METHOD FOR OBTAINING A PREPARATION OF BETA-AMYLASES FROM THE SOLUBLE FRACTIONS OF STARCH PLANTS

Abstract

The present invention relates to a method for obtaining a preparation of -amylases from the soluble fraction of starch plants, characterized in that the soluble fraction of starch plants is selected from the group consisting of the soluble fractions of wheat, pea, broad bean, horse bean, rice, barley, rye, buckwheat, potato and sweet potato, and preferably of wheat and barley, a clarification of said soluble fractions is carried out in such a way as to remove therefrom the insoluble substances and the colloids and, optionally, an ultrafiltration of said clarified soluble fractions is carried out in such a way as to obtain an ultrafiltration retentate containing the concentrated -amylase and an ultrafiltration permeate, said ultrafiltration retentate containing the concentrated -amylase is diafiltered and the resulting -amylase is recovered.

Claims

1-10. (canceled)

11. A method for obtaining a preparation of -amylases from the soluble fraction of starch plants comprising: a) clarification of a soluble fraction selected from the group consisting of soluble fractions of wheat, pea, broad bean, horse bean, rice, barley, rye, buckwheat, sweet potato and potato said clarification comprising the removal of insoluble substances and colloids; b) an optional ultrafiltration of said clarified soluble fraction to obtain an ultrafiltration retentate containing concentrated -amylase and an ultrafiltration permeate; and c) recovery of the resulting concentrated -amylase.

12. The method of claim 11, wherein step b) is carried out, and that, between steps b) and c), a diafiltration of said ultrafiltration retentate containing the concentrated -amylase is carried out.

13. The method of claim 11, wherein the clarification of the soluble fraction is carried out by a filtration technique selected from the group consisting of frontal filtration, filtration under pressure and vacuum filtration.

14. The method of claim 13, wherein vacuum filtration is carried out using belt filters or rotary filters.

15. The method of claim 11, wherein said clarification is carried out by centrifugation.

16. The method of claim 15, wherein the centrifugation is carried out using a disk centrifuge of self-cleaning or nozzle separator type, or a disk centrifuge of high performance vortex nozzle separator type.

17. The method of claim 11, wherein said clarification is carried out by tangential membrane filtration.

18. The method of claim 17, wherein the tangential member filtration is a microfiltration having a cut-off threshold of between 0.1 and 1 rm.

19. The method of claim 11, wherein the ultrafiltration is carried out using a membrane having a cut-off threshold of from 10,000 Da to 50,000 Da.

20. A method of preparing maltose-rich syrups comprising contacting a solution of liquefied starch with a -amylase composition purified according to the method of claim 11 under conditions that permit saccharification of said solution of liquefied starch.

Description

EXAMPLE 1: OBTAINING A FIRST PREPARATION OF -AMYLASES from Wheat SolublesLaboratory Scale

[0111] In the manufacture of starch from wheat, 60 liters of soluble fractions are collected at the inlet of the solubles evaporator, a step conventionally carried out for cattle feed after concentration, sold by the Applicant company under the name Corami.

[0112] These soluble fractions have a pH of 4 and a -amylase activity of about 25 DP/ml.

[0113] These 60 liters are centrifuged in an SA1 desludging disk centrifuge sold by the company Westfalia, at a flow rate of 40 liters/hour.

[0114] 45 liters of supernatant from the centrifugation are recovered. A turbidity of 60 NTU is measured.

[0115] They are then filtered on a Choquenet laboratory chamber sheet filter of 56 cm.sup.2, a cloth filter preloaded with Clar-O-Cel 13/6 (CECA) and Vitacel L10 (J. Rettenmaier und Shne) celluloses.

[0116] Continuous topping-up is also carried out with these celluloses at a rate of 1 g/l.

[0117] 40 liters of filtrate having a turbidity of 8 NTU and a -amylase activity of 22 DP/ml are recovered.

[0118] The filtrate is then ultrafiltered on a Millipore laboratory module with 0.18 m.sup.2 of membranes.

[0119] 39.5 liters of ultrafiltered filtrate and a retentate concentrated by a factor of 75, having a -amylase activity of between 1500 and 1600 DP/ml, are recovered.

[0120] The ultrafiltration retentate is dialyzed at constant volume with 2.5 volumes of water continuously in such a way as to reduce the concentration of impurities of the solubles by a factor of 10.

