MXPA97006706A - Method for liquiding almi - Google Patents

Abstract

According to the invention, there is provided a method for liquefying starch, comprising the steps of treating the starch prior to or simultaneously with the liquefaction of the starch, to inactivate and / or eliminate the enzyme inhibiting composition present in the starch, and form treated starch, the addition of (alpha) -amylase to the treated starch, and reacting the treated starch with an effective time and temperature to liquefy the treated starch. Effective means for treating the starch includes the addition of a phytate degrading enzyme and heat treatment, optionally followed by filtration or centrifugation, of the granular starch or a starch solution.

Description

METHOD FOR LIQUID STARCH BACKGROUND OF THE INVENTION The present invention relates to the modification of the use of α-amylase in the conversion of grain starch to side products, such as dextrose, fructose and alcohol. In particular, the present invention relates to the elimination and / or inactivation of an enzyme inhibitor composition from a granular starch prior to liquefaction. Grains such as corn have been widely used as a source of starch. One of the well-known methods for the separation and purification of starch for use in industrial processes is the wet milling process. This method has been developed in a highly specific and integrated system, designed to separate the main components of a grain or seed as completely as possible (see Stanley A. Watson, Starch: Cheiuistry __ &__ Jechnoloqy Vol. II.) Industrial Aspects, Academic Press, New York, 1967, pp. 30-51). In a common wet milling process, the dry beans used for the production of REF: 25530 starch products, are first subject to a soaking process called maceration. During the maceration the grains are subjected to a backflow of water which separates many soluble materials, including phytate and phytic acid, sugars, salts and proteins, from the granules of the seed. The macerated grains are separated from the soaking water (maceration water) and subject to mechanical breaking and grinding procedures. Flotation and centrifugation techniques are then used to separate the germ from the starch, fiber and protein. The resulting suspension of the endosperm (starch), fiber and protein, is then ground and sieved to separate the fiber. Finally, the protein and components related to the endosperm are separated based on the density through countercurrent rinsing and centrifugation to separate the starch from the protein / gluten stream. The isolated starch stream is freshly rinsed to remove any soluble materials related to the non-granular starch, including soluble materials such as inorganic salts, and compounds such as phytate and salts of phytic acid. The resulting product is a highly purified suspension of insoluble granular starch which serves as the initial product for conversion to fructose. In general, the processing of starch to fructose consists of four steps: liquefaction of the granular starch, saccharification of the liquefied starch in dextrose, purification, and isomerization to fructose. The objective of a starch liquefaction process is to convert a concentrated suspension of polymeric starch granules to a solution of short chain length dextrins, soluble, low viscosity. This step is essential for convenient handling with standard equipment and for efficient conversion to glucose or other sugars. To liquefy the granular starch it is necessary to gelatinize granules by raising the granular starch temperature above about 72 ° C. The heating process instantaneously breaks insoluble starch granules to produce a water soluble starch solution. The solution of solubilized starch is then liquefied by α-amylase (EC 3.2.1.1). A common process of enzymatic liquefaction involves adjusting the pH of a granular starch suspension to between 6.0 and 6.5, the optimum pH of the a-amylase derived from Bacillus le cheniformi s, with the addition of calcium hydroxide, hydroxide sodium or sodium carbonate. The addition of calcium hydroxide has the advantage also of providing calcium ions, which are known to stabilize α-amylase against inactivation. After addition to the α-amylase, the suspension is pumped through a steam jet to instantaneously raise the temperature to between 80 ° -115 ° C. The starch is immediately gelatinized and, due to the presence of α-amylase, depolymerized through random hydrolysis of (1-4) -glycosidic bonds by α-amylase to a fluid mass that is easily pumped. In a second variation of the liquefaction process, the α-amylase is added to the starch suspension, the suspension is maintained at a temperature of 80-100 ° C to partially hydrolyze the starch granules, and the suspension of partially hydrolyzed starch is pumped through a jet at temperatures greater than about 105 ° C to completely gelatinize any remaining granular structure. After cooling of the gelatinized starch, a second addition of the α-amylase can be performed to further hydrolyze the starch.

A third variation of this process is called the dry milling process. In dry grinding, the whole grain is ground and combined with water. The germ is optionally removed by flotation separation or equivalent techniques. The resulting mixture, which contains starch, fiber, protein and other grain components, is liquefied using a-amylase. The general practice in the art is to undertake enzymatic liquefaction at a lower temperature when the dry milling process is used. In general, low temperature liquefaction is believed to be less efficient than high temperature liquefaction in the conversion of starch to soluble dextrins. Typically, after gelatinization the starch solution is maintained at an elevated temperature in the presence of α-amylase until an ED of 10-20 is achieved, usually in a period of 1-3 hours. The dextrose equivalent (DE) is the industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. The non-hydrolyzed granular starch has an ED of virtually zero, while the DE of the D-glucose is defined as 100.

The maximum temperature at which the starch solution containing the α-amylase can be maintained depends on the microbial source from which the enzyme was obtained and on the molecular structure of the α-amylase molecule. The α-amylases produced by wild-type strains of B. subtili s or B. amyl oliquefaci ens, are typically used at temperatures no greater than about 90 ° C due to excessively rapid thermal inactivation above this temperature, while the a-amylases produced by the wild-type strains of B. The cheniformi s can be used at temperatures up to approximately 110 ° C. . It is known that the presence of starch and calcium stabilizes the α-amylases against inactivation. However, α-amylases are used at pH values above 6 to protect against rapid inactivation. At low temperatures, it is known that B-α-amylase. li cheniformi s shows excellent hydrolyzing activity on the starch substrate at pH values as low as 5. However, when the enzyme is used for hydrolysis of the starch at ordinary jet temperatures, for example between 102 ° C and 109 ° C, the pH must be maintained at least above pH 5.7 to avoid excessively rapid inactivation. The pH requirement unfortunately provides a narrow window of processing opportunity because pH values above 6.0 result in undesirable byproducts, for example, maltulose. Therefore, in reality, the pH of the liquefaction must be maintained between 5.9 and 6.0 to achieve a satisfactory yield of the hydrolysed starch. Another problem related to the pH of liquefaction, is the need to raise the pH of the starch suspension from about 4, the pH of a cornstarch suspension as it comes from the wet milling step, to 5.9-6.0. This pH adjustment requires the expensive addition of acid-neutralizing chemicals, and also requires additional refining by ion exchange of the final starch conversion product to remove the chemical. In addition, the next step after liquefaction, typically saccharification of the liquefied starch into glucose, requires a pH of 4-4.5; therefore, the pH must be adjusted below 5.9-6.0 to 4-4.5; requiring additional chemical addition and refining steps.

