CS250262B3 - Method of glucose-fructose syrup's enzyme production from saccharose - Google Patents

Description

Glucose-glucose syrup has a higher sweetness compared to sucrose and is useful in various industries, especially in the food, pharmaceutical, etc.

Sucrose can also be hydrolyzed non-enzymatically either physically chemically cleaved at high temperature ion exchangers while isolating both monosaccharides, or chemically by acid hydrolysis. However, these methods have economic and other limits. In the case of cleavage at ion exchangers, the limit and concentration of sucrose, the quality and availability of ion exchangers, respectively. their regeneration. In the case of chemical cleavage of sucrose, the limit is high consumption of mineral acids, corrosion of the apparatus and, above all, poor organoleptic and visual properties of the obtained glucose-glucose syrup (bitterness and coloring).

Industrial practice also employs either a single administration of a soluble invertase enzyme that is previously isolated from various biological sources, or multiple repeated or continuous administration of the enzyme in various immobilized forms. The single use of soluble invertase or native living cells containing this enzyme is uneconomical despite the poor quality of the final product, especially when using technical enzyme preparations or living cells. Immobilized invertase, on the other hand, allows continuous, respectively. discontinuous multiple uses.

However, for the production of various forms of immobilized invertase, it is necessary to have this enzyme in the technical or pure state. The production of immobilized invertase therefore requires isolation or importation of this enzyme from producer cells. Therefore, the cost of n, and the isolation of the enzyme, are economically disadvantageous in the full cost of producing the immobilized invertase. As known from patent and technical literature (AO 209 743, AO 214 096, AO 234 059, Enzyme Engineering, Vol. 6, I. Chibata, S. Fukui, LB Wingard, eds., Plenum Press, New York and London (1982), various forms of immobilized invertase can be used for relatively low initial sucrose concentrations, of about 250-62% at 3.0 to 40.0% by weight, while the thermostability and operating half-lives of the differently immobilized enzyme are insufficient.

It has now been found that immobilized whole cells or other forms of immobilized cellular materials can be used for enzymatic hydrolysis of not only pure sucrose, but also crystalline (raw sugar, affinity) or liquid intermediates of its production (raw juice, light) juice, heavy juice, clergy, syrups or sugar by-products (molasses), the initial concentrations of which may be in the range of 0.5 to 75.0% by weight. the conversion rate is low and therefore the enzyme hydrolysis with said immobilized materials has to be carried out at higher temperatures, which imposes considerable requirements on their thermostability.

In MS. No. 231,458 discloses methods of immobilizing whole native cells, cross-linked cells, cell walls, and disintegrated cell suspensions with various enzymatic activities using various prokaryotic and eukaryotic cells as a starting biological material, including invertase activity. In MS. No. 249,325 describes a method of consolidating and stabilizing yeast cell biocatalyst particles. This method and the methods disclosed in U.S. Pat. Nos. 209 265 and 231 458 permit the application of these biocatalysts with invertase activity at relatively high temperatures and high initial substrate concentrations.

The method for the enzymatic production of glucose-glucose syrup from sucrose according to the invention is characterized in that whole immobilized cells, immobilized cell walls or immobilized disintegrated cell suspensions and β-D-fructofuranosidase activity prepared according to AO 231 458 and AO 249 325 are used for enzymatic hydrolysis.

The substrates used are aqueous solutions of pure sucrose, technical sucrose in the form of natural sugar, raw sugar, affinity sugar, aqueous sucrose solutions in the form of liquid sugar, sucrose solutions formed as an intermediate of sucrose production such as raw juice, light juice, heavy juice, clergy , ores and further by-products of production, in particular molasses from the production or diluted molasses.

The enzymatic hydrolysis is carried out at temperatures in the range of 10 to 90 ° C, preferably 60 to 75 ° C, at the pH of the reaction mixture in the range of 4.0 to 7.0, preferably 4.5 to 5.5, at initial sucrose concentrations of 0, 5 to 75.0 weight percent, preferably 40 to 65 weight percent.