[0121] The resulting preparation of -amylases is then stored at +4 C.

EXAMPLE 2: OBTAINING A FIRST PREPARATION OF -AMYLASES FROM WHEAT SOLUBLESLABORATORY SCALE WITH FLOCCULANT

[0122] In the manufacture of starch from wheat, 120 liters of soluble fractions are collected at the inlet of the solubles evaporator at a pH of 4.3.

[0123] They are then treated with 20 ppm of Fe.sup.3+ and 25 ppm of polymeric Flopam AN 923 PWG anionic flocculant (SNF) in such a way as to flocculate the insoluble and colloidal particles.

[0124] The soluble fractions thus treated are then centrifuged under the conditions of example 1 at a flow rate of 40 liters/hour.

[0125] 90 liters of soluble fractions thus centrifuged are obtained. They have a turbidity of about 17 NTU and a -amylase activity of 21 DP/ml.

[0126] In the same way as in example 1, this fraction is subjected to fine filtration on a chamber sheet filter.

[0127] 80 liters of a filtered solution having a turbidity of 5 NTU are obtained.

[0128] Ultrafiltration is then carried out on a Millipore laboratory module with 0.18 m.sup.2 of membranes having a cut-off threshold of 30 kDa.

[0129] 79 liters of ultrafiltered filtrate and a retentate concentrated by a factor of 80 and having a -amylase activity of between 1500 and 1600 DP/ml are recovered.

[0130] The ultrafiltration retentate is dialyzed at constant volume with 2.5 volumes of water continuously in such a way as to reduce the concentration of impurities of the solubles by a factor of 10.

[0131] The resulting preparation of -amylases is then stored at +4 C.

EXAMPLE 3: OBTAINING A FIRST PREPARATION OF -AMYLASES FROM WHEAT SOLUBLESPILOT SCALE

[0132] The pilot test consists of a test in which 10 m.sup.3 of soluble fractions of wheat are collected and prepared as in example 2.

[0133] The insoluble and colloidal particles are removed by centrifugation on an NA7 centrifuge in self-cleaning operating mode.

[0134] 7 m.sup.3 of supernatant having a turbidity of 22 NTU are recovered.

[0135] This supernatant is subsequently filtered by frontal filtration on a 0.3 m.sup.2 AMAFILTER filter loaded with the previously defined celluloses. It is topped up with 1 g/l of cellulose.

[0136] Once filtered, the solution has a turbidity of 5 NTU.

[0137] It is ultrafiltered on equipment consisting of polysulfonated spiral membranes having a cut-off threshold of 30 kDa.

[0138] 75 liters of retentate concentrated by a factor of approximately 95 and having a -amylase activity of approximately 2000 DP/ml are then obtained.

[0139] The ultrafiltration retentate is dialyzed at constant volume with 2.5 volumes of water continuously in such a way as to reduce the concentration of impurities of the solubles by a factor of 10.

[0140] In order to stabilize the enzyme, 50 kg of sorbitol powder sold by the Applicant company under the trademark Neosorb, containing 99.5% of dry matter, are added.

[0141] The final preparation then has a -amylase activity of 1550 DP/ml.

EXAMPLE 4: OBTAINING THE PREPARATION OF -AMYLASES FROM BARLEY SOLUBLES

[0142] 100 kg of barley flour and 72 liters of water are added to a kneading machine so as to obtain a hard dough.

[0143] After standing at ambient temperature for 30 minutes, the dough is washed in countercurrent manner, in a rotary sieve made by the applicant company, with water so as to separate the starch from the gluten.

[0144] 200 liters of a suspension of starch are collected, and are sieved on a first vibrating sieve of 100 m, then a second of 63 m, in order to remove the fibers, the gums and the residual gluten.

[0145] The filtrate is then centrifuged at high flow rate (200 l/h) on a decanter centrifuge sold under the name SEDICANTER by the trademark FLOTTWEG in such a way as to isolate the A-starches (large granule) in the concentrate and the B-starches (small granule) in the supernatant.

[0146] The B-starch suspension overflow is again centrifuged on the desludging SA1 diskseparator (WESTFALIA) at reduced flow rate (40 l/h) in such a way as to isolate the B-starch in the concentrate, and soluble fraction, in the supernatant. The pH is 4.5.

