Method for producing stable aqueous solution of beta amylase, aqueous solution obtained and use thereof

Abstract

The present invention relates to a method for stabilizing an aqueous solution of beta amylase, in particular by using glycerol, potassium sorbate and sodium carbonate. The admixture of the additives is particularly effective over time in maintaining the enzymatic activity of the beta amylase. Another object of the invention consists of the use of said mixture for the specific function of maintaining the enzymatic activity of beta amylase. Another object of the present invention is to provide an aqueous solution of beta amylase comprising the above mixture. The final object of the invention consists of the use of said aqueous beta amylase solution for use in bread making, in the malt industry, as a food additive, as a digesting agent, for sweetener production, in pharmacy, and finally for the production of maltose and maltose-rich syrups.

Description

[0001] This application is a divisional application of the patent application with the application date of June 15, 2015, application number 201580032316.1, and invention title "Method for manufacturing a stable aqueous solution of β - amylase, the obtained aqueous solution and its uses".

[0002] The present invention relates to a method for manufacturing an aqueous solution of β - amylase (in particular by using glycerol, potassium sorbate and sodium carbonate). This combination of additives has proven to have particularly high performance aimed at maintaining the enzymatic activity of β - amylase over time.

[0003] Another subject of the present invention lies in the use of said combination having the specific function of maintaining the enzymatic activity of β - amylase. Another subject of the present invention consists of an aqueous solution of β - amylase containing the above - mentioned combination. The last subject of the present invention lies in the uses of said aqueous solution of β - amylase in bread - making, in the malt industry, as a food additive, as a digestive agent, for the production of sweeteners, in pharmacy, and finally for the production of maltose and maltose - rich syrups.

[0004] β - amylase is an exohydrolase that releases maltose units from the non - reducing β - end of α1→4 - linked glucose polymers or oligomers, and this reaction stops at the first α1→6 branch point encountered. During germination (artificial germination of grains), β - amylase activity isolated from this enzyme cocktail, which is the main component of the "saccharifying power" (corresponding to the combined activity of α - amylase, β - amylase, α - glucosidase and debranching enzyme), is essential for the production of maltose or other fermentable sugars derived from starch.

[0005] Thus, the saccharifying activity of β - amylase alone is used in a large number of applications: in bread - making, in the malt industry, as a food additive, or even as a digestive agent, for the production of sweeteners, in pharmacy for the production of vaccines, and finally for the production of maltose and maltose - rich syrups (precursors of maltitol and maltitol syrup).

[0006] There are many methods for manufacturing β - amylase. Thus, it is known that grains of ungerminated barley, rye or wheat are all choices of biomaterials for large - scale commercial preparation of β - amylase. In addition, it is known to those skilled in the art that about half of the β - amylase that can be extracted from ungerminated barley, wheat or rye grains can be easily obtained in the form of free enzyme by extraction with water and salt solutions. The other half is in a "bound" form, and for its extraction, the addition of a reducing agent or a proteolytic enzyme is required. Another part of β - amylase that cannot be directly extracted, called "latent", has also been described: detergents are required to extract it from the grains. In addition, depending on the target application, the methods for extracting β - amylase described in the prior art have been adjusted.

[0007] In this regard, the Applicant has developed and protected in Application EP 2 414 379 an original method for producing β - amylase, which, in this sense, relies on a starting material that has hitherto been little exploited: the "soluble fraction". The latter has hitherto only been used as a nitrogen source in fermentation and as a nutritional feed for livestock, provided that the said fraction is rich in fibre.

[0008] Such soluble fractions are produced during the wet extraction of starch - containing plant components, which starch - containing plants are, for example, maize, potato, sweet potato, wheat, rice, pea, broad bean, horse bean, cassava, sorghum, konjac, rye, buckwheat and barley. The components produced during the extraction and termed "valuable" are, in particular, starch, protein or other fibres. On the one hand, the "soluble fraction" represents the "non - valuable" components: these are the liquid residues resulting from the said extraction, although these residues may still contain traces of rare insoluble substances and contain diverse and variable particles and colloids.