As is common in many plant seeds, phytic acid, the myoinositol hexaphosphate ester, is known to be present in the grains in the form of phytate salts, such as potassium phytate, calcium phytate and magnesium phytate. As indicated above, it has been the general belief that all significant amounts of phytic acid present in corn seeds are leached from the seeds during the maceration process to be removed from the liquefaction stream before "further processing." Surprisingly, as described herein, the Applicants have discovered that a form of phytate appears to be present in the granular starch subsequent to maceration and intensive rinsing. While not wishing to be bound by any theory, the Applicants believe that the residual phytate is effectively bound within the starch granule itself, and thus is not separated from the starch during the intensive rinsing processes. In U.S. Patent No. 5,322,778, liquefaction between pH 4.0 and 6.0 was achieved by the addition of an antioxidant such as bisulfite or a salt thereof, ascorbic acid or a salt thereof, erythorbic acid, or phenolic antioxidants such as butylated hydroxyanisole. -, butylated hydroxytoluene, or α-tocopherol to the liquefaction suspension. According to this patent, sodium bisulfite must be added in a concentration higher than 5 mM. In U.S. Patent No. 5,180,669, liquefaction between a pH of 5.0 to 6.0 was achieved by the addition of carbonate ions in excess of the amount needed to buffer the solution to the ground starch suspension. Due to an "increased pH" effect which occurs with the addition of carbonate ion, the suspension is generally neutralized by the addition of a source of hydrogen ion, for example, an inorganic acid such as hydrochloric acid or sulfuric acid. PCT Publication No. WO 94/02597, a mutant α-amylase having improved oxidative stability is described, wherein one or more methionines are replaced by any amino acid except cysteine or methionine. In PCT Publication No. 94/18314, it is described a mutant amylase having improved oxidative activity, wherein one or more of the residues of methionine, tryptophan, cysteine, histidine or tyrosine is replaced with a non-oxidizable amino acid.

In PCT Publication No. WO 91/00353, the problems associated with liquefaction are confronted by the genetic engineering of α-amylase to include features including increased thermal, acid and alkaline stability. In US Pat. No. 4,914,029 the phytase is added to the corn steep liquor to reduce the amount of phytic acid in the corn steep liquor, and thus to more efficiently use the maceration liquor of corn in feed. animals. Despite the advances made in the prior art, there is a need for an efficient medium for the liquefaction of starch at low pH levels using commercially available α-amylase. Similarly there is a need in the art for a method which allows the liquefaction of dry milled grain at higher temperatures. However, none of the methods described above provides the important advantages of allowing the elimination and / or inactivation of a composition responsible for the liquefaction of the starch at low pH, aided by inefficient or non-existent enzyme. Furthermore, none of the methods described above allows a flexible process for liquefaction, which does not require the addition of antioxidant or a neutralizing acid, the preparation of a genetically engineered enzyme, or the discovery of a new α-amylase, the which has exceptional stability characteristics at low pH.