[0147] All these prior steps make it possible to obtain a soluble fraction in accordance with the present invention, i.e. freed of the noble components, in particular freed of the starch, whichever starch it is, the proteins, the fibers and the gums. 60 liters of said soluble fraction, which has a turbidity of 120 NTU, are taken.

[0148] The soluble fraction is treated according to the flocculation method of example No. 2.

[0149] Centrifugation is carried out on the previous desludging centrifuge at 40 l/h and 40 liters of soluble fraction having a -amylase activity of 28DP/ml and a turbidity of 21 NTU are recovered.

[0150] This soluble fraction is filtered on a chamber sheet filter preloaded with Clar-O-Cel 13/6 (CECA) and Vitacel L10 (J. Rettenmaier und Shne) celluloses.

[0151] The soluble fraction was topped up with 1 g/l of celluloses.

[0152] 40 liters of filtrate having a turbidity of 6 NTU and a -amylase activity of 27 DP/ml are thus obtained.

[0153] Ultrafiltration is carried out on a Millipore laboratory module with 0.18 m.sup.2 of 30 KDa membranes, described in example 2.

[0154] Concentration by a factor of approximately 65 is carried out.

[0155] 0.6 liters of a solution of concentrated enzymes having a -amylase activity of 1700DP/ml is finally obtained.

[0156] The ultrafiltration retentate is dialyzed at constant volume of 2.5 volumes of water continuously in such a way as to reduce the concentration of impurities of the solubles by a factor of 10.

[0157] It is stabilized with 400 g of sorbitol powder sold by the Applicant company under the trademark Neosorb containing 99.5% of dry matter.

[0158] The enzymatic preparation then has a -amylase activity of 1400 DP/ml.

EXAMPLE 5: OBTAINING MALTOSE-RICH SYRUPS, WITH A COMMERCIAL PURIFIED -AMYLASE AS CONTROL

a) Obtaining the Maltose Syrup Through the Action of -Amylase Alone

[0159] A compared saccharification is carried out in the laboratory using a commercial purified -amylase from the company GENENCOR (OPTIMALT BBA) and using the preparation of -amylases in accordance with the invention, prepared according to example 1.

[0160] The -amylase, at the concentration of 4% (commercial enzyme on dry starch), is added to a solution of liquefied starch containing 30% of dry matter, having a Dextrose Equivalent level of 6 (dissolution of maltodextrin of DE 6).

[0161] The doses are intentionally high in order to detect the possible presence of parasitic enzymatic activities.

[0162] The reaction is carried out at a temperature of 58 C. and at pH 5 for 24 hours.

[0163] The results are expressed as % of glucose monomers and oligomers generated by the -amylase action, oligomers having a degree of polymerization equal to 2 and more, determined by High Pressure Liquid Chromatography of sodium type.

[0164] The DP2s correspond to maltose, the DP1s to glucose and the DP3s to maltotriose.

TABLE-US-00001 DP > 3 DP3 DP2 DP1 Starting solution 96.5 1.5 1.2 0.3 (t = 0) GENENCOR -amylase 44 5.3 49.9 0.4 (t = 24 h) (-Amylase example 1 42.6 5.7 51.4 0.4 (t = 24 h)

[0165] The results demonstrate that the preparation of -amylases in accordance with the invention can effectively replace the commercial -amylase.

b) Obtaining a Maltose-Rich Syrup Through the Action of a -Amylase Preparation Combined with a Pullulanase and a Maltogenase in the Laboratory

[0166] The tests were carried out under the previous conditions, with the difference that 2 enzymes are added simultaneously with the addition of the -amylase preparation.

[0167] They are the PULLUZYME pullulanase sold by the company ABM, which specifically hydrolyzes the 1.fwdarw.6 linkages of starch, and the maltogenic -amylase sold by the company NOVOZYMES under the name MALTOGENASE, which specifically hydrolyzes the 1.fwdarw.4 linkages.

[0168] These enzymes are also added in proportions of 4% (commercial enzyme on dry starch).

[0169] As a control, a -amylase preparation originating from a malt extract (=cocktail of enzymes predominantly composed of -amylases) is used as -amylase source.

