METHOD FOR MANUFACTURING A STABLE AQUEOUS SOLUTION OF BETA-AMYLASE, AQUEOUS SOLUTION OBTAINED AND USES THEREOF

Abstract

The present invention relates to a method for stabilising an aqueous solution of β-amylase, in particular by the use of glycerol, potassium sorbate and sodium carbonate. Said cocktail of additives is particularly effective at maintaining the enzymatic activity of β-amylase over time. Another aim of the present invention consists of using said cocktail for the specific function of maintaining the enzymatic activity of the β-amylase. Another aim of the present invention is to provide an aqueous solution of β-amylase containing the aforementioned cocktail. A final aim of the present invention consists of using said β-amylase aqueous solution in bread-making, in malting, as a food additive, as a digestive agent, for sweetener production, in pharmacy and, finally, for maltose and maltose-enriched syrup production.

Claims

1-10. (canceled)

11. A method for the production of maltose or maltose enriched syrups, comprising adding the aqueous solution of β-amylase to maltose or a maltose enriched syrups, said aqueous solution of β-amylase, said solution containing: a) potassium sorbate; b) glycerol; and c) sodium carbonate.

12. The method as claimed in claim 11, characterized in that said solution contains: a) from 0.05 to 1% potassium sorbate; b) from 30 to 60% glycerol; and c) from 0.05 to 1% sodium carbonate these % being expressed as % by dry weight of each constituent relative to the total weight of said aqueous solution.

13. The method as claimed in claim 11, characterized in that said solution contains: a) from 0.05 to 0.5% potassium sorbate; b) from 30 to 50% glycerol; c) from 0.05 to 0.5% sodium carbonate; these % being expressed as % by dry weight of each constituent relative to the total weight of said aqueous solution.

14. The method as claimed in claim 11, characterized in that said solution contains: a) from 0.2 to 1% of potassium sorbate; b) from 40 to 60% of glycerol; c) from 0.2 to 1% of sodium carbonate; these % being expressed as % by dry weight of each constituent relative to the total weight of said aqueous solution.

15. The method as claimed in claim 11, characterized in that said solution contains: a) from 0.1 to 0.3% of potassium sorbate; b) from 35 to 45% of glycerol; c) from 0.1 to 0.3% of sodium carbonate; these % being expressed as % by dry weight of each constituent relative to the total weight of said aqueous solution.

16. The method as claimed in claim 11, characterized in that said solution contains: a) approximately 0.2% potassium sorbate; b) approximately 40% glycerol; c) approximately 0.2% sodium carbonate; these % being expressed as % by dry weight of each constituent relative to the total weight of said aqueous solution.

17. The method as claimed in claim 11, characterized in that said solution has a content by dry weight of β-amylase of between 5 and 20% of the total weight thereof.

18. The method as claimed in claim 11, characterized in that said solution has a content by dry weight of β-amylase of between 10 and 20% of the total weight thereof.

19. The method as claimed in claim 11, characterized in that said solution has a content by dry weight of β-amylase of approximately 15% of the total weight thereof.

20. The method as claimed in claim 11, characterized in that the β-amylase is obtained from a soluble fraction of starch plants.

21. The method as claimed in claim 11, characterized in that said aqueous solution of β-amylase is obtained by the steps consisting in: providing a soluble fraction of starch plants; carrying out a microfiltration step on said soluble fraction, in order to obtain a microfiltration permeate; carrying out an ultrafiltration step on the microfiltration permeate, in order to obtain an ultrafiltration retentate.

Description

EXAMPLES

Manufacture of Aqueous Solutions of Beta-Amylase

[0067] Firstly, in the production of starch from wheat, a soluble fraction is taken off at the inlet to the solubles evaporator, which step is conventionally carried out to manufacture products intended for feeding livestock, once concentrated. These products are sold by the applicant company under the name Corami®. These soluble fractions have a pH of between 4 and 5 and a β-amylase activity of the order of 30° DP/ml.

[0068] Here, the soluble wheat fractions are microfiltered on pilot scale equipment. The microfiltration unit is fitted with ceramic membranes made of titanium oxide, the cut-off threshold of which is equal to 0.2 μm. The permeate flow rate is fixed at 12 l/(h m.sup.2). The volume concentration factor is equal to 1.5. The temperature and the pH of the permeate are equal to 45° C. and approximately 4.5, respectively.

[0069] 0.8 l Neutrase (Novozymes) protease is added to the soluble fraction, at a fixed concentration of 0.1% by volume relative to the total volume of said composition. This protease is left to act beforehand for 1 hour at room temperature.

[0070] An ultrafiltration is then carried out as described above.