BRIEF DESCRIPTION OF THE INVENTION An objective of this invention is to provide efficient low pH liquefaction of the starch, using readily available wild-type or mutant a-amylase enzymes. A further objective of the present invention is to provide the elimination and / or inactivation of an enzyme inhibiting composition, present in granular starch, which is primarily responsible for inefficient liquefaction by α-amylase at low pH. A further object of the present invention is to provide a method for liquefying the starch without the addition of expensive antioxidants. A further objective of the present invention is to provide a simple and efficient way to liquefy the starch, which allows flexibility in the method and does not require the use of a-amylase manipulated by genetic engineering. According to the invention, there is provided a method for liquefying the starch, comprising the steps of treating the starch before or simultaneously with the liquefaction of the starch to inactivate and / or remove an enzyme inhibiting composition, present in the starch, and form treated starch; the addition of α-amylase to the treated starch; and the reaction of the treated starch for an effective time and temperature to liquefy the treated starch. According to another embodiment of the invention, there is provided a composition of interest comprising a mixture of α-amylase and aqueous starch at a pH of less than 5.7, said composition containing either the enzyme inhibitory composition, inactivated or substantially free of enzyme inhibiting inactivating composition. As noted in more detail below, the practice of the present invention confers significant advantages to commercial processes of starch liquefaction. While not wishing to be bound by any theory, the Applicants believe that their discovery that a specific composition present in granular starch, unidentified to date as a substituent thereof, is responsible for the problems associated with low liquefaction. pH of the starch with α-amylase. From an elementary analysis of the isolated composition, this composition seems to comprise a phytate form. The surprising identification of the composition responsible for the problems of low pH liquefaction, allows the possibility of inactivating and / or eliminating the responsible agent and in this way efficiently liquefying the granular starch at low pH values, and as low as pH 4.5, with well-known and characterized α-amylases. As shown below, the Applicant's invention allows, for the first time, a process for liquefaction of the starch at low pH, which can be used with commercially viable systems, including α-amylase. In addition, the present invention does not require the use of a specially designed mutant or wild-type enzymes that exhibit exceptional stability characteristics at low pH, or costly measures such as the addition of antioxidants or acid neutralization. The invention itself, together with the additional objects and the advantages in question, will be better understood with reference to the following detailed description, taken in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION "Liquefaction" or "Liquify" means a process by which the starch is converted to shorter chain dextrins and less viscous. In general, this process involves the gelatinization of the starch simultaneously with or followed by the addition of α-amylase. "Maceration liquor" means a liquid that is extracted from the macerated grains during the maceration process. The maceration liquor contains a significant portion of the soluble components of the grain. "Granular starch" or "starch granules" means a water-insoluble component of edible grains, which remains after the removal of the husk, fiber, protein, germ, and soluble materials through the steps of maceration, mechanical breaking, separations, sifting, countercurrent rinsing and centrifugation, typical of the wet grinding process of the grains. The granular starch comprises intact starch granules which almost exclusively contain packaged starch molecules (eg, amylopectin and amylose). In corn, the granular starch component comprises approximately 99% starch; the remaining 1% is comprised of protein, ash, fiber, and trace components expressly associated with the grains. The packing structure of the granular starch severely repairs the ability of α-amylase to hydrolyze the starch. The gelatinization of the starches is used to break up the granules, to form a soluble starch solution and facilitate enzymatic hydrolysis. "Starch solution" means the water-soluble gelatinized starch, which results from the heating of the granular starch. After heating the granules above about 72 ° C, the granular starch dissociates to form an aqueous mixture of loose starch molecules. This mixture comprising, for example, about 75% amylopectin and 25% amylose in yellow-tilled corn forms a viscous solution in water. In commercial processes to form glucose or fructose, it is the starch solution that is liquefied to form a soluble dextrin solution. "Enzyme inhibition composition" or "EIC" means a composition in the granular starch which acts to inhibit the hydrolysis by α-amylase of a starch solution during liquefaction at low pH. The chemical analysis of a composition (EIC) extracted from the granules of gelatinized starch which acts to inhibit α-amylase at low pH, has revealed that the EIC comprises a form of phytate. The phytate forms comprising the inhibition composition of the enzyme are believed to be salts of phytate of magnesium, iron, potassium, manganese, zinc and / or calcium. "Treatment" is defined to mean the treatment of the granular starch or a starch solution, to decrease or eliminate an effect caused by the enzyme inhibiting composition during the enzymatic hydrolysis of the starch at low pH, for example, below pH 5.7 . The treatment includes, for example, the addition of a compound or compounds to the granular starch or a starch solution which acts to prevent the enzyme inhibition composition from destabilizing, inactivating or otherwise decreasing the hydrolyzing activity of starch characteristic of the -amylase; attaching the granular starch or a starch solution to conditions or separation techniques to eliminate or significantly decrease the inhibitory property of the enzyme inhibition composition prior to liquefaction at low pH, or the addition of a compound or compounds that eliminates the enzyme inhibitory composition of the solution, through the chemical modification of EIC to a non-EIC compound. "α-amylase" means an enzymatic activity that breaks down or hydrolyzes the (1-4) glycosidic bond, for example, that in starch, in amylopectin or in amyloid polymers. The appropriate α-amylases are the a-amylases of natural origin as well as the recombinant or mutant amylases, which are useful in the liquefaction of starch. Preferred amylases in the present invention are α-amylases derived from Bacillus, and particularly Bacillus li cheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus. The treatment of the starch according to the present invention allows the liquefaction reaction, for example enzymatic hydrolysis of the starch, amylopectin or amylose, to be carried out efficiently at a pH less than 6.0, or even lower than 5.0, in contrast to the liquefaction methods of the prior art. Preferably, the liquefaction reaction is carried out at a pH of between about 4.5 and about 5.7, more preferably, between about 4.5 and about 5.5 and more preferably between about 4.5 and about 5.2. In a preferred embodiment of the invention, the granular starch or a starch solution is treated to inactivate an enzyme inhibitor composition present therein, by heat treatment prior to the addition of the α-amylase. In this embodiment, α-amylase is preferably added to the granular starch or to the starch solution subsequent to heating, to ensure inactivation of the enzyme inhibiting composition, without first affecting the α-amylase. However, the addition of α-amylase simultaneously with the treatment step is considered within the scope of the present invention. The suspension is then incubated for an appropriate time at an appropriate pH and at an appropriate temperature, as is well known in the art, to liquefy the starch. According to the present invention, the enzyme inhibitor composition can be significantly decreased in its ability to inhibit α-amylase by heating the starch solution prior to liquefaction, for example, prior to the addition of α-amylase. Alternatively, it is considered within the scope of the invention to first incubate the starch solution with amylase at a lower temperature than, for example, 60 ° C to 90 ° C, to release the enzyme inhibitory composition of the starch or of the .solutions of gelatinized starch, before determining the liquefaction. Subsequently, the temperature is raised to liquefy the starch, raising the temperature to an appropriate temperature and for an appropriate sufficient time to substantially liquefy the starch. Preferably, the temperature is raised to about 80 ° C to about 115 ° C. Inactivation of the enzyme inactivating composition can occur during incubation at a lower temperature or during the increase and maintenance of temperature during liquefaction. In this embodiment, additional α-amylase can be added during incubation with α-amylase at low temperature or during or after subsequent liquefaction. As with the other embodiments of the invention, this mode will allow efficient liquefaction at pH values below 5.7. In another preferred embodiment, the granular starch or the starch solution is treated with a composition that chemically modifies or degrades the enzyme inhibitor composition, to eliminate the enzyme inhibiting characteristic thereof, and thereby eliminate the inhibitory composition of the enzyme. starch enzyme. Preferably, an enzyme is added which degrades the phytate to the starch granules or to the starch solution before liquefaction. A preferred phytate degradation enzyme comprises phytase or acid phosphatase. Many of the enzymes produced by microorganisms that catalyze the conversion of phytate to inositol and inorganic phosphate, are widely known as phytases. The phytase producing microorganisms comprise the filamentous fungi and bacteria, including the Bacillus subtilis, Pseudomonas, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus ni ger vari age to wamori, Aspergillus terreus, Aspergill usufum. Preferably, the phytase is derived from Aspergill usufum. The purification of such phytase enzymes from microbial sources is carried out by techniques known in the art. For example, in Ullah, and collaborators. Preparative Biochemistry, 18 (4), pp. 443-458 (1988) describes the purification of an aggregated phytase from Aspergill usufum, the disclosure of which is incorporated by reference herein. The concentration of the phytate degrading enzyme added to the granular starch of the starch solution must be effective to significantly degrade the enzyme inhibitor composition in the solution. Of course, the determination of a concentration of the phytate-degrading enzyme is dependent on the pH, the temperature, the reaction time, the specific enzymatic activity and, in addition, the type of grain from which the granular starch is obtained. or the starch solution. However, the optimal conditions for the activity of the phytate-degrading enzyme are readily assessable by one of skill in the art. Preferably, the concentration of the phytate-degrading enzyme is from about 0.1 to about 100 units of phytase (phytase unit) per gram of starch. More preferably, the concentration of phytate-degrading enzyme is from about 1 to about 25 phytase units per gram of starch. A phytase unit (phytate degradation activity) is defined as the amount of enzyme that will release 1 μ ol of inorganic phosphorus (P) of phytate Mg-K 0.042 M per minute at 37 ° C (Sigma Chemical Co., St. Louis , Mo.). It is contemplated that the phytate-degrading enzyme be added either to the granular starch or to the starch solution. It is believed that phytate degrading enzymes are effective for purposes of the present invention, however that if the starch is in granular form or soluble in solution. In fact, the Applicants have discovered that the addition of a phytate-degrading enzyme to granular starch, for example, prior to gelatinization, is effective for the solution treatment according to the present invention. Alternatively, the phytase can be added to the starch solution during or after gelatinization. According to another preferred embodiment, the inhibitory enzyme composition is inactivated and / or eliminated from the granular starch or from the starch solution before liquefaction. The removal of the enzyme inhibitor composition can be by any chemical or mechanical separation method recognized in the art, which is effective in removing the compounds including phytic acid or phytate salts from the solution. Suitable separation methods include chromatography, ion exchange, microfiltration and centrifugation. An especially preferred process comprising the removal by precipitation of phytate dependent on pH or heat treatment followed by filtration or centrifugation. Also preferably, removal of the enzyme inhibitor composition is achieved through high temperature centrifugation or high temperature filtration. The pH of the granular starch or of the starch solution during the treatment is any pH that allows the elimination or inactivation of the enzyme inhibiting composition. While the treatment step can be performed at any pH level, the treatment is efficient at a pH of between about 4 and about 6, because the undesirable need to adjust the pH level of the granular starch stream is eliminated. coming from the wet milling process. Preferably, the pH is between about 4.5 and about 5.7; more preferably, the pH is between about 4.5 and about 5.5; and more preferably, between about 4.5 and about 5.2. It should be noted that by maintaining the pH of the treatment step in these intervals, the pH of the treatment step will correlate well with that of the starch processing stream in wet ground grain, thus avoiding the excessive cost associated with adjusting the pH of the wet ground corn. The temperature of the treatment step is a suitable temperature for the elimination or "inactivation of the inhibitory composition of the enzyme, and may depend on the specific mode of the treatment chosen.Where the treatment step comprises the addition of the phytase, it must if a temperature is chosen which is appropriate for the hydrolysis activity for the specific enzyme For the microbial phytase, a suitable temperature will generally be between about 20 ° C and about 60 ° C and preferably between about 30 ° C and about 40 ° C. C. However, the development of temperature resistant phytase enzymes, which are capable of hydrolyzing the phytate at temperatures of, for example 100-110 ° C, are especially contemplated as being within the scope of the present invention, and are preferred, where the treatment step comprises heat treatment, optionally followed by filtration or centrifugation, temperature d It must be greater than the gelatinization temperature of the starch; preferably between about 80 ° C and about 150 ° C, more preferably between about 90 ° C and about 110 ° C, and more preferably between about 95 ° C and about 110 ° C. The time for the treatment step can also vary with the specific treatment-chosen type. Where the treatment step comprises the addition of phytase, the treatment time will depend on the specific activity of the added phytase enzyme, and on the temperature of the incubation. With microbial phytase, the treatment time is preferably from about 1 to about 24 hours and more preferably from about 3 to about 6 hours, depending on the conditions. Where the treatment step contains the removal of the enzyme inhibitory composition from the granular starch or from the starch solution of the heat treatment, optionally followed by filtration or centrifugation, the treatment time is preferably from about 10 seconds to about 60 minutes , and more preferably from about 3 to about 10 minutes. It is believed that the inactivation of the phytate from the granular starch can be essentially instantaneous under appropriate conditions, such as heat, and thus, the treatment time can be limited only by the thermal constraints. After the heat treatment, centrifugation or filtration can be used to separate the inactivated phytate from the solution by means recognized in the art. Where centrifugation is used to remove the phytate from the starch during the treatment, the centrifugation can be carried out at a force g of at least 2000 x g and preferably at a force g of between about 5000 x g and about 10,000 x g. The treatment conditions can also be affected by the type of grain or grind used. For example, a grain containing a relatively high concentration of enzyme inhibitor composition may require a longer treatment time than that of a grain having a lower concentration of the enzyme inhibitor composition. Variable levels of phytic acid content in different plant material are described in Lehrfield, J. Agrie. Food Chem., Vol. 42, pp. 2726-2731 (1994), incorporated by reference herein. When the grain is prepared for liquefaction by the dry milling process, it is likely that a much greater amount of phytate is present than in the wet milling process, due to the presence of the fiber and protein fraction. In this way, the treatment of dry milled starch may require more stringent conditions than wet milled starch. Subsequent or, simultaneously with the treatment of the granular starch or the starch solution to inactivate and / or remove the enzyme inhibiting composition, the α-amylase is added to the starch to liquefy the starch to lower molecular weight dextrins. Thus, it is contemplated as within the scope of the invention to either treat the granular starch or the starch solution before or simultaneously with the liquefaction using α-amylase. The liquefaction can be carried out according to any well-known liquefaction technique, which uses a-amylase. The pH within the liquefaction step according to the invention is preferably less than about 5.7, more preferably less than 5.3, and more preferably between about 4.5 and about 5. The following examples are representative, and not limiting, of the advantages conferred to through the use of the present invention. However, someone of ordinary skill in the art could be able to substitute the conditions, grains, temperature, enzymes and the like according to the above description.