TABLE-US-00002 DP GREATER DP3 DP2 DP1 Starting solution 96.5 1.5 1.2 0.3 (t = 0) GENENCOR -amylase 7 2 85.3 5.5 (t = 24 h) -Amylase according 7.2 1.5 85.5 5.2 to example 1 (t = 24 h) -Amylase extracted 6.3 3.3 83.3 7.0 from malt T (t = 24 h)

[0170] While the carbohydrate profile obtained using the purified -amylase and the carbohydrate profile obtained using the preparation of -amylases in accordance with the invention are equivalent, thereby demonstrating the excellent behavior of said preparation according to the invention, the carbohydrate profile obtained using malt extract produces a much higher glucose content.

c) Obtaining a Maltose-Rich Syrup Through the Action of a -Amylase Preparation Combined with a Pullulanase and a Maltogenase on the Industrial Scale

[0171] The preparation of example No. 3 is used to carry out the industrial test.

[0172] The saccharification is carried out on 200 m.sup.3 of a solution of liquefied starch containing 30% of dry matter, having a Dextrose Equivalent level of 6. The -amylase preparation (1% on a dry basis), the pullulanase and the maltogenase are added simultaneously under the following conditions: pH 5, 55 C.

[0173] The following results are obtained after 66 hours (expressed as % DP generatedmeasurement carried out by sodium HPPLC):

TABLE-US-00003 DP > 3 DP3 DP2 DP1 6 1.1 88.3 4.3

[0174] This maltose-rich syrup can be purified, hydrogenated and crystallized so as to obtain a maltitol of quality identical to that obtained by the conventional routes.

d) Obtaining Maltose Syrup Through the Action of the Ultrafiltered and Diafiltered -Amylase Preparation

[0175] A compared saccharification is carried out in the laboratory using the preparation of -amylases in accordance with the invention, prepared according to example 1 (-amylase example 1), and using a preparation of -amylases clarified according to example 1 but not subjected to the ultrafiltration and diafiltration steps (-amylase without UF).

[0176] For the saccharification with the -amylase example 1 preparation, said -amylase example 1 preparation is added, at the concentration of 4% (commercial enzyme on dry starch), to a solution of liquefied starch containing 30% of dry matter, having a Dextrose Equivalent level of 6 (dissolution of maltodextrin of DE 6). The enzymatic preparation then has a -amylase activity of 1.8DP/ml. The reaction is carried out at a temperature of 58 C. and at pH 5 for 24 h.

[0177] For the saccharification with the -amylase without UF preparation, l 1 of filtrate at 8 NTU of example 1 (filtrate neither ultrafiltered nor diafiltered) was taken. Said filtrate at 8 NTU is diluted to 1/15.sup.th so as to obtain a concentration of -amylase at 1.8DP/ml. The pH is adjusted to 5. 700 ml of said diluted filtrate are taken and 300 g of maltodextrin of DE 6 are added. The reaction is carried out at a temperature of 58 C. for 24 h.

[0178] The following results are obtained (expressed as % DP generatedmeasurement carried out by sodium HPPLC):

TABLE-US-00004 DP > 3 DP3 DP2 DP1 Starting solution 96.5 1.5 1.2 0.3 (t = 0) -Amylase without UF 41.5 3.8 48.5 2.2 (t = 24 h) -Amylase example 1 42.6 5.7 51.4 0.4 (t = 24 h)

[0179] The results demonstrate that the nonultrafiltered and nondiafiltered preparation of -amylases (-amylase without UF) makes it possible to obtain a maltose-rich syrup containing 2.2% of glucose. Unlike the -amylase example 1 preparation (maltose-rich syrup obtained containing 0.4% of glucose), the -amylase without UF preparation is therefore contaminated with enzymatic impurities.

[0180] In order to evaluate the degree of purity of the maltose-rich syrups obtained using the two preparations, -amylase example 1 and -amylase without UF, in particular the amount of salts that they contain, the conductivity of said syrups was measured at 20 C. (CDM210 instrument sold by the company Mettler) after filtration of the syrups on a disc filter with a porosity of 1 m from the company Millipore.

[0181] The results are the following:

TABLE-US-00005 Conductivity (S) -Amylase without UF 200 -Amylase example 1 20

[0182] The ultrafiltered and diafiltered preparation (-amylase example 1) makes it possible to obtain a maltose-rich syrup which contains a much lower amount of salts than the maltose-rich syrup obtained by means of the -amylase without UF preparation. Consequently, the purification (filtration, discoloration, demineralization) of the syrup obtained by means of the -amylase without UF preparation will be more difficult than that of the syrup obtained by means of the -amylase example 1 preparation.