[0009] The method which is the subject of Application EP 2414379 is based on an initial selection of the soluble fraction to be treated, then on a step of clarification by microfiltration and finally on a step of purification by ultrafiltration. In this case, it has been demonstrated that the β - amylase obtained is very well suited for the preparation of maltose syrup, just like β - amylases produced according to various techniques according to the prior art, but using more complex and more expensive methods.

[0010] Thereafter, the Applicant has also protected, by Patent Application FR 2 994 440 and French Patent Application No. 1356022, improvements to this method, which were not published at the filing date of the present application. These improvements are, in particular, based on the use of a protease during the microfiltration step, which makes it possible to greatly reduce the clogging of the microfiltration membrane and thus to increase the production time before washing during the ultrafiltration step, and also on the use of a pectinase during the ultrafiltration step, which makes it possible to limit the clogging of the ultrafiltration membrane, to reduce the viscosity of the retentate at the end of this step and at the same time to increase its abundance in the β - amylase.

[0011] Now, before being used in the applications already mentioned, the ultrafiltration retentate containing β - amylase in concentrated form can be stored for weeks or even months. Then, it seems that its enzymatic activity decreases over time. It will be recalled that the activity of an enzyme is defined by the amount of substrate (or product formed) that is transformed per unit time and under the optimal operating conditions of the enzyme (temperature, pH, etc.). This value thus quantifies the effectiveness of the enzyme.

[0012] Conventionally, enzyme activity is measured by determining another parameter, the saccharifying activity. The latter is expressed as degrees of saccharifying power (°DP) and is defined as the amount of enzyme contained in a 0.1 ml sample solution of a 5% enzyme preparation by weight that is sufficient to reduce 5 ml of Fehling’s solution when the sample is placed in 100 ml of substrate at 20 °C for 1 h.

[0013] Currently, several documents are known that describe methods for obtaining β - amylase to improve its stability in terms of its enzyme activity.

[0014] Document CN 102965358 discloses a method for obtaining β - amylase from soybeans by precipitation, followed by draining, clarification, and ultrafiltration. The method uses an optional salt of calcium chloride and sulfuric acid in the precipitation step.

[0015] Document CN 102399763 describes the production of β - amylase from bran, where calcium chloride and sodium hydrogen phosphate are added, then concentrated and stabilized in the presence of sorbitol and potassium sorbate, followed by sterilization.

[0016] Document CN 101544967 discloses a method for manufacturing β - amylase by precipitation, separation, and centrifugation, and then recommends adding calcium chloride, orthophosphoric acid, diatomaceous earth, and glycerol.

[0017] Document CN 1225943 describes a method for preparing β - amylase that includes the steps of ultrafiltration, concentration, and precipitation of an extract of soybean flour, preceded by the addition of sodium sulfate and adjustment of the pH between 3.6 and 5.

[0018] Document US2496261 describes a method for obtaining β - amylase from sweet potatoes, which includes a step of precipitation in the presence of ammonium sulfate, followed by acidification with hydrochloric acid.

[0019] Document US 4024000 describes a method for preparing β - amylase that uses divalent or trivalent ions selected from calcium hydroxide, magnesium hydroxide, barium hydroxide, and aluminum hydroxide and their salts, and adjusts the pH in the range between 4.5 and 8.

[0020] However, it should be noted that none of these solutions make it possible to obtain a preparation of β - amylase in the form of an aqueous solution that is sufficiently stable over time, particularly over a period of several weeks, and more specifically for at least 70 days. In the course of its research, the applicant has successfully demonstrated that only a very specific selection of additives makes it possible to achieve such a goal. This combination of additives consists of potassium sorbate, glycerol, and sodium carbonate.

[0021] Thus, the first subject of the present invention consists of a method for stabilizing an aqueous solution of β - amylase obtained from the soluble part of starch plants, the method comprising at least one step of introducing the following into the aqueous solution of β - amylase:

[0022] a) potassium sorbate;

[0023] b) glycerol; and

[0024] c) sodium carbonate.

