose (amylose entangled with available azurin from Megazyme, Ireland) in 50 mM of MES (2- ( N-morpholino) ethanesulfonic)), 2 mM calcium chloride, pH 6.0 at 50 ° C. After 5, 10, 15, 20 and 25 minutes, respectively 1.0 ml of reaction mixture is transferred to 4.0 ml of stop solution consisting of 4% (w / v) of TRIS (Tris (hydroxymethyl) amnomethane) . The stopped sample is filtered through a Whatman No. 1 filter and its optical density at 590 nm is measured against the distilled water. The enzyme solution tested must be diluted so that the optical density obtained is a linear function of time. The slope of the line for optical density against time is used to calculate the endoamylase activity in relation to the GRINDAMYL ™ A1000 standard (available from Danisco Ingredients), which is defined as having 1000 endoamylase units (EAU) per g.

Tests for measuring retrogradation (rancidity) For the evaluation of the anti-rancidity effect of the non-maltogenic exoamylase of the present invention, the firmness of the crumb can be measured 1, 3 and 7 days after baking by means of an Instron 4301 Universal Food Texture Analyzer or similar equipment known in the art. Another method that is traditionally used in the art and that is used to evaluate the effect on retrograde starch of a non-maltogenic exoamylase according to the present invention is based on DSC (differential scanning calorimetry). Here, the enthalpy of fusion of amylopectin retrograde in bread crumb or crumb of a mass of model system baked with or without enzymes (control) is measured. The DSC equipment is applied in the described examples is a Mettier-Toledo DSC 820 with a temperature gradient of 10 ° C per minute from 20 to 95 ° C. For the preparation of the samples 10-20 mg of crumb are weighed and transferred to Mettier-Toledo aluminum pans which are then hermetically sealed. The model system masses used in the described examples contain standard wheat flour and optimum amounts of water or pH buffer with or without the maltogenic exoamylase according to the present invention. They are mixed in a Brabender Farinograph apparatus of 10 or 50 g for 6 or 7 minutes, respectively. The samples of the masses are placed in glass test tubes (15 * 0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath starting with 30 minutes of incubation at 33 ° C followed by heating from 33 to 95 ° C with a gradient of 1.1 ° C per minute, and finally an incubation of 5 minutes at 95 ° C. Subsequently, the tubes are stored in a thermostat at 20 ° C before analysis by DSC.

Conclusion In summary the present invention is based on the surprising discovery that maltogenic exoamylases - which hydrolyze starch by cutting linear maltooligosaccharides on the scale of four to eight D-glucopyranosyl units of non-reducing amylopectin chain ends and which preferably have a sufficient degree of thermostability - are highly effective in retarding or reducing the damaging retro-degradation in baked goods.

Deposits The following sample was deposited in accordance with the Budapest Treaty in the well-known depository DSMZ (Deutsche Sammlung von Mikrooganismen und Zellkulturen GmbH of Mascheroder Weg 1 b, D-38124 Braunschweig) on March 12, 1999: BT-21 number of DSM : DSM 12731 The present invention also encompasses derivable and / or expressible sequences of those deposits and modalities comprising them, as well as active fragments thereof.

Introduction to the Examples Section and the Figures The present invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 shows a graph; Figure 2 shows a graph; Figure 3 shows a graph; Figure 4 shows a graph; Figure 5 shows a graph; Figure 6 shows a trace and Figure 7 shows a graph. In more detail: Figure 1 shows the extracellular amylolytic activity (mU / mL) in liquid cultures of B. clausii BT-21 grown on 2% starch substrates at 45 ° C. * soluble starch, • amylopectin, * corn starch, B whole brown rice. The bars indicate the standard deviation. Figure 2 shows the effect of pH on the activity of the product specific amylase. Effect of pH at 55 ° C. Figure 3 shows the effect of temperature on the activity of the product specific amylase. Effect of temperature at pH 9.5 B with 5 mM CaCl2 D without CaCl2. Figure 4 shows the thermostability tested as a residual activity of the product specific amylase after incubation at increasingly high temperatures at pH 9.5 with 5 mM CaCl2. Figure 5 shows products (in mM) formed by incubating the product specific amylase (505 mU / mL) with 1% soluble starch and 5 mM CaCl2 at 55 ° C and pH 9.5 O glucose, x maltose, D maltotriose, A maltotetraose, • maltopentaose, B matohexaose. Figure 6 shows the trace of HPAEC-PAD obtained by incubating the product specific amylase (505 mU / mL) with 1% soluble starch at pH 9.5 and 55 ° C. A) soluble starch without enzyme, B) incubation with enzyme for 30 minutes. Figure 7 shows the determination of the endo and exo activity of amylase specific for BT-21 product of B. clausii in comparison with known starch action amylases. The formation of blue color (% of maximum) is plotted against the production of mM maltose. The slope of the curves indicates the prevalence of endo or exo activity.