Example 1 Assay for the Determination of α-Amylase Activity The α-amylase activity was determined through an assay that depends on the ability of the starch to form a colorful blue iodine complex, and the disappearance of this color, when the starch is hydrolyzed to shorter dextrin molecules. The activity of α-amylase was defined in terms of the digestion time required to produce a color change denoting a defined state of starch dextrinization. The reagents used were as follows: Amount of phosphate to phosphate diacid potassium (340 g) and sodium hydroxide (25.3 g) were dissolved in water and diluted to approximately 2 liters. The buffer was cooled to room temperature and the pH adjusted to 6.2 ± 0.1. The buffer was diluted to 2 liters in a volumetric flask. Substrate substrate - Ten grams (dry substance) of "soluble lintner starch were suspended in 50 ml of water and washed in approximately 300 ml of boiling water, the suspension was boiled again and boiled for 5 minutes. With constant stirring, the starch solution was cooled with constant stirring to room temperature and 125 ml of phosphate buffer was added The solution was diluted to 500 ml with water.The starch substrate was freshly processed daily. of reserve iodine - iodine crystals were dissolved (5.5 g) and potassium iodide (11.0 g) in water and were diluted volumetrically to 250 ml. The solution was protected from light. Diluted iodine solution - potassium iodide (20 g) and 2 ml of stock iodine solution were dissolved in water and diluted volumetrically to 500 ml. The solution was made fresh daily. Dilution Solvent in Enzyme - Calcium chloride (11.1 g) was dissolved in 4 liters of water. The water used for all the reagents was either distilled or deionized water.

The unknown a-amylase sample was diluted to between 10-15 LU / ml (as defined below) with enzyme dilution solution. For many commercial preparations of α-amylase, an appropriate dilution was found to be diluted at 2000. Aliquots of five milliliters of diluted iodine solution were filled into 13 x 100 mm test tubes and 10 ml of the substrate was placed starch in a 23 x 200 mm test tube All tubes were placed in the water bath at 30 ° C. A Hellige comparator equipped with a special a-amylase disc (number of 620-s5 catalog) 5 milliliters of the diluted enzyme (also at 30 ° C) was mixed with the starch substrate and time was taken in. At appropriate time intervals, for example 1 minute intervals at 15 minute intervals. seconds after the reaction, aliquots of 1 ml of the enzyme mixture of a substrate were transferred to a tube containing the diluted iodine solution quenched.The solution of iodine and starch was mixed and transferred to a square tube of precision of 13 m m and the color was compared with the standard a-amylase color disc in the Hellige comparator. When the time of the end point approached, the samples were taken at intervals of 0.25 minutes.

The time required for the colors of the samples and the color disc to appear was recorded and the activity was calculated (in liquefons per gram or mi) according to the formula: ^ 70 LU / ml or LU / g =? - x D Where LU = unid i liquefón V volume of enzyme (5 ml) t = time of dextrinization (minutes) D = dilution factor: volume of dilution -í- milliliters or grams of diluted enzyme.

Example 2 Starch Liquefaction Condition - Determining the DE (Dextrose Equivalent) of Liquefied Starch The liquefaction of the starch was performed using a reactor composed of stainless steel pipe of 15.24 m (50 feet) in length of 6.1 mm (0.24 inches) (5.3 mm (0.21 inches) in "internal" diameter) flexed in a 25.4 cm coil (10 inches) in diameter and approximately 14 cm (5.5 inches) in height The coil was equipped in a 29.2 cm (11.5 inch) in line static mixer (ColeParmer # G-04669-60) mounted approximately 1. 22 m (4 ft) from the front end. The rear end of the coil was equipped with an adjustable Swagelo in-line pressure relief valve (# SS-4CA-3) regulated at a disintegration pressure of approximately 1.4 kg / cm '(20 psi). The starch suspension was fed to the coil at a rate of about 70 ml / minute with a piston metering pump. The coil was heated by immersion in a glycerol-water bath heated to 105.5 ° C. The temperature in the bath was maintained using a circulation heater / temperature controller (Fisher Scientific model 7305). The granular starch was obtained from a wet mill for corn and used within two days. As another source of starch, LO-DEX ™ 10 (a purified, water soluble dextrin produced by limited hydrolysis of corn starch) was purchased from American Maize-products Company, Hammond, Indiana. The LO-DEX ™ 10 used herein had an initial SD of about 9.5. The starch or maltodextrin was diluted to a level of approximately 30-35% dry solids with deionized water, and the pH was adjusted with 2.5% NaOH or 6% HCl as required. Calcium was added in the desired form of CaCl2 »2H20. The typical conditions of liquefaction were: Starch or LO-DEX ™ 10 30% -35% solids Calcium 40-60 ppm (30 ppm added) pH 5.0 - 6.0 a-Amylase 12 - 14 LU / g carbohydrate (dry base) Starch or LO-DEX 10 containing enzyme and calcium in the form of CaCl2 »2H20 was introduced into the reactor at approximately 70 ml / min. The temperature of the reactor was maintained at 105.5 ° C by immersion of the reactor, in a glycerol-water bath. The starch samples were transferred from the reactor to a second liquefaction bath stage at 95 ° C and maintained for 90 minutes. The degree of liquefaction of the starch was measured immediately after the second stage of liquefaction by the determination of dextrose equivalent (DE) of the sample according to the method described in Standard Analyti cal Methods of the Member Compani es of the Com Refiners Associa ti on, Inc , 6a. ed., Analitical Procedure Committee (1980).