[0025] Advantageously, potassium sorbate, glycerol and sodium carbonate are introduced into the aqueous solution of β - amylase in the following proportions:

[0026] a) from 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably approximately 0.2% potassium sorbate;

[0027] b) from 30% to 50%, preferably from 35% to 45%, and very preferably approximately 40% glycerol;

[0028] c) from 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably approximately 0.2% sodium carbonate;

[0029] These % are expressed as % of the dry weight of each component relative to the total weight of the aqueous solution.

[0030] Advantageously, the aqueous solution of β - amylase has, relative to the total weight of the aqueous solution, a β - amylase content of between 5% and 20% by dry weight, preferably between 10% and 20% by dry weight, and very preferably equal to approximately 15% by dry weight.

[0031] Potassium sorbate, glycerol and sodium carbonate are preferably in the form of an aqueous solution. Those skilled in the art will know how to adjust the solid extract of these solutions relative to the solubility of the product, and in order to limit the viscosity of these solutions, and in order to make them easy to handle, and especially pumpable.

[0032] According to one embodiment, an aqueous solution of β - amylase is obtained by the following steps, which consist of:

[0033] - providing the soluble part of starch plants;

[0034] - subjecting the soluble part to a microfiltration step to obtain a microfiltration permeate;

[0035] - subjecting the microfiltration permeate to an ultrafiltration step to obtain an ultrafiltration retentate.

[0036] Thus, in this embodiment, potassium sorbate, glycerol and sodium carbonate are introduced into the ultrafiltration retentate constituting the aqueous solution of β - amylase.

[0037] In a more detailed manner, upstream of the method according to the invention, it is suitable to select the soluble part of the starch plant to be processed. This selection is made in particular from the group consisting of the soluble parts of the following: corn, potato, sweet potato, wheat, rice, pea, broad bean, horse bean, cassava, sorghum, konjac, rye, buckwheat and barley.

[0038] The microfiltration of the soluble part of the starch plant especially aims to eliminate insoluble substances, colloids, and microbiological materials in order to obtain a transparent composition containing β-amylase. Thus, the latter composition is the microfiltration permeate. According to a particularly advantageous variant of this embodiment, the microfiltration step is carried out in the presence of at least one protease. Before microfiltration, the protease is placed in contact with the soluble part of the starch plant to be processed: those skilled in the art will know how to adjust the contact time necessary for the enzymatic action.

[0039] The protease used in the present invention is preferably selected from serine proteases, thiol proteases, aspartyl proteases and metalloproteases, and more specifically is selected from metalloproteases. By name, the preferred protease in the present invention is the product sold under the following names: Sumizyme TM APL, Lypaine TM 6500L, Neutrase TM 0.8L, Brewlyve TM NP 900, Brewers Clarex TM A protease in an amount by volume between 0.01% and 0.1% relative to the volume of the soluble part of the starch plant to be processed will preferably be used.

[0040] The microfiltration step of this embodiment is preferably carried out according to tangential membrane microfiltration. The applicant more specifically recommends carrying out tangential microfiltration with a ceramic membrane having a porosity of 0.1 μm to 1 μm.

[0041] By any technique additionally known to those skilled in the art, the microfiltration step may optionally be preceded by a flocculation step of the insoluble particles contained in the soluble part of the starch plant.

[0042] For this first microfiltration step, the applicant recommends operating at a pH between 4 and 5 and a temperature between 40 °C and 50 °C.

[0043] Specifically, the microfiltration step is controlled by an increase in the transmembrane pressure (TMP) over time with a fixed permeate flux.

[0044] In this embodiment, ultrafiltration follows microfiltration. The first aim is to concentrate the microfiltration permeate containing β-amylase while removing any potentially remaining contaminating salts, sugars, and proteins from it. Thus, ultrafiltration is carried out on the microfiltration permeate to obtain an ultrafiltration retentate containing β-amylase.