The enzyme hydrolysis is carried out batchwise at a concentration of immobilized materials of 1 to 200 g of wet mass. liter ^{-1 of the} reaction mixture, preferably 100 g wet mass. liter ^{-1 of the} reaction mixture, either by leaving the particles of immobilized materials permanently in the apparatus and separating the solution containing the reaction products from the reaction mixture, or separating the biocatalysts after the conversion is carried out outside the reaction apparatus, apply.

The enzymatic hydrolysis is carried out continuously, either in fixed bed devices of immobilized materials by flowing substrate solutions through a fixed bed of particles, or preferably in buoyant bed devices of immobilized materials, either by pumping substrate solutions so that the biocatalyst particles are in continuous fluid flow or The method of claim 1, wherein the particles are raised by other means, for example by stirring.

The resulting enzyme reaction products are further treated by filtration, centrifugation, bleaching, thickening, optionally and preferably enriched with additives such as vitamins, flavorings, colorings and others.

The reaction rate and efficiency of enzyme hydrolysis was monitored by refractometric measurement of the dry matter of the solution and by the polarometric measurement of the sucrose content of the solution according to Friml and Tichá (Laboratory Control of Sugar Production, published by STI Food Industry, Prague 1977). From the obtained analytical results the theoretical calculation of the so-called invert quotient (Q; j, which is a value that indicates the ratio of invert sugar concentration (i) to total raw material (Sj; dimension - weight percent):

Qi = -j- · 100

The method of determination of β-D-fructofuranosidase activity, definition of activity and specific activity are given in MS. AO 249 325

Compared to the use of immobilized invertase for sucrose hydrolysis, the immobilized cellular materials obtained by the processes of AO 231 458 and AO 249 325 have substantially higher thermostability and are capable of enzymatic hydrolysis of high substrate concentrations not only in pure sucrose but also in various technical forms and its intermediates production, which will be demonstrated in the following examples of the invention.

Example 1

1000 g of wet mass of whole cross-linked cells of brewer's yeast, cross-linking of native cells with 1% glutaraldehyde and their washing was carried out according to MS. AO

231 458, was suspended in 9000 g cler with a refractometric dry matter of 65.8% by weight. The specific activity of the cross-linked cells was 600 μπιοί glucose. min ^- !. mg ^-1 dry matter. After homogenization crosslinked cells in the substrate solution with a vortex mixer, the suspension was transferred into a thermostated vessel equipped with a stirrer, pH adjusted with concentrated acetic acid to pH 4.5 and the suspension heated to 60 ^C C. Continuous Stirring (300 rpm. Min ^"1) carried for 3 hours. After this time, the conversion rate was 94.6%. The suspension was centrifuged on a Sharples centrifuge, the hot mixture was filtered through an asbestos cellulose plate, and the crosslinked cell mass was reused as described above for enzymatic hydrolysis of a fresh cleric batch, under the same conditions as described above. This procedure was repeated a total of 15 times, except that after every fifth repeated cycle of enzyme hydrolysis, 100 g of wet mass of cross-linked cells was supplemented with a storage unused biocatalyst to compensate for handling losses during centrifugation. The average degree of enzyme conversion over fifteen repeated cycles of enzyme hydrolysis was 92.3%.

Example 2

5 kg of mutually immobilized whole cells of baker's yeast were prepared according to the procedure of MS. AO 231 458 and the particles obtained were strengthened according to the art. AO 249 325. The specific activity of the immobilized cells was 500 μΐηοΐ glucose. min ^-1 dry matter particles.

g of wet, well aspirated and washed mass of the biocatalyst particles were suspended in 450 g of cler with an initial refractometric dry matter of 66.0%. The cler was pretreated with concentrated acetic acid to pH 4.6. The suspension was transferred to a plastic container which was designed so that the biocatalyst could be reused in this container many times. Above the bottom of the vessel was placed a filter baffle with fixed textile material (mill nylon sail, the porosity of which did not allow the biocatalyst particles to escape from the reaction vessel. vessel was thermostated in a water bath at 75 degrees C. the reaction mixture was continuously stirred with an anchor stirrer speed of 120 to 150 minutes' ^1. the reaction time was 3 hours. After the end of conversion (Qi = 92.3 ° / o J the hydrolysis products from the described apparatus were vacuum aspirated and fresh feed (450 g of substrate solution (clone at S = 66.0 ° / o and pH = 4.6) was run) and this procedure was repeated two hundred times as described.