Example 3 HPLC analysis of Fitato The analysis for the phytate was carried out through HPLC (High Liquid Chromatography Resolution) as follows. An HPLC system consisting of a Millipore / Waters Automated Gradient Controller, model 510, Waters a column of 250 mm by 4.6 mm (of internal diameter) packed with a Poros 20 PI / M resin (PerSeptive Biosystems) and a detector of Dionex conductivity equipped with a Dionex anion suppressor and a Dionex SRS controller was used. Samples of commercial phytate, EIC derived from the maize gluten stream, whole maize milled, maceration, ground whole wheat flour, ground rice, and EIC of the granular starch, were diluted with deionized water to between 10-200 mg / L of phytate and filtered to remove any insoluble material. Between 20 and 500 ml of the sample ^ (depending on the concentration of the phytate) were injected into the column and the column was washed with water for 2 minutes at a flow rate of 1.3 ml / min. After 2 minutes, a linear gradient from 0 to 40 mM NaOH was started and the next 20 minutes proceeded at a flow rate of 1.3 ml / min. Phytate eluted from the column after approximately 15 minutes. A linear series of dilutions of sodium phytate (Sigma Chemical Company, #P 8810) was used to calibrate the response of the conductivity detector. It was found that the EIC of each source resulted in elution peaks identical to the commercial phytate.

Example 4 Isolation of EIC from a Corn Gluten Current A composition that inactivates or inhibits α-amylase was isolated from a protein-rich starch stream (termed the corn gluten stream) generated during the fractionation of the corn endosperm in a corn wet mill, as follows. The insoluble protein and grams of starch were removed from the corn gluten fraction (approximately 18.6% solids) by centrifugation (approximately 6000 x g for 15 min.). The supernatant was further clarified by vacuum filtration through a Whatman # 3 filter paper. The filtrate was fractionated by ultrafiltration using a cut polysulfone hollow fiber cartridge of 5,000 molecular weight (A / G Technology Corp., model UFP-5-D-4). Approximately 1200 ml of the filtrate from the ultrafiltration step were adjusted to pH 9 by the addition of 1 N NaOH. The precipitate that formed was recovered by centrifugation (approximately 6000 x g for 10 minutes) and washed by resuspension in water.

After the recovery of the precipitate from the washing water by centrifugation (approximately 6000 xg for 10 minutes), the precipitate was resuspended in approximately 500 ml of water and dissolved by adjusting the pH slowly to 5, by dropwise addition of 3 M HCl. The solution was filtered through Whatman filter paper. # 3 to remove any undissolved material and the filtrate was cooled to approximately 4 ° C. Two volumes of ethanol (at 4 ° C) were added to the filtrate and the resulting precipitate was recovered by filtration through a sintered glass filter. The precipitate was washed with cold ethanol, recovered and placed under vacuum at room temperature to dry it. The 1200 ml ultrafiltrate produced 3.04 g of EIC.

Example 5 Inhibition of α-Amylase by EIC During Liquefaction at low pH The EIC (from 0 to 200 mg / liter) isolated as in Example 4, was added to LO-DEX ™ 10 at 35% (pH 5.2) containing 50 ppm of calcium, α-amylase was added (SPEZYME® AA20, produced by B. licheniformi s and commercially available from Genencor International, Inc., South San Francisco, CA) at a ratio of 12 LU / g of carbohydrate and the pH of the solution was adjusted and maintained at pH 5.2 by the addition of NaOH 2.5% or 6% HCl, as required. The solution was hydrolyzed using the reactor system described in Example 2. The degree of hydrolysis of LO-DEX ™ 10 was measured by dextrose equivalent (DE) immediately after secondary retention. Table 1 illustrates that increasing concentrations of EIC reduce the final ED of the liquefied LO-DEX ™ 10 (initial SD of 9.5), indicating increased inhibition of α-amylase.

TABLE 1 Effect of EIC on the hydrolysis of LO-DEX ™ 10 Catalyzed by α-Amylase, at pH 5.2 EIC (mg / liter) DE 0 18.7 50 17.5 100 15.8 EIC (mg / liter) DE 150 13.9 200 12.9 As can be seen from this example, the presence of EIC significantly reduces the effectiveness of α-amylase at pH 5.2.

Example 6 Dependence of the pH of the EIC Inhibition of α-Amylase The EIC (200 mg / liter) isolated as in the Example 4, LO-DEX ™ 10 was added to 35% containing 50 ppm of calcium, and the pH was adjusted to about 6.0 or 5.2, α-amylase was added (SPEZYME® AA20, produced by B. licheniformis and commercially available from Genencor International, Inc.) at a ratio of 12 LU / g of carbohydrate, and the pH of the solution was adjusted and maintained either at pH 5.2 or 6.0 by the addition of 2.5% NaOH or 6% HCl, as required. The solution was hydrolyzed using the reactor system and the procedure described in Example 2. Identical controls but not containing EIC were performed at pH 5.2 and 6.0 at the same time as the test samples.

The degree of hydrolysis of LO-DEX ™ 10 was measured by dextrose equivalent (DE) immediately after secondary retention. The results are given in Table 2. As can be seen, the inhibition of α-amylase by EIC during the hydrolysis of LO-DEX ™ 10, is pH dependent. At pH 6.0, the addition of 200 mg / L of EIC caused only a reduction of approximately 6% in the development of ED. At pH 5.2, the development of ED was reduced to approximately 65%.

Table 2 Effect of pH on the inhibition of the EIC of a-amylase during the hydrolysis of the LO-DEJff110 pH EIC (mg / L) DE 6. 0 0 19.2 6.0 200 18.6 5.2 0 18.7 5.2 200 12.9 Example 7 Elemental Analysis of EIC The EIC isolated from the corn gluten fraction, as in Example 4, was subjected to elemental analysis by atomic adsorption spectroscopy (Galbraith Laboratories, Inc. Knoxville, TN). The results are given in Table 3.

Table 3 Elemental Analysis of EIC Carbon 7.41% Magnesium 10.32 Hydrogen 2.48 Manganese 0.07 Nitrogen < 0.05 Zinc 0.05 Phosphorus 22.38 Iron 0.04 Calcium 0.78 Ashes 69.50 This analysis was consistent with that of a mixture of magnesium, manganese, zinc or iron salts of phytic acid. Subsequently, the EIC was analyzed for the phytate content, by HPLC analysis as described in Example 3. The analysis indicated that the anionic component of EIC was substantially comprised of phytate.

Example 8 Isolation of EIC from Whole Whole Corn 250 g of ground whole corn were suspended in 500 g of deionized water and the pH of the resulting suspension was adjusted to 4 with 6% HCl. The suspension was stirred for approximately 8 hours and then allowed to stand for approximately 10 hours The suspension was separated by filtration on Whatman # 3 filter paper and the filtrate (approximately 310 g) was adjusted to pH 9 with 1 M NaOH. The generated precipitate was recovered by filtration through a 0.45 m membrane filter and washed with water.The precipitate was suspended in water to form a solution and the pH was adjusted to 5 by slow addition of 6% HCl. filtered to remove any insoluble material and cooled to approximately 4 ° C. Two volumes of cold ethanol were added and the precipitate that formed was recovered by centrifugation.The precipitate was washed once with cold methanol and dried overnight under vacuum at room temperature.