[0071] After 1 hour of microfiltration, a microfiltration permeate with a DP degree of 25° DP/ml is obtained, this degree reflecting the enzymatic activity of the solution containing the β-amylase. The enzymatic activity is measured by the diastatic activity. The latter is expressed in degree of diastatic power (° DP), defined as the amount of enzyme contained in 0.1 ml of a 5% by weight solution of a sample enzyme preparation sufficient for reducing 5 ml of Fehling's solution, when said sample is placed in 100 ml of substrate for 1 h at 20° C.

[0072] The microfiltration step is followed by an ultrafiltration step, carried out on the microfiltration permeate. The main aim of this step is to concentrate said permeate and to remove from it any possible residual contaminating salts, sugars and proteins. The ultrafiltration pilot equipment is fitted with organic polysulfone membranes with a cut-off threshold of 25 kDa (Alfa Laval membranes). The filtration temperature is fixed at 25° C. to limit bacterial growth as much as possible and preserve enzymatic activity. The transmembrane pressure (TMP) is fixed at 4 bar maximum.

[0073] An aqueous solution of β-amylase is thus obtained which consists of the ultrafiltration retentate, having a content by dry weight of β-amylase equal to 15% of the total weight thereof.

[0074] Different cocktails, as indicated in tables 1 to 3, were tested. All the % are expressed as % by dry weight of product relative to the total weight of the aqueous solution. Once the preparations have been produced, an enzymatic assay is carried out on each sample (contained in sterile 100 ml containers) according to the method described in patent application FR 2 994 440 (measure of the beta-amylase activity). This value serves as reference for the whole study. The different samples are then placed in a temperature-controlled oven at 37° C. for the desired period; a sample is then taken to measure the residual beta-amylase activity at different times (the days on which samples are taken are indicated in tables 1 to 3). The results are given in tables 1 to 3 and are expressed as % residual beta-amylase activity. The temperature of 37° C. is chosen so as to accelerate the phenomena which bring about the drop in enzymatic activity.

[0075] Table 1a demonstrates that the best result is obtained with the mixture of 40% glycerol, 0.2% potassium sorbate and 0.2% Na.sub.2CO.sub.3. It also demonstrates that compared to other cocktails using other ingredients, it is indeed the solution according to the invention which makes it possible to develop the best degree of stability. This is therefore indeed a non-obvious selection of ingredients to produce a cocktail which leads to surprising and entirely advantageous results in terms of limiting loss of enzymatic activity. Table 1a demonstrates that the cocktails as described in claim 1 of the present application make it possible to develop very high degrees of stability. The greatest stability is, moreover, obtained with the final cocktail described in this table, produced with the optimal doses of each ingredient, as described in claim 2 of the present application.

[0076] Table 2 demonstrates that glycerol, used alone and even at a high dose, does not make it possible to achieve the satisfactory degree of stability. Table 3 demonstrates that the substitution of glycerol with other sugars also does not make it possible to achieve a satisfactory degree of stability.

TABLE-US-00001 TABLE 1 50% glycerol + 40% glycerol + 40% sorbitol + 40% sorbitol + 50% glycerol + 50% glycerol + 0.2% PS + 0.2% PS + 0.2% PS + 0.2% PS + 0.2% PS + Days 0.2% PS 1% Na.sub.2HPO.sub.4 1% Na.sub.2CO.sub.3 1% Na.sub.2HPO.sub.4 1% CaCO.sub.3 1% CaCO.sub.3 0 100 100 100 100 100 100 20 98 98 30 72 34 89 89 77 85 92 60 60 72 48 70 75 66 69 70 90 45 50 44 45 47

TABLE-US-00002 TABLE 1a 60% glycerol + 40% glycerol + 40% glycerol + 40% glycerol + 40% glycerol + 0.2% PS + 1% PS + 0.2% PS + 0.4% PS + 0.2% PS + days 0.4% Na.sub.2CO.sub.3 0.4% Na.sub.2CO.sub.3 0.4% Na.sub.2CO.sub.3 0.2% Na.sub.2CO.sub.3 0.2% Na.sub.2CO.sub.3 0 100 100 100 100 100 72 74 73 76 76 80 90 49 48 54 54 60

TABLE-US-00003 TABLE 2* 0% 30% 40% 50% glycerol glycerol glycerol glycerol 0 100 100 100 100 30 0 53 65 69 60 0 27 44 56 90 0 7 16 28 PS: potassium sorbate *the formation of a large insoluble deposit is also noted in the case of calcium carbonate

TABLE-US-00004 TABLE 3 40% mixture (45% glucose, 50% 10% glycerol + 20% glycerol + 40% glucose + 40% glucose + 40% 10% fructose, Days glucose 30% glucose 20% glucose 0.5% Na.sub.2HPO.sub.4 3% NaCl maltose 45% maltose) 0 100 100 100 100 100 100 100 30 66 53 61 82 42 46 39 60 43 35 37 43 15 30 20 90 28 22 22 22 7

Manufacture of Maltose Syrups

[0077] Two tests are then carried out, relating to the manufacture of maltose syrups from two aqueous solutions of beta-amylase stabilized by a cocktail according to the invention or by a cocktail not according to the invention, these 2 solutions having been kept for 90 days at 25° C. before being used.