[0045] The Applicant more specifically recommends carrying out ultrafiltration using a membrane with a cut-off threshold of 10,000 Da to 50,000 Da, preferably with a cut-off threshold of 30,000 Da. These soluble fractions can be ultrafiltered, for example, on a module equipped with a laboratory-scale cassette of a polysulfone membrane with a 30,000 Da cut-off threshold and a pilot-scale polysulfone spiral membrane with a 30,000 Da cut-off threshold. Thus, the enzyme is concentrated in the retentate over time.

[0046] This ultrafiltration can be carried out in the presence of pectinase. In the present application, the term pectinase denotes an enzyme capable of decomposing pectin, which is one of the components of polysaccharide polymers and plant cell walls. They consist of a backbone of 1-4 linked uronic acids. In this regard, they should not be grouped together with cellulases and hemicellulases in such a way that they are wrongly associated with them: cellulases are enzymes directly involved in the reaction of decomposing cellulose (a straight chain of D-glucose molecules), while hemicellulases hydrolyze hemicellulose (generally a branched sugar polymer, such as glucose, xylose, etc.).

[0047] Pectinase is typically introduced into the microfiltration permeate and then the ultrafiltration step is carried out, and it is left to act.

[0048] A person skilled in the art will know how to adjust the contact time necessary for the action of the enzyme. Pectinase typically remains to act for a period ranging from 30 minutes to 4 hours, preferably from 30 minutes to 2 hours, at a temperature between 25 °C and 60 °C, preferably between 25 °C and 50 °C.

[0049] Pectinases particularly suitable for the use according to the invention are the products RapidaseTM ADEX D (pectinase; DSM), Peclyve TM ESP (pectinase; Lyven), or Sumizyme TM ARS (pectinase and arabinase; Takabio), and these examples are not limiting in any way.

[0050] Preferably, an amount of pectinase is introduced that is between 0.05% and 1% by volume relative to the total volume of the microfiltration permeate.

[0051] After the ultrafiltration step, a dialysis step of the ultrafiltration retentate can be carried out in order to reduce the concentration of impurities in said retentate.

[0052] Furthermore, it is desirable to maintain the solution of said β-amylase obtained at a temperature of less than 15 °C, preferably less than 10 °C, ideally around 5 °C, in order to further improve the maintenance of its enzyme activity.

[0053] Another subject of the invention is the use of the following to maintain the enzyme activity of β-amylase in an aqueous solution:

[0054] a) Potassium sorbate;

[0055] b) Glycerol; and

[0056] c) Sodium carbonate.

[0057] "Maintaining enzyme activity" means the ability to limit the decrease in the degree °DP as indicated in the experimental section. Typically, "maintenance" is referred to if after 70 days at a temperature of 37 °C, the degree °DP is still greater than at least 70% of its initial value.

[0058] Another subject of the invention consists of a stabilized aqueous solution of β-amylase, the solution comprising:

[0059] a) Potassium sorbate;

[0060] b) Glycerol; and

[0061] c) Sodium carbonate.

[0062] More specifically, this stabilized aqueous solution of β-amylase comprises:

[0063] a) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably around 0.2% potassium sorbate;

[0064] b) From 30% to 50%, preferably from 35% to 45%, and very preferably around 40% glycerol;

[0065] c) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably around 0.2% sodium carbonate;

[0066] These % are expressed as % of the dry weight of each component relative to the total weight of the aqueous solution of said β-amylase.

[0067] Relative to the total weight of the aqueous solution, it also has a β-amylase content, by dry weight, between 5% and 20%, preferably between 10% and 20%, very preferably around 15%.

[0068] The final subject of the present invention consists of the use of an aqueous solution for stabilizing β - amylase according to the invention in bread - making, in the malt industry, as a food additive, as a digestive agent, for the production of sweeteners, in pharmacy for the production of vaccines, and finally for the production of maltose and maltose - rich syrups (precursors of maltitol and maltitol syrup).