EXAMPLES - SECTION A EXAMPLE 1 Fermentation and production of non-maltogenic exoamylase from Pseudomonas saccharophila Production Cepa IAM No. 1544 of P. saccharophila was obtained from IAM Culture Collection, Inst. Of Molecular and Celllular Biosciences, University of Tokyo, Japan. The fermentation of the strain was carried out in a 3 liter bioreactor Applikon ADI with 2 liters of work volume and under the following conditions: Temperature: 30 ° C Agitation speed: 1000 rpm Aerated: 1 volume of air per volume of medium per minute PH pH 7.4 constant by adjustment with 2M sodium hydroxide and 10% hydrochloric acid (w / v) Medium: Bacto Triptona 20 g / l Bacto yeast extract 20 g / l Starch 20 g / l Na2HPO4 »2H2O 5.6 g / l KH2PO4 1.5 g / l After 1 day of fermentation, the fermentation broth was centrifuged and filtered to remove the cells. The non-maltogenic exoamylase activity in the cell-free broth was 5 units per ml determined as described above. 1. 2 Purification of non-maltogenic exoamylase from P. saccharophila Non-maltogenic exoamylase from P. saccharophila was purified by hydrophobic interaction chromatography using a low sub-column Phenyl Sepharose FF of 150 ml (Pharmacia, Sweden) equilibrated with buffer solution A with 200 mM sodium sulfate, 50 mM triethanolamine, 2 mM calcium chloride, pH 7.2. The filtered fermentation broth (500 ml) was adjusted to 200 mM sodium sulfate and pH 7.2 and loaded onto the column. The non-maltogenic exoamylase was diluted with a linearly descending gradient of sodium sulfate in buffer A. The fractions containing the exoamylase activity were mixed. The mixed fractions were diluted three times with water and further purified by anion exchange chromatography on a Q-Sepharose FF column (Pharmacia) of 150 ml equilibrated with buffer A being 50 mM triethanolamine, 5 mM calcium chloride, pH 7.5. The non-maltogenic exoamylase was diluted with a linear gradient of 0 to 1 M sodium chloride in buffer A. The fractions containing exoamylase activity were mixed. This partially purified preparation was used for the tests described below. This had an activity of 14.7 units per ml and only one band of amylase activity was stained when it was tested in a polyacrylamide gel electrophoresis system for amylase activity. 1. 3 Characterization of non-maltogenic exoamylase from P. sacc 7aroj? / 7 / 7a As an introduction, the DNA sequence for the gene encoding exoamylase from P. saccharophila (which is named PS4) by Zhou et al (Zhou) has been published. JH, Baba T, Takano T, Kobayashi S, Arai Y (1989) FEBS Lett 1989 Sep 11; 255 (1): 37-41"Nucleotide sequence of the maltotetraohyd rolase gene from Pseudomonas saccharophila." In addition, it can be accessed to the DNA sequence in GenBank with accession number X16732. It has now been determined that the molecular weight of the PS4 enzyme purified by mass spectrometry (MALDI-TOFF) is 57500 ± 500 D, which agrees with the molecular weight The theoretical temperature of 57741 D obtained from the sequence The optimum temperature and pH of PS4 are 45 ° C and pH 6.5 in accordance with Zhou ef al (Zhou JH, Baba T, Takano T, Kobayashi S, Arai Y (1992) Carbohydr Res 1992 Jan; 223: 255-61"Properties of the enzyme expressed by the Pseudomonas saccharophila maltotetraohyd rolase gene (mta) in Escherichia coli. ") The hydrolysis pattern of the non-maltogenic exoamylase was determined by analysis of the hydrolysis products generated by incubation of 0.7 units of partially purified non-maltogenic exoamylase for 15 or 300 minutes at 50 ° C in a test tube with 4 ml of mg / ml waxy corn starch as described above. Patterns of the hydrolysis products detected after 15 minutes and 300 minutes are shown in Table 1 and indicate that P. saccharophila produces a non-maltogenic exoamylase as defined in the present invention and that this enzyme liberates maltotetraose as the predominant product for 85.8% and 93.0% by weight after 15 and 300 minutes of hydrolysis, respectively.

TABLE 1 Product of glucose-to-maltodecase hydrolysis of non-maltogenic exoamylase from P. saccharophila EXAMPLE 2 Baking test of non-maltogenic exoamylase from P. saccharophila A baking test was started to evaluate the anti-fever effect of non-maltogenic exoamylase from P. saccharophila. A recipe for Danish toast was used. This contains flour (2000 g), dry yeast (30 g), sugar (30 g), salt (30 g) and water (approximately 1200 g corresponding to a mass consistency of 400 units Brabender (BU) + 60 g of water additional to compensate for the dry yeast used) is mixed in a Hobart mixer (model A-200) for 2 minutes at low speed and for 12 minutes at high speed. The temperature of the dough is 26 ° C at the end of the mixing. The dough is left to stand for 10 minutes at 30 ° C after which the dough is divided into 750 g dough pieces. The dough pieces rest for 5 minutes in a fermentation room at a temperature of 33 ° C and a relative humidity of 85%. Then the dough pieces are molded in a Glimek moulder (type LR-67) with the following parameters 1: 4, 2: 2, 3:14, and 4:12, after which the molded dough pieces are transferred to Baking trays and left until they reach a standard lightness in a fermentation cabin for 50 minutes at a temperature of 33 ° C and a relative humidity of 85%. Finally, the dough pieces with standard lightness are baked for 40 minutes at a temperature of 220 ° C, with 10 seconds of steam, in a Wachtel oven (model AE 416/38 COM). A partially purified preparation of non-maltogenic exoamylase from P. saccharophila was added to the dough in doses of 1470 units per kg of flour. After baking the loaves with or without the non-maltogenic exoamylase, these were cooled to 20 ° C and after that they were stored at 20 ° in plastic bags. Firmness was determined by an Instron 4301 universal food texture analyzer on day 3 and day 7 after baking as the average of 10 slices of a bread for day 3 and the average of two loaves with 10 slices per bread measures per day. Table 2 shows that a lower firmness was observed in the loaves with added enzyme for both days.

TABLE 2 Antipyretic effect of non-maltogenic exoamylase of P. saccharophila Table 3 shows that for day 7 the anti-fumidity effect of the non-maltogenic exoamylase from P. saccharophila is statistically significant over a 95% confidence level.

TABLE 3 Statistical analysis of the antipyretic effect of non-maltogenic exoamylase from P. Saccharophila ANOVA table for firmness on day 7 for the enzyme Analysis of variance Source Sum of Df Average of Ratio F Value P squared squares Between groups 144.0 1 144.0 72.00 0.0136 Within the 4.0 2 2.0 groups Total (corrected) 148.0 3 The StatAdvisor The ANOVA table decomposes the variance of the firmness on day 7 into two components: a component between groups and a component within groups the relationship F, which is in this case equal to 72.0, it is a ratio of the estimated calculation between groups to the estimated calculation within groups. Because the P value of the F test is less than 0.5, there is a statistically significant difference between the average firmness on day 7 from one enzyme level to another at a level of 95.0% confidence. To determine what averages are significantly different from one another, select Multiple Range Tests from the list of options in the table.