From 250 g of ground whole corn, 0.772 g of EIC were recovered. EIC was confirmed by HPLC analysis for its presence of characteristic phytate as in Example 3. The identification of the EIC was confirmed by the addition of isolated EIC to a liquefaction mixture of LO-DEX ™ 10 at pH 5.2, and determining if it was inhibited a-amylase EIC (200 mg / liter) from ground whole corn was added to LO-DEX ™ 10 to 35% containing 50 ppm of calcium ion in the form of CAC12 »2H20 and the pH was adjusted to approximately 5.2. Α-Amylase (SPEZYME® AA20, produced by ^ B. licheniformis and commercially available from Genencor International, Inc.) was added at a ratio of 12 LU / g carbohydrate, and the pH of the solution was adjusted to pH 5.2 by addition of 2.5% NaOH or 6% HCl, as required. The solution was hydrolyzed using a reactor system and the procedure described in Example 2. An identical control but not containing EIC was made at pH 5.2 at the same time as the test sample. The degree of hydrolysis of LO-DEX ™ 10 was measured by dextrose equivalent (DE) immediately after the second retention.

The results of the tests, shown in Table 4 below, are consistent with those generated when using EIC isolated from the maize gluten stream shown above.

Table 4 Effect of EIC isolated from Whole Corn Powdered on the Stability of α-Amylase During the Hydrolysis of LO-DEX1 * 10 to pH 5.2 DE sample 200 mg / L of EIC 9.5 Control 16.3 The HPLC analysis of EIC isolated from the whole ground corn was carried out as described in Example 3. One hundred microliters of a 15 mg / L EIC solution was injected into the HPLC column and was only found in the material an anionic peak, identified by the elution time as a phytate. The HPLC profile was substantially identical to that for EIC isolated from the corn gluten fraction.

Example 9 Isolation of EIC from Corn Maceration Liquor The maceration liquor of corn, weighed, obtained from a wet milling plant of corn, for example, in concentrate of evaporated maceration water, having a density of approximately 19 Bé was clarified by centrifugation. One liter of rinse liquor was adjusted from pH 4.2 to 18.1 by the addition of 5 M NaOH. The resulting precipitate was collected by centrifugation, suspended in water to wash the precipitate and again collected by centrifugation. approximately 400 ml of water and the pH of the suspension was adjusted to approximately 4 by the slow addition of 6% HCl After the precipitate had dissolved, the solution was filtered through Whatman # 3 paper to remove any Insoluble material, and the filtrate was cooled to approximately 4 ° C. The EIC was precipitated into the solution by the addition of 2 volumes of ice-cooled ethanol and was collected by centrifugation.The precipitate was washed once with cold ethanol, it was collected by centrifugation and dried under vacuum at room temperature. From 1 liter of heavy maceration liquor, 23.3 g of EIC were recovered. The identification of the EIC was confirmed by evaluation of inactivation of α-amylase during the hydrolysis of LO-DEX ™ 10 at pH 5.2 and by HPLC analysis for phytate. To determine the effect of EIC from corn steep liquor on liquefaction "using α-amylase, 200 mg / liter was added to LO-DEX ™ 10 to 35% containing 50 ppm of calcium ion in the CaCl 2 form * 2H20 and the pH was adjusted to approximately 5.2, α-amylase (SPEZYME® AA20, produced by B. licheniformis and commercially available from Genencor International, Inc.) was added at a ratio of 12 LU / g carbohydrate and the pH of the solution was adjusted to pH 5.2 by the addition of 2.5% NaOH or 6% HCl as required The solution was hydrolyzed using the reactor system and the procedure described in Example 2. An identical control but not containing EIC was used. performed at pH 5.2 for comparison.The degree of hydrolysis for LO-DEX ™ 10 was measured by dextrose equivalent (DE) immediately after secondary retention.An identical control was performed at pH 5.2 except for not having EIC aggregate. results are shown in Table 5 These results are consistent with those generated when using EIC isolated from the maize gluten stream shown above.

Table 5 Effect of Isolated EIC of the Corn Maceration Liquor on the Stability of a-Amylase during the Hydrolysis of LO-DEX ™ 10 to pH 5.2 Sample of 200 mg / L of EIC 10.9 Control 14.9 HPLC analysis of the EIC isolated from the corn steep liquor was performed as described in Example 3. One hundred microliters of a solution at 200 mg / L of EIC was injected to the HPLC column and only one material was found in the material. anionic peak, identified by the elution time as phytate. The HPLC profile was substantially identical with that for EIC isolated from the corn gluten fraction.

Example 10 Identification of EIC in Granular Corn Starch A suspension of granular corn starch from a wet corn mill was filtered on Whatman # 3 filter paper to separate the water from the starch granules. The granular starch was resuspended with deionized water and refiltered to remove any water soluble components of the insoluble starch.The granular starch was then resuspended in water and diluted to 35% solids.Al-amylase was added (12 LU /. g of carbohydrate) from B. licheniformi s (SPEZYME® AA20, commercially available from Genencor International, Inc.) and calcium (50 ppm) and in the granular starch suspension was liquefied at pH 5.2 as described in Example 2. The water recovered from the first filtration of the corn starch suspension was used to dissolve LO-DEX * "10 to produce a 35% sodium solution. The a-amylase (12 LU / g carbohydrate) and the calcium ion added as CaCl2 »2H20 (50 ppm) were added and the solution was liquefied at pH 5.2 as described in Example 2. A control containing LO-DEX ™ 10 dissolved in deionized water, instead of the filtrate water from the corn starch suspension, was liquefied at the same time.

The EIC was not detected in the water of the filtrate from the granular starch suspension when it was analyzed for its presence of characteristic phytate by means of HPLC. HPLC analysis at pH 6 of the liquefied granular corn starch, however, detected an elution peak which indicated the presence of a substance identical to the EIC isolated from the corn gluten in Example "4. The HPLC analysis showed the presence of between 30 and 40 mg of phytate per liter of 30% by weight solids of liquefied granular starch Comparing the liquefaction results of α-amylase in the maltodextrin mixed with granular starch filtrate water and α-amylase in LO -DEX ™ 10 mixed with deionized water, confirmed that the EIC present in the granular starch was not washed out.As a result, the results of these experiments, shown in Table 6 below, suggest that the EIC responsible for the inactivation of the α-amylase during the liquefaction of starch, is associated with starch granules, not free in solution.

Table 6 Liquefaction with Filtering Water from the Corn Starch Granules Washed Shows 90 min. FROM Washed Corn Pellets ~ 0 LO-DEX ™ 10 in Deionized Water 17.9 LO-DEX ™ 10 in Filtered Water 18.1 Derived from Starch Suspension Example 11 Inactivation of EIC Fitaea ml of EIC isolated as in Example 4, were treated with 500 units of phytase (from A. ficuum, Sigma Chemical Company, P 9792) for 30 minutes at pH 2.5, at 37 ° C. 10 ml of the treated EIC was added to LO-DEX ™ 10, to produce 1 liter of 35% solution of LO-DEX ™ 10 containing 50 ppm of added calcium ion as CaCl2 »2H20 and 200 mg / 1 of EIC. The a-amylase derived from B. li cheniformi s (SPEZYME® AA20, commercially available from Genencor International, Inc.) was added at a ratio of 12 LU / g carbohydrate and the solution was liquefied at pH 5.2 using the reactor system and the process described in Example 2 above . Consistent controls of LO-DEX ™ 10 without non-added EIC and LO-DEX ™ 10, containing 200 mg / ml untreated EIC, were liquefied at the same time. As shown in Table 7, the treatment with inactive phytase EIC thus preventing its ability to inhibit α-amylase.