Number Average Qi Cycles (%)

1— 10 91.0 11— 20 89.5 21— 30 88.9 31— 40 86.8 41— 50 85.2 51— 60 84.1 61— 70 83.7 71— 80 82.9 81— 90 81.5 91—100 80.2 101—110 78.3 11.1—120 76.2 121—130 72.3 131—140 70.1 141—150 67.8 151—160 65.1 .161—170 61.3 171—180 57.3 181—190 53.8 191—200 50.0

The results show that under the selected reaction conditions (refractometric dry matter of the substrate 66.0%, reaction temperature 75 ° C, pI-4.6, biocatalyst concentration 10% by weight), the biocatalyst can be used at least 100 times at an average conversion rate of 80 % theory / 100 repeated cycles of use. The operating half-life of the biocatalyst (monitored specific activity of the biocatalyst particles between cycles of reuse) is 200 repeat cycles under the reaction conditions indicated, which correlates with the degree of conversion efficiency.

Example 3 g of wet, well aspirated and washed mass of biocatalyst particles, the preparation method and specific activity of which are given in Example 2, were suspended in 450 g of a 50 percent refractometric dry matter sugar solution prepared from pure sucrose (refined sugar). The pH of this solution was adjusted to 4.5 with concentrated acetic acid prior to mixing with the biocatalyst. Further, the enzyme hydrolysis was carried out as described in Example 2, including the apparatus described, except that the reaction temperature was 68 ° C. The reaction time for one cycle was 3 hours. In the first hydrolysis cycle, Qi = 99.2% was achieved.

Under the above reaction conditions and method, the same biocatalyst batch was used a total of 30 times with an average Qi / 30 degree of reuse of 94.5%,

250282

Example 4

Molasses having a refractometric solids of S = 74.0 percent and a pH of 8.1 were diluted with drinking water to S = 65.0% and the pH adjusted to 5.5 with concentrated hydrochloric acid. 9 kg of this molasses solution was mixed with 1 kg of wet mass of the biocatalyst, whose specific activity and preparation method was mentioned in Example 2. The suspension was stirred continuously for 3 hours at 75 ° C. The degree of hydrolysis in the first cycle was 77.1%. After the first cycle of enzyme hydrolysis, the biocatalyst particles were separated by vacuum filtration through hot textile material, and the separated biocatalyst was resuspended in fresh molasses solution feed and 5 cycles of enzyme hydrolysis were performed as described substrate concentration and otherwise reaction conditions and handling.

The average degree of enzyme hydrolysis (5 cycles of reuse of the same biocatalyst feed was 75% of theory. Example 5

The heavy juice, an intermediate from sucrose production, with a refractometric dry matter of 55.0% and a pH of 8.5 was acidified with concentrated acetic acid to pH 4.6. An amount of 0.5 kg of the wet mass of the biocatalyst, whose specific activity and method of preparation was mentioned in Example 2, was transferred to 4.5 kg of the so prepared heavier juice, the suspension heated to 75 ° C and was stirred for 60 min. conversion intervals monitored over time intervals. The results are shown as follows.

Hydrolysis time (min)

Degree of hydrolysis Qi (%)

120

180

240

280

66.4

92.12

94.7

96.2

96.2

After 4 hours of conversion, the biocatalyst was separated by hot filtration through a filter press and resuspended in a fresh batch (4.5 kg) of heavy juice and a total of 10 repeated conversion cycles were performed with the same biocatalyst batch and otherwise in the same reaction conditions.

The average conversion rate / 10 repeated cycles of using the same biocatalyst feed was 93.7% of theory. Hydrolyzed heavy juice was collected in a storage vessel. A total of 43.8 kg of hydrolyzed heavy juice was obtained and treated as follows. 0.5 kg of activated carbon and 2 kg of diatomaceous earth were mixed into the heated solution (80 ° C) and the slurry was vacuum filtered hot through a filter press and after adjusting the pH to 8.1 using concentrated sodium hydroxide solution and concentrated to a refractometric evaporator. dry matter 75 ° / o.