EXAMPLE 3 The cloning and expression of the mta gene coding for non-maltogenic exoamylase from Pseudomonas saccharophila in Escherichia coli MC1061 is described below. In this regard, P. saccharophila IAM 1520 was cultured in two ml of LB medium and the cells were harvested by centrifugation 10 min 20,000 x g. Total DNA was isolated using a slightly modified miniprep protocol. The cells were resuspended in 300 μl of resuspension buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 μg / ml RNase A) after which the cells were used using a Fastprep FP120 (BIO101; ). After lysis, 300 μl of lysis buffer (200 mM NaOH, 1% SDS) and 300 μl neutralization buffer (3.0 M potassium acetate, pH 5.5) were added. After centrifugation at 20,000 x g for 15 minutes at 4 ° C, the supernatant was collected and 0.6 volumes of isopropanol were added. The DNA was precipitated by centrifugation at 20,000 x g for 30 minutes at 4 ° C, washed with 70% ethanol and redissolved in 100 μl of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). For PCR amplification, 4 different PCR primers were designed: # 1 ATG ACG AGG TCC TTG TTT TTC pos 213-233 # 2 GCT CCT GAT ACG ACA GCG pos 2403-2386 # 3 GCC ATG GAT CAG GCC GGC AAG AGC CCG pos 663-683 # 4 TGG ATC CTC AGA ACG AGC CGC TGGT pos 2258-2238 The positions refer to the sequence for mta found in access number GenBank X16732. The primers with the highest number first are antisense primers and the sequences are the complementary sequences. In bold, the nucleotides that are complementary to the DNA template are represented, and the restriction sites introduced are underlined. Initiator # 3 introduces a unique? / Co1 site, and initiator # 4 a BamH site? Which are used for the next cloning in the pBAD / gIII expression vector (Invitrogen). A first PCR amplification was performed using the following primer combination: reaction 1 # 1 + # 2 giving a 2190 bp fragment with 50-150 ng of IAM1520 genomic DNA as a template, using Expand DNA polymerase (Boehringer Mannheim, Germany) in accordance with the manufacturer's instructions and the following amplification protocol: 94 ° C 2 min. (94 ° C 1 min, 58 ° C 2 min, 72 ° C 2 min) for 35 cycles and finally 72 ° C, 5 min. A 2190 bp fragment was isolated from the gel using the 'gene clean kit' (BIO101; California). The fragment was used as template DNA in a second PCR with the following primer combination: reaction 2 # 3 + # 4 giving a 1605 bp fragment using the same amplification protocol described above.

A 1605 bp fragment was purified and cloned into the pCR-BLUNT vector (Invitrogen) in accordance with the manufacturer's instructions. The sequence of the cloned fragment was confirmed by sequence determination using single-dye sequencing technology and an ALF sequence determinant (Pharmacia, Sweden) using the universal and inverse primers, and the four labeled internal primers. CAT CGT AGA GCA CCT CCA 999-982 GAT CAT CAA GGA CTG GTC C 1382-1400 CTT GAG AGC GAA GTC GAA C 1439-1421 GAC TTC ATC CGC CAG CTG AT 1689-1708 The positions refer to the sequence for mta found in GenBank with access number X16732. The primers with the highest number first are antisense primers and the sequences are the complementary sequences. After confirming the sequence, the mta gene was cloned into the expression vector pBAD / glII (Invitrogen). The mta gene was released from pCR-BLUNT by digestion with BamH \ followed by softening with the Klenow and Ncol fragment, and a 1602 bp fragment was purified. The expression vector pBAD / glII was digested with Ncol and Pmel and purified. After ligating, the expression construct obtained was transformed into Escherichia coli MC1061 cells, and the protein was expressed in accordance with the pBAD / glII manual (Invitrogen).

EXAMPLE 4 Comparison of the effect of a non-maltogenic exoamylase and a maltogenic exoamylase on the retrogradation of starch Sweet potato ß-amylase (EC 3.2.1.2, which can be obtained from Sigma with a product number A705) is a maltogenic exoamylase that liberates maltose from the non-reducing ends of the starch. The thermal stability of this maltogenic exoamylase is similar to that of the non-maltogenic exoamylase from P. saccharophila as indicated by the residual activities after incubation for 15 minutes at temperatures from 45 to 75 ° C in 50 mM sodium citrate, 5 mM calcium chloride, pH 6.5 (table 4).

TABLE 4 Residual activities of sweet potato ß-amylase and non-maltogenic exoamylase of P. saccharophila after incubation at increasing temperatures (%) Activity after incubation at 45 ° C adjusted to 100%.

The effects of both enzymes on starch retrodegradation have been tested by DSC analysis of the baked and stored products from the model system masses as described in "Tests to measure retrodegradation and rancidity". For this test, 485 units of non-maltogenic exoamylase from P. saccharophila and 735 units of sweetpotato ß-amylase evaluated in accordance with the "amylase test protocol" in the masses were used. The doughs were prepared from 50 g of standard Danish wheat flour (Danisco 98022) with 30.7 ml of 50 mM sodium citrate solution, 5 mM calcium chloride, pH 6.5 without (control) or with added enzymes. After 7 days of storage the amount of retrograde amylopectin was quantified by measuring its enthalpy of fusion. It was found by statistical analysis that both enzymes significantly reduce the retrogradation of starch (Table 5); that is, the non-maltogenic exoamylase from P. saccharophila reduces the amount of amylopectin retrograde on day 7 to 86% (1.77 J / g) while the sweet potato β-amylase decreases it to 96% (1.96 J / g). ) of the control (2.05 J / g). In conclusion, the non-maltogenic exoamylase of P. sccharophila is clearly much more effective in reducing retrogradation and rancidity than maltogenic amylase with comparable thermostability.

TABLE 5 Effect of non-maltogenic exoamylase of P. saccharophila (PS4) and sweet potato 3-amylase (SP2) on starch degradation based on the measurement of enthalpy of fusion of retrograde degraded amylopectin (in J / g) 7 days after baking Multiple interval test for enthalpy by treatment Method 95.0 percent LSD Treatment Average Count PS4 13 1, 77154 SP2 22 1, 96273 Control 6 2.05167 Contrast Difference Limits +/- Control - PS4 * 0.280128 0.0941314 Control - SP2 * 0.0889394 0.087841 PS4 -SP2 * -0.191189 0.06672 * Indicates a statistically significant difference.

This table applies a multiple comparison procedure to determine what averages are significantly different from the others. The lower half of the result shows the estimated difference between each pair of averages. An asterisk has been placed near the three pairs, which indicates that those pairs show statistically significant differences at a confidence level of 95%. The method that is currently being used to discriminate between averages is Fisher's minimum significant difference procedure (LSD).