[0069] The following examples make it possible to better understand the invention without, however, limiting its scope.

[0070] Examples

[0071] Production of aqueous solution of β-amylase

[0072] First, in the production of starch from wheat, the soluble fraction is removed at the inlet of the soluble matter evaporator. Once concentrated, this step is routinely carried out to manufacture products for feeding livestock. These products are sold by the company applying for this patent under the name These soluble fractions have a pH between 4 and 5 and a β - amylase activity level of 30° DP / ml.

[0073] Here, the soluble wheat fraction is microfiltered on a pilot - scale device. The microfiltration unit is equipped with a ceramic membrane made of titanium dioxide, and its retention threshold is equal to 0.2 μm. The permeate flow rate is fixed at 12 L / (h m 2 ). The volume concentration factor is equal to 1.5. The temperature and pH of the permeate are equal to 45 °C and approximately 4.5, respectively.

[0074] 0.8 L of neutral protease (Novozymes) is added to the soluble fraction, and this neutral protease is added at a concentration of 0.1% by volume relative to the total volume of the composition. At room temperature, this protease is left to act for 1 hour in advance.

[0075] Then, ultrafiltration is carried out as described above.

[0076] After 1 hour of microfiltration, a microfiltered permeate with a DP degree of 25° DP / ml is obtained, and this degree reflects the enzyme activity of the solution containing β - amylase. The enzyme activity is measured by the saccharifying activity. The latter is expressed as the degree of saccharifying power (°DP), which is defined as the amount of enzyme contained in a 0.1 - ml sample solution of a 5% enzyme preparation by weight that is sufficient to reduce 5 ml of Fehling’s solution when the sample is placed in 100 ml of substrate at 20 °C for 1 h.

[0077] After this microfiltration step, an ultrafiltration step is carried out with the microfiltration permeate. The main purpose of this step is to concentrate the permeate and remove from it any possible residual contaminated salts, sugars and proteins. The ultrafiltration pilot plant is equipped with an organic polysulfone membrane (Alfa Laval membrane) which has a cut-off threshold of 25 kDa. The filtration temperature is fixed at 25 °C to limit bacterial growth as much as possible and maintain enzyme activity. The transmembrane pressure (TMP) is fixed at a maximum of 4 bar.

[0078] An aqueous solution of β-amylase is thus obtained, which solution consists of the ultrafiltration retentate and which, on a dry weight basis, has a β-amylase content equal to 15% of its total weight.

[0079] Different formulations as shown in Tables 1 to 3 were tested. All % are expressed as % of the dry weight of the product relative to the total weight of the aqueous solution. Once the formulations have been produced, an enzyme assay is carried out on each sample (contained in a sterile 100 ml container) according to the method described in patent application FR 2 994 440 (measurement of β-amylase activity). This value is used as a reference for the whole study. The different samples are then placed in a temperature-controlled oven at 37 °C for the desired period; the samples are then taken out in order to measure the residual β-amylase activity at different times (the days on which the samples are taken out are indicated in Tables 1 to 3). The results are given in Tables 1 to 3 and are expressed as % residual β-amylase activity. The temperature of 37 °C was chosen in order to accelerate the phenomena causing the decrease in enzyme activity.

[0080] Table 1a demonstrates that the best results are obtained with a mixture of 40% glycerol, 0.2% potassium sorbate and 0.2% Na2CO3. This also demonstrates that, compared with other formulations using other ingredients, it is indeed the solution according to the invention that enables the best stability to be developed. Thus, this is indeed a non-obvious choice of the ingredients used to produce a formulation which results in unexpectedly and entirely advantageous results in terms of limiting the loss of enzyme activity. Table 1a demonstrates that the formulation as described in claim 1 of the present application enables a very high degree of stability to be developed. Furthermore, the maximum stability is obtained with the last formulation described in this table, produced with the optimal doses of the various ingredients, as described in claim 2 of the present application.

