METHOD FOR EXTRACTING B-AMYLASES FROM A SOLUBLE FRACTION OF A STARCH PLANT AND IN THE PRESENCE OF A PROTEASE

Abstract

The present invention concerns an improved method for extracting -amylases from a soluble fraction of a starch plant, said method comprising a step of clarification by microfiltration and a step of concentration and purification by ultrafiltration. This method is characterised in that it uses a protease during the microfiltration step, making it possible to greatly reduce the fouling of the membranes used, which increases the production time before washing. Simultaneously, perfectly clear permeates are obtained, which assists with the subsequent ultrafiltration step and makes it possible to improve the transmission of -amylase. Finally, the protease has no negative effect on the -amylase activity.

Claims

1-10. (canceled)

11. A method for extracting -amylases from a soluble fraction of a wheat, comprising the steps of: a) microfiltration of a soluble fraction of a wheat; and b) an ultrafiltration step, wherein the microfiltration step is carried out in the presence of at least one protease.

12. The method according to claim 11, wherein the protease is chosen from serine proteases, thiol proteases, aspartyl proteases and metalloproteases.

13. The method according to claim 12, wherein the protease is a metalloprotease.

14. The method according to claim 11, wherein microfiltration step is carried out by tangential membrane microfiltration.

15. The method according to claim 14, wherein the tangential microfiltration is carried out with ceramic membranes having a porosity of 0.1 m to 1 m.

16. The method according to claim 11, wherein the microfiltration step is preceded by a step of flocculation of the insoluble particles contained in the soluble fraction of starch plants.

17. The method according to claim 11, wherein the microfiltration step is carried out at a pH of between 4 and 5 and at a temperature of between 40 C. and 50 C.

18. The method according to claim 11, wherein the ultrafiltration is carried out using membranes which have a cut-off threshold of 10 000 Da to 50 000 Da.

19. The method according to claim 18, wherein the ultrafiltration is carried out using polysulfone membranes having a cut-off threshold of 30 000 Da in cassettes on a laboratory scale and polysulfone spiral membranes having a cut-off threshold of 30 000 Da on a pilot scale.

20. The method according to claim 18, wherein the ultrafiltration step is carried out using membranes which have a cut-off threshold of 30 000 Da.

Description

EXAMPLES

[0033] In this example, the preparation of -amylase is first of all carried out according to the method described in application EP 2 414 379, i.e. on the basis of a step of microfiltration of soluble fraction without using protease: it is the control test. The preparation of -amylase is then carried out according to the method which is the subject of the present invention, i.e. by microfiltration of soluble fraction in the presence of a protease: these tests illustrate the present invention.

[0034] In the manufacture of starch from wheat, a soluble fraction is collected at the inlet of the solubles evaporator, a step conventionally carried out to produce 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 4 and a -amylase activity of about 30 DP.

[0035] The microfiltration of soluble fractions of wheat is carried out here on pilot-scale equipment. The microfiltration unit is equipped with ceramic membranes made of titanium oxide, the cut-off threshold of which is equal to 0.2 m. 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 respectively equal to 45 C. and 4.5.

[0036] The test protease is added to the soluble fraction, said test protease being at a concentration fixed at 0.1% by volume relative to the total volume of said composition. This protease is left to act beforehand for 1 hour.

[0037] For each of the tests, the change in the TMP pressure is monitored, as already previously described, during the microfiltration step: the TMP increases over time as the membrane becomes fouled. For each test, the degree of diastatic power ( DP) is also determined at the beginning of the test (Feed DP), after 1 hour ( DP 1 hour) and at the end of the method after approximately 8 hours ( DP 8 hours); it is thus possible to calculate the transmission of the -amylase activity after 1 hour (T 1 hour) and after 8 hours (T 8 hours). Finally, the turbid or clear nature of the permeate is determined at the end of each test, visually.

[0038] FIG. 1/1 illustrates the change in the TMP pressure as a function of time, for the control test carried out without protease, and for each test representative of the invention which uses a protease. It is very clearly apparent that the use of a protease makes it possible to limit the increase in the TMP pressure over time: the membrane fouling phenomena have therefore indeed been successfully slowed down. It is even noted that, for some proteases, including in particular the Brewlyne NP 900 product, the transmembrane pressure is perfectly stable: in this case, the membrane fouling phenomenon is virtually absent.

[0039] Furthermore, it is noted for each test according to the invention that the permeate obtained is perfectly clear. Finally, as demonstrated in table 1, the transmission is improved in the case of the invention compared with the control test carried out without protease.

TABLE-US-00001 TABLE 1 Protease Feed DP 1 T 1 DP 8 T 8 used DP hour hour hours hours None 31 25 81 19 61 (control) Sumizyne 31 28 90 22 71 APL Lypaine 38 33 87 26 68 6500 L Brewlyne 36 32 89 27 75 NP 900 Brewers 37 35 95 25 68 Clarex Neutrase 32 27 84 24 75 0.8 L

[0040] The microfiltration step is followed by an ultrafiltration step, carried out on the microfiltration permeate. The main objective thereof is to concentrate said permeate and to remove from it any contaminating residual salts, sugars and proteins. 40 liters of microfiltration permeate are recovered for each of the tests apart from the control test. Said permeate is ultrafiltered on a MILLIPORE laboratory module with 0.18 m.sup.2 of membranes having a cut-off threshold of 30 000 Da. 39.5 liters of ultrafiltered permeate and a retentate concentrated by a factor of 75, having a -amylase activity of between 1500 and 1600 DP, are recovered. The ultrafiltration retentate is continuously dialyzed at constant volume with 2.5 volumes of water so as to reduce the concentration of impurities of the solubles by a factor of 10. The -amylase preparation thus obtained is then stored at +4 C.

Example 2

[0041] The same procedure as previously is carried out, but starting from soluble fractions having a pH of 4 and a -amylase activity of about 30 DP.

[0042] The test enzyme is added to the soluble fraction: [0043] Neutrase 0.8 L (protease) at a concentration fixed at 0.1% by volume relative to the total volume of said composition; [0044] Optiflow (hemicellulase) at a concentration fixed at 1% by volume relative to the total volume of said composition; [0045] Rapidase (pectinase) at a concentration fixed at 1% by volume relative to the total volume of said composition; [0046] Finizyme (lysophospholipase) at a concentration fixed at 1% by volume relative to the total volume of said composition.

[0047] Each enzyme is left to act beforehand for 1 h.

[0048] For each test, the degree of diastatic power ( DP) is also determined at the beginning of the test (Feed DP), after 1 hour ( DP 1 hour) and at the end of the method after approximately 8 hours ( DP 8 hours); it is thus possible to calculate the transmission of the -amylase activity after 1 hour (T 1 hour) and after 8 hours (T 8 hours). The results appear in table 2.

[0049] It is noted that the protease according to the invention makes it possible to improve the transmission of the -amylase activity much more significantly than the other enzymes. In addition, after 8 hours, only the permeate obtained with the protease according to the invention is clear, the others having a very marked cloudy appearance.

TABLE-US-00002 TABLE 2 Enzyme Feed DP 1 T 1 DP 8 T 8 used DP hour hour hours hours None 32 25 79 21 70 (control) Neutrase 32 29 92 25 86 0.8 L Optiflow 31 20 65 9 30 Rapidase 31 21 68 12 39 Finizyme 31 26 84 23 74