EXAMPLE -SECTION B Materials and methods Materials Amylopectin and amylose from corn, corn starch, carboxymethylcellulose (CMC), bovine serum albumin (BSA), dextran, pullulan, maltose, maltotriose and a mixture of maltotetraose to maltodecaose were obtained from Sigma Chemical Co., St. Louis E.U.A. Soluble starch was obtained from Merck KGaA, Darmstadt, Germany. Yeast and tryptone extract were obtained from Difco Laboratories, Detroit, USA. Brown rice was used from Neue Allgemeine Reisgesellschaft mbH, Hamburg, Germany. The pharmaceutical grade a-, β-, and β-cyclodextrins were obtained from Wacker Chemie Danmarck Aps, Glostrup, Denmark. The maltotetraose was prepared as previously described [32]. All chemical compounds were, unless otherwise indicated, of analytical grade.

Isolation of ß. clausii BT-21 The strain was isolated from a soil sample collected in Assens, Denmark, identified by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) Production of the enzyme β was cultured. BT-21 in an optimized liquid medium consisting of 2.0% soluble potato starch, corn amylopectin, corn starch, or whole brown rice, 0.5% yeast extract, 0.5% tryptone, 0.1% KH2PO4, 0.1 % Na2HPO4, 0.02% MgSO4, -7H2O, 0.02% CaCl2-2H2O, and 0.1% (NH4) SO4. After autoclaving, a sterile Na2CÜ3 solution was added to a final concentration of 1% (pH about 10). One ml of spore suspension in glycerol (stored at -80 ° C) was used to inoculate 100 ml of the real medium and incubated at 45 ° C for 18 hours in a shaking incubator (New Brunswick Scientific, Edison, NJ, USA) ) at 200 rpm. 2 ml of this culture was used to inoculate a shake flask with 200 ml of medium and incubated at 45 ° C in a shaking incubator. Aliquots were taken at regular intervals and the OD at 600 mm was measured to determine the growth of the strain in the medium. Samples were centrifuged (4 ml) at 9600 rpm for 10 minutes at 4 ° C and the pH and amylase activity were determined. All the culture experiments were performed in triplicate. The average value was determined (X = (Sni =? Xi) / n) and the values of standard deviation (dev.std. = V (Sn¡ =? (X¡-X) / (n-1)).

Purification of the product specific amylase After culturing B. clausii BT-21 on whole brown rice for 52 hours, the whole rice cells and grains were removed from the extracellular fluid (1000 ml) by centrifugation at 9600 rpm for 15 minutes at 4 ° C. The product specific amylase was purified using an affinity gel prepared by covalent attachment of β-cyclodextrin to a 6B matrix of epoxide-activated sepharose (Pharmacia Biotech, Uppsala, Sweden) [33]. The extracellular free supernatant was incubated with 12 g of gel while stirring for 1 hour at 4 ° C. The supernatant was then removed by centrifugation at 9600 rpm for 10 minutes at 4 ° C. The unbound protein was removed by washing the gel with 75 ml of 50 mM phosphate buffer, pH 8.0, followed by centrifugation. The washing step was repeated 7 times. The bound protein was eluted with 45 ml of 50 mM phosphate buffer pH 8.0, containing 10 mM of a-cyclodextrin followed by centrifugation. The elution step was repeated 4 times. A-cyclodextrin was used to elute the enzyme, since ß- and? -cyclodextrins interfered with the protein determination method of Bradford (1976) [34]. The a-cyclodextrin was then removed by dialysis (Spectra / Por dialysis membrane of 6-8 kDa, The Spectrum Companies, Gardena, CA, E.U.A.) against 5 liters of 10 mM triethanolamine pH 7.5, while stirring at 4 ° C. The regulatory solution was changed after 2 hours, followed by another 12 hours of dialysis. The dialysis bags were placed in CMC to concentrate the sample. 10 mM was applied to a HiTrap Q column (5 ml pre-packaged, Pharmacia Biotech, Uppsala, Sweden) using an FPLC system (Pharmacia, Uppsala, Sweden). Proteins were eluted at the rate of 1.0 ml / minute with 25 ml of 10 mM triethanolamine, pH 7.5, followed by a gradient of 20 mM NaCl / minute in 10 mM triethanolamine, pH 7.5. The enzyme was eluted up to 0.5 MNaCl. The protein content was estimated by the method of Bradford, (1976) [34] using the BIO-RAD protein test (Bio-Rad Laboratories, Hercules, CA, USA). BSA was used as a standard.

Gel electrophoresis 15 μL samples were analyzed by 10% native tris-glycine gel, as described by [35]. The gel was then placed in 50 mM phosphate buffer at pH 6.5 and stirred for 30 minutes. A 1% (w / v) solution of soluble starch was incubated with the gel while at the same time being stirred for 45 minutes. After washing in buffer, the gel was incubated with an iodine solution (4 mM l2, 160 mM Kl) and decolorized with buffer solution. The faded bands indicated the hydrolysis activity of starch. SDS-PAGE (10%) was performed in accordance with [36] followed by silver staining [37]. A broad-range molecular weight standard SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, E.U.A.) was used.

Enzyme test Two ml of soluble starch solution (1.25%) in 0.1 M borate buffer pH 10.0 were incubated with 0.5 ml of enzyme solution for 2 hours at 45 ° C. The reaction was stopped by boiling the mixture for 10 minutes. The formation of reducing sugars was determined with the CuS? 4 / bichinchonate test [38] and was calculated as mM maltose equivalents formed. One unit of activity corresponded to the amount of enzyme that produced 1 μmol maltose equivalents / minute at pH 10.0 and 45 ° C.