Table 7 Treatment with Fitasa on the inactivation of EIC of α-Amylase Shows 90 min. FROM Free Control of EIC 15.4 200 mg / L of EIC 9.5 200 mg / L of EIC Treated with Phytase 14.4 Example 12 Phytase Treatment of Granular Corn Starch Phytase (from A. fi cum, 5 ml of 250 units / ml obtained from Sigma Chemical Co. , St. Louis, MO., Product No. P9792) to 1 liter of 34% granular corn starch containing approximately 50 ppm of added calcium ion as CaCl2 »2H20, and incubated for 5 i, oras at 37 ° C , pH 4.0. After incubation the pH of the granular starch suspension was adjusted to pH 5.2 and 12 LU / g carbohydrate α-amylase derived from B. licheniformis (SPEZYME® AA20, commercially available from Genencor International) was added to the granular starch suspension. , Inc.). The mixture was liquefied using the reactor system described in Example 2. A control liquefaction at pH 5.2 was performed at the same time using a suspension of 34% granular corn starch which had not been treated with the phytase. In a second test, the phytase (wheat, Sigma Chemical Company P1259, 160 units) was incubated with 1 liter of 35% corn granular starch suspension containing approximately 50 ppm of added calcium ion as CaCl2 »2H20 for 6 hours at 55aC, pH 5.15. After incubation, the pH of the granular starch suspension was adjusted to pH 5.5 and 12 LU / g of α-amylase carbohydrate derived from B. licheniformi s (SPEZYME® AA20, commercially available from Genencor International) was added to the suspension. , Inc.). The mixture was liquefied using the reactor system and the process as described in Example 2. Control liquefaction at pH 5.5 was performed at the same time using a 35% suspension of corn granular starch, not treated with phytase. As shown in Table 8, the phytase treatment of granular corn starch before liquefaction increases the ability of α-amylase to hydrolyze the starch at low pH.

Table 8 Effect of Phytase on the Stability of a-Amylase During Liquefaction at Low pH 90 min. , PH of the Liquefaction Control Starch Treated with Phytase 5.2 ~ 0 1.4 5.5 6.4 8.6 Example 13 Hot Filtration of Maltodextrin Containing EIC EIC (200 mg / liter) isolated as in Example 4, was added to LO-DEX ™ 10 at 35% w / w containing 50 ppm of added calcium in the form of CaCl2"2H20 and the pH was adjusted to approximately 5.2 . The solution was divided into two parts. One part was heated to approximately 100 ° C and immediately filtered under vacuum through a Whatman # 3 filter paper while hot. The second part was filtered under vacuum through a Whatman filter paper # 3, while it was at room temperature (for example, 20-25 ° C). Α-Amylase (SPEZYME® AA20, produced by B. Li cheniformis and commercially available from Genencor International, Inc.) was added to each solution at a ratio of 12 LU / g carbohydrate, and the pH of each solution was adjusted to pH 5.2 by the addition of 2.5% sodium hydroxide or 6% hydrochloric acid as required. Each solution was hydrolyzed using the reactor system and the procedure described in Example 2. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (DE) immediately after secondary retention. Samples of both solutions taken before hydrolysis were analyzed by the phytate as described in Example 3. As shown in Table 9, the filtration of LO-DEX ™ 10 containing EIC at about 100 ° C, eliminates approximately 75% of EIC coming from the solution. As a result, less α-amylase was inactivated during liquefaction at pH 5.2 and the DE of the resulting hydrolyzate was substantially higher.

Table 9 Effect of Hot Filtration on the EIC Inactivation of a-Amylase During Liquefaction at Low pH Sample Conc. Of EIC DE Cold Filtered Maltodextrin 184 mg / L 11.5 Hot Filled Maltodextrin 45 mg / L 14.5"Non-Hydrolyzed" Maltodextrin - 9.5 Example 14 Isolation of EIC from Brown Rice Basmati brown rice (commercially available from Lundburg Mills) was ground to a flour consistency, using a small coffee mill. Two hundred grams of ground rice were added to 500 ml of deionized water and the pH adjusted to approximately 3 with 6% HCl. The suspension was allowed to stand for 30 hours with occasional stirring and then diluted by vacuum filtration through Whatman # 3 filter paper. The pH of the filtrate was adjusted to 9 by addition of 1M NaOH and the precipitate that was developed was recovered by centrifugation. The precipitate was washed once with water, collected by centrifugation and resuspended in water. The pH of the suspension was slowly adjusted to about 5 by the dropwise addition of 6% HCl, and the suspension was stirred to dissolve the precipitate. The dissolved material was removed by filtration through a 5 m membrane filter and the filtrate was cooled to about 4 ° C. Two volumes of cold ethanol were added to the filtrate and the precipitate that formed was recovered by centrifugation. The precipitate was washed once with cold ethanol and dried under vacuum at room temperature. Two hundred grams of ground rice produced 0.3571 g of EIC. The identification of EIC was confirmed by evaluation of inactivation of α-amylase during hydrolysis of LO-DEX ™ 10 at pH 5.2 and by HPLC analysis for phytate. The EIC (200 mg / liter) from ground rice was added to LO-DEX ™ 10 to 35% containing 50 ppm of added calcium ion as CaCl2 »2H20 and the pH is adjusted to approximately 5.2. B-derived amylase was added. li cheniformi s (SPEZYME® AA20, commercially available from Genencor International, Inc.) at a ratio of 12 LU / g carbohydrate and the pH of the solution was adjusted to pH 5.2 by the addition of 2.5% sodium hydroxide or acid hydrochloric acid at 6%, as required. The solution was hydrolyzed using the reactor system and the procedure as described in Example 2. An identical control was performed but did not contain EIC at pH 5.2 at the same time as the test sample. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (SD) immediately after secondary retention. The results of the tests are shown in Table 10.

Table 10 Effect of Isolated EIC of Ground Rice on the Stability of a-Amylase During the Hydrolysis of LO-DEX? * 10 to pH 5.2 Control Sample 16.3 200 mg / L of EIC 11.7 HPLC analysis of EIC isolated from the milled rice was carried out as described in Example 3. One hundred microliters of a solution at 15 mg / L of EIC was injected into the HPLC column and was only found in the shows an anionic peak, identified by the elution time as phytate. The HPLC profile was substantially identical to that of EIC isolated from the corn gluten fraction.

Example 15 Isolation of EIC from Whole Wheat Flour Two hundred grams of commercial whole wheat flour (commercially available from Arrowhead Mills) were added to 500 ml of deionized water containing 12 M D, L-dithiothreitol (Sigma Chemical Co.). The pH of the suspension was adjusted to approximately 3 with 6% HCl, and the suspension was allowed to stand at room temperature for approximately 30 hours with occasional stirring. The suspension was divided by centrifugation.

The pH of the filtrate was adjusted to 9 by the addition of 1 M NaOH and the precipitate that was developed was recovered by centrifugation. The precipitate was washed once with water, collected by centrifugation and resuspended in water. The pH of the suspension was slowly adjusted to about 5, by the dropwise addition of 6% HCl and the suspension was stirred to dissolve the precipitate. The undissolved material was removed by filtration through a 5 m membrane filter and the filtrate was cooled to about 4 ° C. Two volumes of cold methanol were added to the filtrate and the precipitate that formed was recovered by centrifugation. The precipitate was washed once with cold methanol and dried in vacuo at room temperature.

Two hundred grams of whole corn flour produced 0.385 g of EIC. The identification of the EIC was confirmed by evaluation of the inactivation of α-amylase during the hydrolysis of LO-DEX ™ 10 at pH 5.2 and by HPLC analysis for phytate.