Example 6

An aqueous affinity sugar solution having a refractometric dry matter content of 65.0% was prepared and the pH of the solution adjusted to 4.5 with concentrated acetic acid. Into 450 g of this solution was suspended 50 g of the wet mass of the biocatalyst, the specific activity and preparation of which were mentioned in Example 2. Subsequently, batch fresh hydrolysis of the always fresh batch of this technical substrate was carried out as also in Example 2.

In the first cycle of enzyme hydrolysis, after 3 h Q = 90% of theory was reached. A total of 10 repeat cycles of conversion with the same biocatalyst feed were performed and the average degree of enzyme hydrolysis / 10 repeat cycles was 88.4%.

Example 7

An aqueous solution of raw sugar of 65% refractometric solids was prepared and the pH of the solution adjusted to 4.7 with concentrated acetic acid. Into 450 g of this solution was suspended 50 g of the wet mass of the biocatalyst, the specific activity and preparation of which were mentioned in Example 2. Subsequently, batch fresh hydrolysis of the always fresh batch of this technical substrate was carried out as also in Example 2.

In the first cycle of enzyme hydrolysis, Q was achieved; = 88.6% in 4 h conversion. A total of 15 repeat cycles of conversion with the same biocatalyst feed were performed and the average degree of enzyme hydrolysis / 15 repeat cycles was 85.0%. Example 8

From the waste product after the production of cell-free yeast extract from baker's yeast, which is essentially dried yeast walls, the specific value of which ranges from 400 to 700 μπιοί glucose. min ” ¹ . mg < ^-1 & gt ^; dry mass, an immobilized biocatalyst was prepared according to the procedure of Art. AO 231 458 and the resulting particles are solidified and stabilized by the procedure according to U.S. Pat. AO 249 325. Biocatalyst particles with a specific value of 480 μΐηοΐ glucose were obtained. min ” ¹ . mg ” ¹ dry matter with a size distribution of 0.08 to 3.5 mm.

100 g of wet mass of this immobilized material was suspended in 900 g of a refined sugar aqueous solution and 34.2% refractometric dry matter, the pH of which was pre-adjusted to 4.6 with conc. Particle suspension was introduced into the fluidized bed via anchor stirrer (150 rpm. Min ^"1) and allowed to cool to 60 ^C C. The reaction time for one cycle was 3 hours. In the first cycle of the enzymatic hydrolysis has been reached the value Q = 99.0%.

A total of 20 repetitive enzyme hydrolysis cycles were performed with the same biocatalyst feed under the reaction conditions and handling method described in Example 2. The average degree of enzyme hydrolysis / 20 repetitive cycles of use was 97.3%.

Example 9

The cooled aqueous suspension of whole yeast cells (baker's yeast) was mechanically disintegrated in an amount of 10 liters by passing four times through a slotted pressure disintegrator at an overpressure of 6.10 ⁷ Pa. The disintegrated cell suspension was immobilized according to the procedure of Art. AO 231 458 and the particles solidified and stabilized according to the procedure of Art. AO 245 325. A total of 7 kg of a large mass of solid particles of biocatalyst with a specific activity of 600 μΐηοΐ glucose was obtained. min ^-1 . mg ^-1 dry matter with an average size distribution of 0.1 to 5.0 mm.

500 g of the wet mass of this biocatalyst was suspended in 4,500 g of light juice (intermediate from sucrose production) with a refractometric dry matter of 17.5%. Before introducing the biocatalyst into this substrate solution, the pH was adjusted from 9.0 to 4.6 with concentrated hydrochloric acid. The particle suspension was allowed to warm to 60 degrees C and the particles were raised using a 200 rpm stirrer. min “ ¹ . After one hour of reaction the degree of conversion was quantitative (100% of theory).

Under the reaction conditions, the same batch of this biocatalyst was used a total of thirty times at an average theoretical conversion rate of 30 repetitive cycles using 99.0% of theory.

The quantitatively hydrolyzed light juice solution was collected in a storage tank. A total of 130 L of glucose-glucose syrup was obtained. The yellowish solution was bleached by mixing 3 kg of activated carbon and hot filtration through an asbestos cellulose plate. The colorless solution was adjusted to pH 7.8 with concentrated sodium hydroxide solution to a vacuum evaporator and concentrated to 68% solids. The concentrated sweetening colorless syrup was flavored by adding 0.3 g / kg lemon syrup and 0.1 g / kg syrup ascorbic acid.