Characterization of the enzyme To determine the optimum temperature, the purified enzyme was incubated in a final concentration of 1% soluble starch in 0.1 M borate buffer pH 10.0 (with or without the addition of 5 mM CaCl2) for 15 minutes at temperatures from 30 ° C to 90 ° C. The determination of the temperature stability was performed by incubating the purified enzyme in 50 mM glycine / NaOH buffer, pH 9.5 containing 5 mM CaCl2 for 30 minutes at 30, 40, 50, 55, 60, 70, 80 and 90 ° C. Residual activity was determined by incubating the heat-treated enzyme in a final concentration of 1% soluble starch in 50 mM glycine / NaOH buffer, pH 9.5 at 55 ° C for 15 minutes. The optimum pH was determined by incubating the purified enzyme in soluble starch of final concentration 1% in different buffer solutions at 55 ° C for 15 minutes. The regulatory solutions used were 50 mM citrate (pH 4.0 to 6.0), 50 mM tris-maleate (pH 6.5 to 8.5), and 50 mM glycine-NaOH (pH 9.0 to 11.0). A solution of 12-KI (0.02% of 12 and 0.2% of Kl) was prepared in accordance with Fuwa, 1954 [39]. The blue color formation of starch-iodine was measured in duplicate with the following modifications. A 500 μL sample was withdrawn from the enzymatic hydrolysis of soluble starch at different time intervals. Then 250 μL of HCl and 250 μL of 12-KI solution were added and mixed. Deionized water (4.0 ml) was added and mixing repeated. The formation of a blue color was measured spectrophotometrically at 600 nm. The hydrolysis of different substrates was evaluated by means of the purified enzyme with soluble starch of potato, corn amylopectin, dextran, pullulan (1%) amylase (0.1%) amylase (0.1%), and 10 mM of a-, β-, and? -cyclodextrin. The substrates were dissolved in 50 mM glycine-NaOH buffer solution with 5 mM CaCl2 at pH 9.5 and the purified enzyme was added (505 mU / mL). The different substrates were incubated at 55 ° C and samples were removed at different time intervals. The reaction was stopped by boiling for 10 minutes and the samples were analyzed as described below. The hydrolysis of malto-oligosaccharides was evaluated by the enzyme purified with maltose, maltotriose and maltotetraose in a final concentration of 2 mM and a mixture of maltotetraose to maltodecaose (5 mM). The malto-oligosaccharides were dissolved in buffer solution of 50 mM borate with 5 mM CaCl 2 at pH 9.5 and the purified enzyme (147 mU / mL) was added. The substrates were incubated at 55 ° C and samples were removed at different time intervals. The reaction was stopped by boiling for 10 minutes and the samples were analyzed as described below.

Analysis of the hydrolysis products The hydrolysis products were detected using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PADD). A CarboPac PA-1 column (Dionex Corporation, Sunnyvale, CA, USA) was used with a gradient of 1.0 M sodium acetate from 0 to 60% over a period of 30 minutes in 100 mM NaOH and at a flow rate of 1.0 mL / minute on a Dionex DX-300 or DX-500 system. The hydrolysis products of the starch were identified by comparing their retention times with glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose. Since the retention times of homologous linear malto-oligosaccharides increase with the degree of polymerization, the linear malto-oligosaccharides of intermediate DP could be easily identified [40, 41].

RESULTS AND ANALYSIS Identification of ß. clausii BT-21 According to its composition of fatty acids, the strain showed similarity with the genus Bacillus. A partial sequencing of 16SrDNA showed a similarity of 99.4% with B. clausii. The physiological properties of the alkali tolerant strain confirmed this identification.

Production of amylase activity by B. clausii BT-21 Soluble potato starch, corn starch, corn amylopectin and whole brown rice yielded different levels of extracellular amylase activity in the medium. Although, corn starch contains more lipids and does not contain phosphorus compared to potato starch, amylopectin from corn has a highly branched structure containing a-D- (1-6) O-glycosidic bonds. These three types of starch are accessible to the enzymes after gelatinization, with heat, while whole brown rice contains a less accessible starch encapsulated in the rice grains. The amylase activities in the extracellular fluid or in the liquid cultures with the different starch substrates are shown in Figure 1. The highest amylolytic activity was obtained with whole brown rice as a substrate. This indicates that the presence that a less accessible starch substrate results in an increased production of extracellular amylolytic activities by B. clausii BT-21. Similar results were obtained with wheat bran, which however has difficulty in removing the extracellular fluid before purifying the enzyme. Carbon sources such as galactose, glycogen and inulin have previously been reported as appropriate for β-amylase production. licheniformis [27] and it has been found that soluble starch is the best substrate for the production of an amylase by means of β. stearothermophilus [28]. However, none of these studies has included a less accessible starch substrate.

Purification of product-specific amylase The enzyme was purified by affinity chromatography with β-CD Sepharose 6B followed by anion exchange chromatography (table 6).

TABLE 6 Purification of product-specific amylase from B. clausii BT-21 Volume Activity Activity Protein Activity Total Factor n (mL) (mU / mL) Total (mU) specific recovery (mg) (mU / mg protein purification Fluid 4000 155 620,000 424 731 100 1 extracellular chromatography 704.5 88 62,137 8.5 3,647 10.0 5 affinity with ß-CDD Concentration 172.6 254 43,840 4.8 4.581 7.1 6.3 and dialysis Chromatography 215 251 54.051 2.0 13.493 8.7 18.5 ammonium exchange Native PAGE stained by activity indicated the presence of three amylolytic activities in the extracellular fluid. The product-specific enzyme was completely separated from the other amylolytic activities after affinity chromatography with β-CD followed by anion exchange chromatography. The SDS-PAGE of the purified enzyme preparation indicated that the product specific amylase had been purified to homogeneity and having an apparent molecular weight of approximately 101 kDa. Affinity chromatography cyclodextrin sepharose 6B has previously been used to purify an α-amylase as a final purification step after removal of other amylases by anion exchange chromatography [29]. The 8.7% enzyme recovery and the purification factor of 18.5 obtained for the specific amylase products were similar with the values reported by the authors.