[0081] Table 2 demonstrates that glycerol (used alone and even at high doses) does not enable a satisfactory degree of stability to be achieved. Table 3 demonstrates that replacing glycerol with other sugars also does not enable a satisfactory degree of stability to be achieved.

[0082]

[0083] Table 1

[0084]

[0085] Table 1a

[0086] 0% glycerol 30% glycerol 40% glycerol 50% glycerol 0 100 100 100 100 30 0 53 65 69 60 0 27 44 56 90 0 7 16 28

[0087] Table 2*

[0088]

[0089] Table 3

[0090] PS: Potassium sorbate

[0091] * In the case of calcium carbonate, the formation of large insoluble deposits was also noted

[0092] Production of maltose syrup

[0093] Two tests were carried out involving the production of maltose syrup from aqueous solutions of two β - amylases stabilized with or without the complexing agent according to the invention. These two solutions had been kept at 25 °C for 90 days before use

[0094] At a pH of 5.7 to 6.5, with the aid of 0.2% α - amylase (TERMAMYL 120L sold by Novozymes), a starch milk with 31% dry matter was liquefied in a conventional manner until a DE of approximately 6 was reached

[0095] The reaction medium was then heated at 140 °C for a few seconds to inhibit the α - amylase, and then the pH was adjusted to between 5 and 5.5 and the temperature was adjusted to 55 °C

[0096] In the presence of pullulanase (PULLUZYME 750L sold by ABM), maltogenic α - amylase (MALTOGENASE 4000L sold by Novozymes), and an aqueous solution of β - amylase in a dose equal to 0.1% of the dry matter, saccharification was carried out at 35% dry matter or slightly below this

[0097] The aqueous solution of β - amylase consisted of the ultrafiltration retentate and had an α - amylase content equal to 15% of its total weight by dry weight, as described in the above example

[0098] In the first test not according to the invention, this solution was stabilized with the complexing agent (50% glycerol + 0.2% PS + 1% Na2HPO4) described in the second column of Table 1. As described above, the solution was kept at a temperature of 25 °C for 90 days before use

[0099] In a second test according to the invention, this solution was stabilized with a mixture (40% glycerol + 0.2% PS + 0.2% Na2CO3) as described in the last column of Table 1a. As described above, the solution was maintained at a temperature of 25 °C for 90 days before use.

[0100] For both tests, saccharification lasting approximately 72 hours gave hydrolysis products showing the following composition:

[0101] Not according to the invention:

[0102] DP1: 2%, DP2: 77.9%, DP3: 5.6%

[0103] According to the invention:

[0104] DP1: 5%, DP2: 88%, DP3 < 1.5%

[0105] Production of maltose syrup

[0106] Four tests were then carried out, involving the production of maltose syrup from four stabilized aqueous solutions of β - amylase. Three tests according to the invention and one reference test were carried out, using stabilized solutions that had been stored at 25 °C for 90 days before use.

[0107] At a pH of 5.7 to 6.5, with the aid of 0.2% α - amylase (TERMAMYL 120L sold by Novozymes), a starch milk with 31% dry matter was liquefied in a conventional manner until a DE of approximately 6 was reached.

[0108] The reaction medium was then heated at 140 °C for a few seconds to inhibit the α - amylase, then the pH was adjusted to between 5 and 5.5 and the temperature was adjusted to 55 °C.

[0109] Saccharification was carried out at 35% dry matter or slightly below, in the presence of pullulanase (PULLUZYME 750L sold by ABM), maltogenic α - amylase (MALTOGENASE 4000L sold by Novozymes) and an aqueous solution of β - amylase in a dose equal to 0.1% of the dry matter.

[0110] The aqueous solution of β - amylase consisted of the ultrafiltration retentate and had a β - amylase content equal to 15% of its total weight by dry weight, as described in the above example.