Example 10

Mutually immobilized whole yeast cells (baker's yeast), the method of immobilization, consolidation and the specific activity of this material are mentioned in Example 2, were divided by sieving on sieves of defined mesh size into individual size classes. The biocatalyst fraction, where the particle size distribution after separation was 1.0-5.0 mm, was packed into a 5 cm diameter and 150 cm high glass column in an amount of 2 kg wet mass. The glass column was provided with a tempered water jacket and provided with rigid, perforated filter baffles at both ends, which were covered with a textile material (mill nylon sheet) to prevent particles from escaping from the column. A 35% aqueous solution of refined sucrose, pH 4.6, was pumped from the bottom of the reactor from the reservoir at a rate of 2 ^L / h to bring the particles to a fluid flow. The column was tempered to 65 ^° C.

The degree of conversion at the effluent from the column was quantitative. The column was operated continuously for 5 days to obtain a total of 240 L of glucose-glucose syrup with an average degree of hydrolysis of 97.6%.

Example 11

Mutually immobilized whole yeast cells, whose specific activity, method of immobilization and consolidation is mentioned in Example 2, were used for repeatedly hydrolyzing the clergy solution under the conditions and method described in Example 2 as well. enzyme in the immobilized cell particles (operating half-life of 200 repetitive three-hour cycles at pH 4.6, 75 ° C and substrate concentration of 66% dry matter), the reaction time was extended to 4 hours between the 51st and 100th cycles and between 101 and 150 cycle for 5 to 6 hours. With this programmed reaction time extension, while maintaining a high degree of conversion (more than 30%), the same biocatalyst feed was utilized perfectly during 150 repetitive cycles. The average conversion rate / 150 cycles of reuse in the described process of extended reaction time was 82.3%.

Claims (4)

SUBJECT Process for the enzymatic production of glucose-glucose syrup from sucrose, characterized in that whole immobilized cells, immobilized cell walls or immobilized disintegrated cell suspensions with β-D-fructofuranosidase activity prepared according to author's certificates Nos. 231 458 and 249 325 are used for enzymatic hydrolysis. using aqueous solutions of pure sucrose, industrial sucrose in the form of natural sugar, raw sugar, affinity sugar, aqueous sucrose solutions in the form of liquid sugar, sucrose solutions formed as sucrose intermediates such as raw juice, light juice, heavy juice, clergy, syrups and further by-products of production, in particular molasses from production, possibly diluted molasses and enzyme hydrolysis are carried out at temperatures in the range of 10 to 90 ° C, preferably 60 to 75 ° C, at pH of the reaction mixture in the range 4; 0 to 7.0, preferably 4.5 to 7.0 5.5 at an initial sucrose concentration of 0.5 to 75.0 weight percent, preferably 40 to 65 weight percent. 2. A process according to claim 1, wherein the enzyme hydrolysis is carried out batchwise at a concentration of immobilized materials of 1 to 200 grams of wet mass. OF THE INVENTION. ¹ liter of reaction mixture, preferably 100 grams of wet mass. liter ^{-1 of the} reaction mixture, either by leaving the particles of immobilized materials permanently in the apparatus and separating the solution containing the reaction products from the reaction mixture, or separating the biocatalysts after the conversion is carried out outside the reaction apparatus, in both cases immobilized materials at least twice reuse. Method according to claim 1, characterized in that the enzymatic hydrolysis is carried out continuously, either in fixed bed devices of immobilized materials by flowing the substrate solutions through the fixed bed of particles, or preferably in buoyant bed devices of immobilized materials either by pumping the substrate solutions so. that the particles of biocatalysts are sustained by fluid flow or by introducing the particles in a different manner, for example by stirring. 4. Process according to claim 1, 2 or 3, characterized in that the resulting enzymatic reaction products are further treated by filtration, centrifugation, decanting, thickening, optionally and preferably enriched with additives such as. are vitamins, flavorings, colorants and others.