Characterization of the product specific amylase The purified enzyme showed an activity value at pH 9.5 (figure 2) while the optimum temperature for this activity was 55 ° C with or without the presence of 5mM CaCl2 (figure 3). The thermostabi of the enzyme at pH 9.5 in the presence of 5mM CaCl2 is shown in Figure 4. At temperatures above 55 ° C, the enzyme loses 75% of its maximum activity during an incubation period of 30 minutes. Five other product-specific amylases that form maltohexaose [23], maltopentaose [24] and maltotetraose [26] also show an optimum alkaline pH. It was estimated that the molecular weight of these enzymes is 59, 73 and 80 kDa [23], 180 kDa [24], and 97 kDa [26] while the product specific amylase from B. clausii BT-21 showed a weight estimated molecular weight of 101 kDa. The majority of product-specific amylases and α-amylase showed a lower molecular weight in the range of 50-65 kDa [3, 16, 17, 19, and 21]. The optimum temperature of approximately 55 ° C was similar to that reported for the product specific amylases above [23, 24 and 26]. The purified product-specific amylase hydrolyzed the soluble starch after one hour of incubation mainly to maltohexaose and maltopentaose (52% and 19% of the total hydrolyzed products) as the main starting products of low DP (Figure 5). After 2 hours of incubation the amount of maltohexaose and after 4 hours the amount maltopentase decreased while the amounts of maltotetraose, maltotriose, maltose and glucose were increased. These products accumulated after prolonged hydrolysis indicating that these were not further hydrolysed. After 24 hours of starch hydrolysis the amounts of malto-oligosaccharides were (3%) of maltohexaose, (4%) maltopentaose, (41%) maltotetraose, (13%) of maltotriose, (16%) maltose and (4%) ) of glucose. High performance anion exchange chromatography traces with pulsed amperometric detection obtained after 30 minutes of starch hydrolysis (Figure 6) show that the starch hydrolysis products greater than maltohexaose (DP6) were absent. A study of the time course of hydrolysis of soluble starch by a product-specific amylase that forms maltohexaose has also shown that maltohexaose occurred preferentially in the initial stage of hydrolysis [23]. Kim et al (1995 [26] found that the initial product after 1 hour of hydrolysis of starch with a product-specific amylase that forms maltotetraose was mainly maltohexaose (54%) followed by a gradual increase in the amounts of maltotetraose and maltose while that the amount of maltohexaose decreases After 20 hours, the composition of the malto-oligosaccharides has changed to 0.6% to maltohexaose, 1.3% maltopentaose, 52.2% maltotetraose, 8.3% maltotriose, 27.6% maltose and 9% glucose. To further examine the mode of action of the enzyme during the hydrolysis of starch, different substrates were incubated with the purified enzyme (table 7).

TABLE 7 Malto-oligosaccharides in the DP1 to DP6 range formed by hydrolysis of different starch substrates by the specific enzyme of purified product (67 mU / mL). The data indicate as% percent by weight of glucose formed compared to the initial amount of substrate Time Product formed (%) hydrolysis Substrate (min) DP1 DP2 DP3 DP4 DP5 DP6 Starch 15 0 0 0 < 0.1 0.9 7.2 soluble (1%) 240 0 0 0 0.6 7.0 17.8 Amylose 15 0.2 0 0 0 2.4 13.6 (0.1%) 240 1.5 0 4.1 7.7 6.3 20.0 Amylopectin (1%) 240 < 0.1 0.5 0.1 0.9 9.2 24.8 Starches are composed of amylose (20-30%) and amylopectin (80-70%). Amylose is a linear glucan with a-D- (1? 4) O-glycosidic bonds, whereas amylopectin is a branched glucan due to the presence of a-D- (1? 6) O-glycosidic bonds in the molecule. The product-specific amylase more readily hydrolyzes amylopectin indicated by the formation of maltopentaose (9.2%) and maltohexaose (24.8%) compared to soluble starch (7% and 17.8%) and amylose (6.3% 9 and 20%). The enzyme does not hydrolyze by swarming, a glucan with a- (1? 6) O-glycosidic bonds constituted by a structure of maltotriose, or dextran, a glucan with α- (1? 6) O-glycosidic bonds with branches attached to the base structure chain units 0-3. The α-, β-, and β-cyclodextrins, which are cyclic malto-oligosaccharides consisting of 6.7 and 8 glucose units, are also not hydrolyzed even after 24 hours of incubation. The results obtained on dextrin indicate that the α- (1-6) O-glucosidic bonds can not be cut with the product specific amylase. The lack of activity on pullulan indicates that the product-specific amylase can not pass continuous a- (1? 6) O-glycosidic bonds to 3 glucose units or attack any of these three glucose units. The lack of hydrolysis on a-, β-, or β-cyclodextrins indicates that the product-specific amylase hydrolyzed the starch by an exo-type cutting mechanism (30). The HPAEC-PAD trace (figure 6) also indicated an exo-type cleavage mechanism, since starch hydrolysis products greater than DP6 are absent.

To further examine the cutting action of the enzyme on the soluble starch, blue starch-iodine formation against the production of reducing sugars was plotted (figure 7). The slope of the curve indicates the prevailing type of mechanism for cutting the hydrolysis of amylolytic starch [31]. An enzyme with endo activity will produce a slope with a smaller value compared to an enzyme with exo action. A small value of the slope is the result of a rapid reduction of the blue color of the starch-iodine due to the random amylolytic activity, indicated by α-amylase from A. oryzae (the slope is -61). The extracellular enzyme preparation from P.stutzeri showed evidence of an exo-prevailing action rupture mechanism indicated by a larger slope value (the slope is -13). The specific amylase of purified product showed a slope value of -6, which indicates exo activity. The mode of action of hydrolysis of substrates with low DP for the product specific amylase was examined by incubating it with said substrates. Maltose, maltotriose and maltotetraose were not hydrolyzed by the amylase from B. clausii BT-21. This confirms the results obtained on soluble starch, that these products accumulate and are therefore considered as final products of hydrolysis. The product-specific amylase activity on a mixture of malto-oligosaccharides from DP4 to DP10 was studied by a time course experiment. The change of the maximum areas obtained by HPAEC-PAD corresponded to the formation or hydrolysis of malto-oligosaccharides. The formation of maltohexaose (DP6) and the simultaneous decrease in the amount of DP7, DP8, DP9 and DP10 confirmed the maltohexaose formation capacity of the enzyme. However, conditions were achieved in a continuous state and it was found that further degradation of DP6 by hydrolysis of starch was not detected even after 7 days of hydrolysis. The concentration of DP6 was much lower than that obtained in the hydrolysis of starch and indicates that a certain amount of maltohexaose was required for the formation of maltotetraose and maltose to proceed. It was found that the hydrolysis of starch by product-specific amylase from B. clausii BT-21 resembles a two-step procedure. This procedure includes an initial hydrolysis of starch mainly to maltohexaose and small amounts of maltopentaose, which were subsequently hydrolyzed mainly to maltotetraose and maltose accumulating after intense hydrolysis. It seems that the second step of hydrolysis to maltotetraose and maltose is limited to the preliminary hydrolysis of the larger substrate towards maltohexaose, since it seems that there is a concentration dependence for a regulator for the second step to advance.

