CS249526B2 - A method for the selective recovery of sugar, which is either glucose or maltose, from digested liquefied starch - Google Patents

Abstract

The method of selectively recovering sugar, which is either glucose or maltose, from digested liquefied starch according to the proposed solution consists in that a batch of digested liquefied starch is hydrolyzed by the action of a soluble or mobilized enzyme providing glucose or maltose to form a stream leaving the reaction zone containing 50 to 92% by weight of the above-mentioned sugar, this stream is divided into a product stream enriched with sugar containing at least 90% by weight of sugar and a stream depleted of sugar, the stream enriched with sugar is isolated and the stream depleted of sugar is recycled to the hydrolysis stage. The purpose of the proposed solution is to achieve a significant reduction in the reaction time, a reduction in the content of reverse products and a significant increase in the productivity of the entire process.

Description

The invention relates to a method for producing glucose or maltose from starch.

Naturally occurring carbohydrates in the form of starch are renewable sources of commercially important sugars, including the monosaccharide glucose and the disaccharide maltose. Glucose and maltose are food sweeteners and nutrients. Maltose is also useful for culture media. Glucose is particularly useful because it can be isomerized to the even sweeter monosaccharide, fructose.

The invention relates to a two-step process for obtaining highly pure glucose or maltose from starch-containing raw materials obtained from plants such as tapioca, corn, potato, rice and wheat.

A block diagram of a conventional process for producing glucose from digested liquefied starch is shown in Fig. 1. The term "digested liquefied starch", as used in this description and in the figures, means liquefied starch that has been partially hydrolyzed by the enzyme alpha-amylase. Various types of alpha-amylase enzyme are used to produce digested liquefied starch. The digested liquefied starches obtained have different but analogous properties depending on whether maltose or glucose is to be obtained.

Digested liquefied starch has a dry matter content of 30 to 45% and contains a smaller proportion of monosaccharides (up to 10%, but usually less than 4%), 20 to 70% of disaccharides to heptasaccharides (DP2-DP2) and 30 to 80% of octasaccharides and higher molecular weight polysaccharides. One measure of the degree of hydrolysis is the dextrose equivalent. Digested liquefied starch has a higher dextrose equivalent, i.e. it contains a higher proportion of dextrorotatory glucose compared to unprocessed starch. Unprocessed starch contains no or only a small number of free dextrose units; digested liquefied starch may have its dextrose concentration increased to a low value ranging from 5 to 25.

In the conventional process illustrated in Figure 1, the digested liquefied starch 1 enters a saccharification or hydrolysis reaction zone 2 where it is subjected to enzymatically catalyzed hydrolysis using a glucose-forming enzyme such as amyloglucosidase (glucoamylase), hereinafter referred to as AG.

The main feature of the current processes is that the hydrolysis is continued in one stage until the maximum glucose formation is reached, which corresponds to a content of about 94 to 96% glucose in the product stream 3, if a raw material containing about 30% dry matter is used. Although only one reaction zone is shown for saccharification in the figure, this variant represents only one possible embodiment. In other embodiments, it is possible to use a larger number of reaction zones connected in series.

Stream 3 leaving saccharification zone 2 containing more than about 94% glucose (based on dry matter) is then concentrated, if necessary, in evaporation zone 4 to yield product stream 5 containing from about 35 to about 50% dry matter. Product stream 5 is typically used as feedstock for isomerization reaction zone 6, in which glucose is enzymatically converted to fructose by glucose isomerase.

The usual process for producing maltose from starch is analogous to that shown in Fig. 1, the main difference being that the digested liquefied starch is hydrolyzed by an enzyme yielding maltose. Beta-amylase is predominantly and almost exclusively used as the enzyme producing maltose.

The present invention is directed to both the production of glucose and the production of maltose. For the sake of brevity, the following description will be limited to the production of glucose. It will be understood, however, that an analogous description would apply to the production of maltose.

The conventional process suffers from several disadvantages. One of the disadvantages, which is characteristic of all processes carried out to maximum conversion, is the increased cost due to the prolonged reaction time required to achieve maximum conversion. It is true that the longer the time required to achieve such conversion, the more expensive and therefore less advantageous the process. Since glucose suppresses the activity of the enzyme by forming a complex with AG, the rate of hydrolysis decreases with the accumulation of glucose, thereby further increasing the reaction time.