[0111] In a first test (CP) not according to the invention, this solution was stabilized with a mixture (50% glycerol + 0.2% PS + 1% Na2HPO4) as described in the second column of Table 1. As described above, the solution was maintained at a temperature of 25 °C for 90 days before use.

[0112] In the second test (EX1) according to the present invention, this solution was stabilized with the mixture (40% glycerol + 0.2% PS + 0.2% Na2CO3) as described in the last column of Table 1a. As described above, the solution was maintained at a temperature of 25 °C for 90 days before use.

[0113] In the third test (EX2) according to the present invention, this solution was stabilized with the mixture (40% glycerol + 0.4% PS + 0.2% Na2CO3) as described in the fourth column of Table 1a. As described above, the solution was maintained at a temperature of 25 °C for 90 days before use.

[0114] In the fourth test (EX3) according to the present invention, this solution was stabilized with the mixture (40% glycerol + 0.2% PS + 1% Na2CO3) as described in the third column of Table 1. As described above, the solution was maintained at a temperature of 25 °C for 90 days before use.

[0115] For these tests, for each of these examples, saccharification lasting approximately 72 hours gave malt syrups having the following composition:

[0116] Malt syrup CP:

[0117] Glucose: 2%, maltose: 77.9%, maltotriose: 5.6%

[0118] Malt syrup EX1:

[0119] Glucose: 5%, maltose: 88%, maltotriose: <1.5%

[0120] Malt syrup EX2:

[0121] Glucose: 3.2%, maltose: 82.1%, maltotriose: 3.7% Malt syrup EX3:

[0122] Glucose: 2.8%, maltose: 81%, maltotriose: 4.6%.

Claims

1. A method for stably obtaining an aqueous solution of β - amylase from the soluble part of starch plants, the method comprising at least one step of introducing the following into the aqueous solution of β - amylase: a) Potassium sorbate; b) Glycerol; c) Sodium carbonate.

2. The method according to the above claims, characterized in that Introducing the following into the aqueous solution of β - amylase: a) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably about 0.2% potassium sorbate; b) From 30% to 50%, preferably from 35% to 45%, and very preferably about 40% glycerol; c) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably about 0.2% sodium carbonate; These % are expressed as % of the dry weight of each component relative to the total weight of the aqueous solution.

3. The method according to any one of the preceding claims, characterized in that The aqueous solution has a β - amylase content of between 5% and 20% by dry weight, preferably between 10% and 20% by dry weight, and very preferably equal to about 15% of its total weight.

4. The method according to any one of the preceding claims, characterized in that Potassium sorbate, glycerol and sodium carbonate are in the form of an aqueous solution.

5. The method according to the above claim, wherein the aqueous solution of β - amylase is obtained by the following steps, which consist of: - Providing the soluble part of starch plants; - Performing a microfiltration step on the soluble part to obtain a microfiltration permeate; - Performing an ultrafiltration step on the microfiltration permeate to obtain an ultrafiltration retentate.

6. Using the following to maintain the enzyme activity of β - amylase in an aqueous solution: a) Potassium sorbate; b) Glycerol; and c) Sodium carbonate.

7. An aqueous solution of β - amylase, comprising: a) Potassium sorbate; b) Glycerol; and c) Sodium carbonate.

8. The aqueous solution according to the above claim, comprising: a) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably about 0.2% potassium sorbate; b) From 30% to 50%, preferably from 35% to 45%, and very preferably about 40% glycerol; c) From 0.05% to 0.5%, preferably from 0.1% to 0.3%, and very preferably about 0.2% sodium carbonate; These % are expressed as % of the dry weight of each component relative to the total weight of the aqueous solution.

9. The aqueous solution according to any one of claims 7 and 8 has a β - amylase content of between 5% and 20% by dry weight, preferably between 10% and 20% by dry weight, and very preferably about 15% of its total weight.

10. The use of the aqueous solution according to any one of claims 7 to 9 as a food additive, as a digestive agent, for the production of sweeteners, in pharmacy for the production of vaccines, and finally for the production of maltose and maltose - rich syrups.

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