BAKING EXPERIMENT A baking experiment was carried out with the product specific amylase. Doughs were prepared with ten grams of standard Danish wheat flour (Danisco 98078) and 6.2 ml of 0.2 M NaOH-glycine buffer, pH 10 without (control) or with 40 units of the enzyme (evaluated at 45 ° C and pH 10 as described in section B of materials and methods). It was baked and analyzed by DSC after storage in accordance with "tests to measure retrogradation and rancidity". As shown in table 8, the enzyme significantly reduces the amount of retrograde degraded amylopectin found on day 7 after baking which indicates that it has a significant anti-milking effect.

TABLE 8 Effect of product-specific amylase from B.clausii on retrogradation of starch based on measurement of enthalpy of fusion of retrograde degraded amylopectin (in J / g) 7 days after baking.

Multiple Interval Tests > for Entalpia by treatmentMethod: LSD at 95.0 percent treatment average count Enzyme 16 2,44375 Control 16 2,56 Contrast Differences Limits +/- Control - enzyme * 0, 11625 0,491094 * Indicates a statistically significant difference.

Samples of crumbs were extracted from the baked baked goods after baking with distilled water (1g of baked product / 10g of water, stirred for one hour and centrifuged) and analyzed by HPAEC-PAD as described above to detect the products of starch formed by the enzyme during the baking of the masses. The accumulation of maltotetraose, maltopentaose, maltohexaose, was found in relation to the control, as a result of the activity of this enzyme.

SUMMARY SECTION The present invention describes a process for making baked products, as well as the appropriate amylases to be used in such a process. Preferred embodiments of the present invention are presented below as numbered paragraphs.

References [1]. K.H. Park, Food Sci. Ind. 25 (1992) 73-82. [2]. M. Okada and T. Nakakuki, Oligosaccharides: production, properties and application, in F.W. Schenck and R.E. Hebeda (Eds.), Starch Hydrolysis products worldwide, production and application, VCH Publishers, New York, 1992, pp. 335-366. [3]. W.M. Fogarty, Microbial amylases, n W.M. Fogarty (Ed.) Microbial enzymes and biotechnology, Applied Science, London, 1983, pp. 1-92. [4]. W.M. Fogarty and C.T. Kelly, Starch-degrading enzymes of microbial origin, n M.J. Bull (Ed.), Progress in industrial microbiology, Vol. 15, Elsevier Scientific 1979, p. 87-150. [5]. K. Kainuma, S. Kobayashi, Y. Ito, and S. Suzuki, FEBS Letters, 26 (1972) 281-285. [6] N. Monma, T, Nakakuki, and K. Kainuma, Agrie. Biol. Chem., 47 (1983) 1769-1774. [7] J.F. Kennedy and C.A. White, Starch / Stárke 31 (1979) 93-99. [8] Y. Takasaki, Agrie. Biol. Chem. 46 (1982) 1539-1547. [9] H. Taniguchi, C.M. Jae, N. Yoshigi, and Y. Maruyama, Agrie.

Biol. Chem. 47 (1983) 511-519. [10] H. Taniguchi, Maltohexaose-producing amylase of Bacillus circulans F-2 in R.B. Friedman (Ed.) Biotechnology of amylodextrin oligosaccharides. ACS Symp. Ser. 458. American Chemical Society, Washington DC, 1991, pp. 111-124. [eleven]. F. Bealin-Kelly, C.T. Kelly, and W.M. Fogarty, Biochem. Soc. Trans., 18 (1990) 310-311. [12] W.M. Fogarty, F. Bealin-Kelly, C.T. Kelly, and E.M. Doyle, Appl, Microbiol. Biotechnol., 36 (1991) 184-489. [13] N. Saito, Archives. Biochem. Biophys., 155 (1973) 290-298. [14] H. Okemoto, S. Kobayashi, M-Monma, H. Hashimoto, K. Hara, and K. Kainuma, Appl. Microbiol. Biotechnol, 25 (1986) 137-142. [fifteen]. O. Shida, T. Takano, H. Takagi, K. Kadowaki, and S. Kobayashi, Biosci., Biotechnol., Biocehm. 56 (192) 76-80. [16] (There is no ref [16]) [17]. Y. Sakano. Y. Kashiwagi, and T. Kobayashi, Agrie. Biol. Chem., 46 (1982) 639-646.

[18] Y. Takasaki, H. Shinohara, M. Tsuruhisa, S. Hayashi, K. Imada, Agrie. Biol. Chem. 55 (1991) 1715-1720. [19] W.M. Fogarty, C.T. Kelly, A.C. Bourke, and E.M. Doyle, Biotechnol. Lett. 16 (1994) 473-478. [twenty]. K. Wako, S. Hashimoto, S. Kubomura, A. Yokota, K.

Aikawa, and J. Kamaeda, J. Jap. Soc. Starch. Sci 26 (1979) 175-181. [twenty-one]. Y. Takasaki, Agrie. Biol. Chem. 49 (1985) 1091-1097. [22] (There is no ref [22]) [23]. T. Hayahi, T. Akiba, and K. Horikoshi, Appl. Microbiol, Biotechnol. 28 (1988b) 281-285. [24] G. Schmid, A. Candussio, and A. Bock, U.S. Patent 5 304 723 (1992). [25] M. A. Me Tigue, C.T. Kelly, E.M. Doyle, and W.M. Fogarty, Enzyme Microb. Technol., 17 (1995) 570-573. [26] YOU. Kim, B.G. Gu, J.Y. Jeong, S.M. Byun, and Y.C. Shin, Appl. Environm. Microbiol. 61 (1995) 3105-3112. [27] A.K. Chandra, S. Medda, and A.K. Bhadra, J. Ferment. Technol., 58, (1980), 1-10- [28]. R.A.K. Sirvastava and J.N. Baruah, Appl. Environ. Microbiol. 52 (1986) 179-184. [29] V. Planchot and P. Colonna, Carbohydr. Res., 272 (1995) 97-109.