EIC (150 mg / liter) from whole wheat flour was added to LO-DEX ™ 10 to 35% containing 50 ppm calcium and the pH was adjusted to approximately 5.2. B-derived amylase was added. li cheniformis (SPEZYME® AA20, commercially available from Genencor International, Inc.) at a ratio of 12 LU / g of carbohydrate and the pH of the solution was adjusted to pH 5.2 by the addition of 2.5% NaOH or 6% HCl , as required. The solution was hydrolyzed using the reactor system and the procedure as described in Example 2.

A control that did not contain EIC was carried out under the same conditions, for comparison. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (SD) immediately after secondary retention. The results of the tests are shown in Table 11.

Table 11 Effect of Isolated EIC of Whole Wheat Flour on the Stability of α-Amylase During Hydrolysis of LO-DEX ™ 10 to pH 5.2 Control sample 16.3 150 mg / L of EIC 14.2 HPLC analysis of EIC isolated from whole wheat flour was performed as described in Example 3. One hundred microliters of a solution at 15 mg / L of EIC, were injected to the HPLC column, and only found in the sample an anionic peak, identified by the elution time as phytate. The HPLC profile was substantially identical to that for EIC isolated from corn gluten fraction.

Example 16 Precipitation of EIC by heating at 95 ° C The EIC (200 mg / L) isolated as in Example 4, was added to LO-DEX ™ 10 at 35% w / w containing 50 ppm of calcium (added as CaCl2 »2H20) and the pH was adjusted to 5.2 with 2.5% NaOH. The solution of LO-DEX ™ 10 was divided into two parts. One part was heated at 95 ° C for about 6 minutes by passing the portion through the reactor solution described in Example 2. After passage through the reactor, the α-amylase was added (SPEZYME® AA20, produced by B. li cheniformis and commercially available from Genencor International, Inc.) to the solution at a ratio of 12 LU / g carbohydrate. The solution was then incubated at 95 ° C for 90 minutes. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (SD) immediately after the 90 minute incubation. The second part of the maltodextrin solution containing EIC was dosed with α-amylase (12 LU / g carbohydrate, SPEZYME® AA20, produced by B. licheniformi s and commercially available from Genencor International, Inc.) and placed through of the reactor system as described in Example 2, except that the temperature was 95 ° C. The solution was then incubated at 95 ° C for 90 minutes. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (SD) immediately after the 90 minute incubation. An identical control but not containing EIC was run at the same time at pH 5.2 for comparison.

The results of the tests shown in Table 12 show that the ability of EIC to inactivate a-amylase during liquefaction can be reduced by heating.

Table 12 Warming effect on the inactivation of EIC of a-Amiiasa Control Sample 17.6 EIC + a-Amylase 14.0 EIC + a-Amylase added after Heating 17.5 Example 17 Precipitation of EIC from Maltodextrin by Heating to 105.5 ° C The EIC (200 mg / L) isolated as in Example 4 was added to LO-DEX ™ 10 at 35% w / w containing 50 ppm calcium ion (added as CaCl 2 »2H_0) and the pH adjusted to 5.2 with 2.5% NaOH. The LO-DEX ™ 10 solution was divided into two parts. A portion was heated at 105.5 ° C for about 6 minutes by passing the solution through the reactor described in Example 2. After passage through the reactor, α-amylase (SPEZYME® AA20, produced by B. licheniformis and commercially available from Genencor International, Inc.) was added to the solution at a ratio of 12 LU / g carbohydrate. The solution was then incubated at 95 ° C for 90 minutes. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (DE) immediately after incubation for 90 minutes. The second part of the maltodextrin solution containing EIC was dosed with α-amylase (12 LU / g carbohydrate, SPEZYME® AA20, produced by B. licheniformi s and commercially available from Genencor International, Inc.) and placed through the reactor system as described in Example 2, at a temperature of 105.5 ° C. The solution was then incubated at 95 ° C for 90 minutes. The degree of hydrolysis of LO-DEX ™ 10 was measured by the dextrose equivalent (DE) immediately after incubation for 90 minutes. An identical control was run simultaneously but that did not contain EIC, at pH 5.2, for comparison.

The results of the tests, shown in Table 3, illustrate that the ability of the EIC to inactivate a-amylase during liquefaction can be reduced by heating before the addition of α-amylase.

Table 13 Effect of Warming on the IAC Inactivation of a-Amylase During Liquefaction at Low pH DE Sample Without EIC Control 15.0 EIC + a-Amylase 10.2 EIC + a-Amylase after heating 17.5 The presence of the EIC in the LO-DEX ™ 10 is believed responsible for the increase in the ED observed after the addition of the EIC and heat treatment when compared to the control, without any added EIC and without heat treatment. Of course, it should be understood that a wide range of changes and modifications can be made to the preferred embodiments described above. It is therefore intended that the above detailed description be understood as expressed in the following claims, including all equivalents, which are intended to define the scope of this invention.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (18)

1. A method for liquefying starch, characterized in that it comprises the steps of: (a) treating the starch before or simultaneously with the liquefaction of starch to inactivate and / or remove an enzyme inhibiting composition present in the starch, to form the treated starch; (b) the addition of the α-amylase to the starch; Y (c) the reaction of the treated starch for an effective time and temperature to liquefy the treated starch.

2. The method according to claim 1, characterized in that step (a) comprises the addition of an enzyme comprising phytate degradation activity, to said starch.

3. The method according to claim 2, characterized in that the enzyme comprising phytate degrading activity is added simultaneously with the α-amylase.

4. The method according to claim 2, characterized in that the enzyme comprising the phytate degrading activity is added at a concentration between about 0.1 and about 100 phytase units per gram of starch.

5. The method according to claim 1, characterized in that step (a) comprises heating the starch at a temperature between about 80 ° C and about 150 ° C before the addition of the α-amylase to inactivate the inhibitory composition of enzyme

6. The method according to claim 5, further characterized by the elimination of the inhibitory composition of the enzyme by centrifugation.

7. The method according to claim 6, characterized in that the centrifugation is carried out subsequent to or simultaneously with raising the temperature of said starch.

8. The method according to claim 1, characterized in that step (b) is carried out simultaneously with step (a).

9. The method according to claim 1, characterized in that step (b) is carried out subsequently to step (a).

10. The method according to claim 1, characterized in that step (c) is carried out at a pH of less than 6.0.

11. The method according to claim 1, characterized in that step (c) is carried out at a pH between about 4.5 and about 5.7.

12. The method according to claim 1, characterized in that step (c) is carried out at a pH between about 4.5 and about 5.2.

13. The method according to claim 9, characterized in that before step (a), the α-amylase is added to the starch at a temperature between about 60 ° C and about 90 ° C, to release the enzyme inhibitory composition, from the starch.

14. The method according to claim 13, characterized in that step (a) comprises the heat treatment subsequent to the addition to the α-amylase, to free the starch from the enzyme inhibitor composition, by heating the starch for a while and a temperature sufficient to inactivate the enzyme inhibitor composition.

15. A method for inactivating a hydrolyzing enzyme, characterized in that the method comprises adding an enzyme inhibiting composition to an aqueous mixture including said hydrolyzing enzyme.

16. The method according to claim 13, characterized in that the hydrolyzing enzyme comprises α-amylase.

17. The method according to claim 14, characterized in that the enzyme inhibitor composition comprises phytate or a salt thereof.

18. A composition of interest, characterized in that it comprises a mixture of α-amylase and aqueous starch at a pH of less than 5.0, said composition being substantially free of the enzyme inhibiting composition.