[0078] A starch milk with 31% dry matter is liquefied in the conventional manner by means of 0.2% of an alpha-amylase (TERMAMYL120L sold by Novozymes) at a pH of 5.7 to 6.5 until a DE of roughly approximately equal to 6.

[0079] The reaction medium is then heated for a few seconds at 140° C. so as to inhibit the alpha-amylase, then the pH is adjusted to between 5 and 5.5 and the temperature to 55° C.

[0080] Saccharification is carried out at 35% dry matter, or slightly below, in the presence of pullulanase (PULLUZYME 750L sold by ABM) and maltogenic alpha-amylase (MALTOGENASE 4000L sold by Novozymes) and an aqueous solution of beta-amylase at doses equal to 0.1% of dry matter.

[0081] The aqueous solution of beta-amylase consists of the ultrafiltration retentate, having a content by dry weight of alpha-amylase equal to 15% of the total weight thereof, as described in the preceding example.

[0082] In a first test not according to the invention, this solution was stabilized with the cocktail according to the second column of table 1 (50% glycerol+0.2% PS+1% Na.sub.2HPO.sub.4). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0083] In a second test according to the invention, this solution was stabilized with the cocktail according to the last column of table 1a (40% glycerol+0.2% PS+0.2% Na.sub.2CO.sub.3). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0084] For these two tests, the saccharification, which lasts approximately 72 hours, gives a hydrolysate showing the following composition:

[0085] Not according to the invention:

[0086] DP1: 2%, DP2: 77.9%, DP3: 5.6%

[0087] According to the invention:

[0088] DP1: 5%, DP2: 88%, DP3: <1.5%

Manufacture of Maltose Syrups

[0089] 4 tests are then carried out, relating to the manufacture of maltose syrups from 4 stabilized aqueous solutions of beta-amylase. 3 tests according to the invention and one reference test were carried out using stabilized solutions having been stored for 90 days at 25° C. before being used.

[0090] A starch milk with 31% dry matter is liquefied in the conventional manner by means of 0.2% of an alpha-amylase (TERMAMYL120L sold by Novozymes) at a pH of 5.7 to 6.5 until a DE of approximately equal to 6.

[0091] The reaction medium is then heated for a few seconds at 140° C. so as to inhibit the alpha-amylase, then the pH is adjusted to between 5 and 5.5 and the temperature to 55° C.

[0092] Saccharification is carried out at 35% dry matter, or slightly below, in the presence of pullulanase (PULLUZYME 750L sold by ABM) and maltogenic alpha-amylase (MALTOGENASE 4000L sold by Novozymes) and an aqueous solution of beta-amylase at doses equal to 0.1% of dry matter.

[0093] The aqueous solution of beta-amylase consists of the ultrafiltration retentate, having a content by dry weight of beta-amylase equal to 15% of the total weight thereof, as described in the preceding example.

[0094] In a first test not according to the invention (CP), this solution was stabilized with the cocktail according to the second column of table 1 (50% glycerol+0.2% PS+1% Na.sub.2HPO.sub.4). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0095] In a second test according to the invention (EX1), this solution was stabilized with the cocktail according to the last column of table 1a (40% glycerol+0.2% PS+0.2% Na.sub.2CO.sub.3). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0096] In a third test according to the invention (EX2), this solution was stabilized with the cocktail according to the fourth column of table 1a (40% glycerol+0.4% PS+0.2% Na.sub.2CO.sub.3). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0097] In a fourth test according to the invention (EX3), this solution was stabilized with the cocktail according to the third column of table 1 (40% glycerol+0.2% PS+1% Na.sub.2CO.sub.3). The solution remained at a temperature of 25° C. for 90 days before being used as indicated above.

[0098] For these tests, the saccharification, which lasts approximately 72 hours, gives a maltose syrup showing, for each of the examples, the following compositions:

[0099] Maltose syrup CP:

[0100] Glucose: 2%, maltose: 77.9%, maltotriose: 5.6%

[0101] Maltose syrup EX1:

[0102] Glucose: 5%, maltose: 88%, maltotriose: <1.5%

[0103] Maltose syrup EX2:

[0104] Glucose: 3.2%, maltose: 82.1%, maltotriose: 3.7%

[0105] Maltose syrup EX3:

[0106] Glucose: 2.8%, maltose: 81%, maltotriose: 4.6%