[30] J.F. Robyt and W.J. Whelan, The a-amylases in J.A. Radley (Ed.) Starch and its derivatives, 4 ed. Chapman and Hall, London, 1968, pp. 430-476. [31] M. Ohnishi and K. Hiromi, General considerations for conditions and methods of amylase assay in The Amylase Research Society of Japan (Ed.) Handbook of amylases and related enzymes. Their sources, isolation methods, properties and applications. Pergamon, Oxford, 1988, pp. -14. [32] L. Duedahl-Olesen, W. Zimmermann, and J.A. Delcour, Cereal Chemistry (1999) In press. [33] K.L. Larsen, L. Duedahl-Olesen, H.J.S. Christensen, F. Mathiesen, L.H. Pedersen, and W. Zimmermann, Carbohydr. Res. 310 (1998) 211-219. [3. 4]. M. M. Bradford, Anal. Chem., 72 (1976) 248-254. [35] Novex, Precast Gel Instructions. NOVEX, San Diego, CA, USES. [36] Mini Protean II Electrophoresis Cell instructions manual. Bio-Rad Laboratories, Hercules, CA, E.U.A. [37] H. Blum, H. Beier, and H.J. Gross, Electrophoresis (1987) 93-99. [38] L.H. Pedersen, H.J.S. Christensen, F. Mathiesen, K.L. Larsen, and W. Zimmermann, Starch, 49 (1997) 250-253. [39] H.J. Fuwa, J. Biochem., 41 (1954) 583-603.

[40] Y.C. Lee, J. Chormatogr. A, 720 (1996) 137-149. [41] R.N. Ammerall, G.A. Delgado, F.L. Tenbarge, and R.B. Friedmann, Carbohydr. Res. 215 (1991) 179-192.

Claims (22)

NOVELTY OF THE INVENTION CLAIMS

1. - A process for making a bread product comprising adding to a starch medium a non-maltogenic exoamylase having the ability to hydrolyze the starch by cutting one or more maltooligosaccharides, consisting predominantly of four to eight D-glucopyranosyl units, from the Non-reducing ends of the amylopectin side chains.

2. A process according to claim 1, further characterized in that the starch medium comprises flour, in which the flour is wheat flour or sorghum flour or mixtures thereof.

3. A process according to claim 2, further characterized in that the non-maltogenic exoamylase is added in an amount that is in the range of 50 to 100,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour , more preferred in an amount that is in the range of 200 to 20,000 units per kg of flour.

4. A method according to any of claims 1-3, further characterized in that the non-maltogenic exoamylase has an endoamylase activity of less than 0.5 units of endoamylase (EAU) per unit of exoamylase activity, preferably further characterized in that the Non-maltogenic exoamylase has an endoamylase activity of less than 0.05 units of endoamylase (EAU) per unit of exoamylase activity, more preferred still, further characterized because the non-maltogenic exoamylase has an endoamylase activity of less than 0.01 units of endoamylase (EAU) per exoamylase activity unit.

5. A method according to any of claims 1-4, further characterized in that the non-maltogenic exoamylase is also characterized in that the non-maltogenic exoamylase has the ability to give hydrolysis product (s), in an incubation test with starch of waxy corn, which could consist of one or more linear malto-oligosaccharides having from two to ten D-glucopyranosyl units and optionally glucose, such that at least 60%, preferably at least 70%, more preferred at least 80% and still more preferred at least 85% by weight of said hydrolysis product (s) consists of linear malto-oligosaccharides having from three to ten D-glucopyranosyl units, preferably malto - linear oligosaccharides consisting of four to eight D-glucopyranosyl units.

6. A method according to claim 5, further characterized by at least 60%, preferably at least 70%, more preferred at least 80% and even more preferred at least 85% of the Hydrolysis product is maltotetraose, maltopentaose, maltohexaose, maltoheptaose or malto-octaose.

7. - A method according to claim 6, further characterized by at least 60%, preferably at least 70%, more preferred by at least 80% and even more preferred by at least 85% of the product of hydrolysis is maltotetraose.

8. A method according to claim 7, further characterized in that the enzyme can be obtained from P. saccharophila or is a functional equivalent thereof.

9. A method according to claim 6, further characterized by at least 60%, preferably at least 70%, more preferred by at least 80% and even more preferred by at least 85% of the Hydrolysis product is maltohexaose.

10. A method according to claim 9, further characterized in that the enzyme can be obtained from Bacillus clausii or is a functional equivalent thereof.

11. A process according to claim 10, further characterized in that the enzyme has a molecular weight of about 101,000 Da (as estimated by electrophoresis in polyacrylamide-sodium dodecylsulfate).

12. A method according to claim 10 or claim 11, further characterized in that the enzyme has optimal activity at pH 9.5 and 55 ° C.

13. - A method according to any of the preceding claims, further characterized in that the bread product is a dough.

14. A method according to any of the preceding claims, further characterized in that the bread product is a baked dough.

15. A method according to any of the preceding claims, further characterized in that the bread product is used to prepare a bread flour baked product.

16. A method according to any of the preceding claims, further characterized in that the bread product is baked.

17. A baked product obtained by the process according to claim 16.

18. An improving composition for a dough, characterized in that the composition comprises a non-maltogenic exoamylase as defined in any of claims 1 to 17, and at least one additional ingredient for dough or a dough additive.

19.- A non-maltogenic exoamylase that can be obtained from Bacillus clausii or a functional equivalent thereof; characterized in that the enzyme has a molecular weight of about 101,000 Da (as estimated by electrophoresis in sodium polyacrylamide-dodecylsulfate) and / or the enzyme has optimal activity at pH 9.5 and 55 ° C.

20. - A non-maltogenic exoamylase according to claim 19, further characterized in that the non-maltogenic exoamylase is also characterized in that the non-maltogenic exoamylase has the ability to give hydrolysis product (s), in an incubation test with waxy maize starch, which could consist of one or more linear maltooligosaccharides having from two to ten D-glucopyranosyl units and optionally glucose; in such a way that at least 60%, preferably at least 70%, more preferred at least 80% and even more preferred at least 85% by weight of said product (s) of hydrolysis consists of linear malto-oligosaccharides having from three to ten D-glucopyranosyl units, preferably linear malto-oligosaccharides consisting of four to eight D-glucopyranosyl units.

21. The use of a non-maltogenic exoamylase in a bread product to retard the rancidity of the bread product.

22. A process, an improving composition, an enzyme or the use substantially as described in the present invention and with reference to claim 1.