Another disadvantage characteristic of the relatively long residence time of the product in the reaction zone in AG catalyzed hydrolysis of digested liquefied starch is that glucose, a monosaccharide, converts back to disaccharides, including isomaltose. Isomaltose is a "stable disaccharide", i.e. it is not easily hydrolyzed, and is bitter, and is therefore a highly undesirable component of the glucose feedstock used for fructose production. The longer the reaction time, the higher the concentration of glucose and also the concentration of isomaltose in the product.

Yet another disadvantage, at least when a soluble enzyme is used in the saccharification zone, is that the enzyme must be continuously replaced as it is lost during production. The cost increases as the amount of glucose produced per unit of enzyme decreases.

Since current industrial processes for producing high fructose corn syrup by glucose isomerization start from a glucose feedstock containing at least 94% glucose, any new or modified method for producing glucose is limited in that it must provide a product with a comparable glucose concentration.

The more efficient process according to the present invention is capable of providing a product stream comprising at least 94% glucose and is not burdened by the above-mentioned disadvantages of the known processes.

The method of selectively obtaining sugar, which is either glucose or maltose, from digested liquefied starch, according to the proposed solution, consists in hydrolyzing a batch of digested or liquefied starch under the action of a soluble or immobilized enzyme providing glucose or maltose to form a stream leaving the reaction zone containing 50 to 92% of the aforementioned sugar, this stream is divided into a product stream enriched with sugar containing at least 90% sugar and a stream depleted of sugar, the stream enriched with sugar is isolated and the stream depleted of sugar is recycled to the hydrolysis stage.

According to a preferred embodiment, the stream leaving the reaction zone contains 70 to 80% sugar.

As regards the separation of the stream leaving the reactor, it is preferably carried out using a membrane or a solid adsorbent.

A characteristic feature of the method for selective glucose recovery is that amyloglucosidase is used as the enzyme. In the selective recovery of maltose, beta-amylase is expediently used.

By carrying out the hydrolysis to a degree substantially lower than the degree corresponding to the maximum glucose formation, a substantial saving of time is achieved according to the invention and glucose with a substantially lower concentration of reversal products is obtained.

The advantage of the invention is therefore that it allows a substantial reduction in the operating time. Another advantage accompanying the reduction in the operating time is that the process according to the invention provides glucose containing fewer reverse products (disaccharides) than known processes.

The method of the invention can take advantage of further advantages resulting from the characteristics of enzymatically catalyzed hydrolysis of digested liquefied starch that cannot be utilized in current known industrial processes. When the dry matter content of the feedstock for AG catalyzed hydrolysis is increased, the stability of the enzyme, as measured by its half-life, also increases. With increasing dry matter content, the rate of glucose formation also increases.

Both of these properties are quite favorable, but nevertheless cannot be used in current industrial processes, since an increase in the dry matter content also leads to a lower maximum achievable glucose concentration accompanied by an increased content of reversal products.

Unlike the processes known heretofore, the method of the invention can advantageously utilize the favorable characteristics of increased enzyme stability and increased glucose production rate without these advantages being accompanied by the disadvantage of increased formation of reversal products. In this sense, the invention is truly synergistic in nature—absorbing beneficial effects but not detrimental effects.

One characteristic feature of the use of immobilized AG in the hydrolysis of digested liquefied starch is that it achieves less than 94% glucose in the product at equilibrium and that the relatively long residence time results in the formation of undesirable reverse products [immobilized AG can therefore be used with difficulty in current industrial processes.

Another advantage of the present invention is therefore that it allows for easy utilization of immobilized AG. When using soluble (or non-immobilized) AG to hydrolyze digested liquefied starch, it is necessary to continuously replace the enzyme that is lost in the production of glucose.

Yet another advantage of the process of the invention is therefore that it provides a substantial increase in productivity, which is defined as the amount of glucose produced per unit of enzyme. The increase in productivity is partly due to the recycling of the enzyme present in the recycle stream (in the case where soluble AG is used) and further due to the extension of the half-life of the enzyme (whether soluble or immobilized AG is used).

The glucose concentration in the stream leaving the process according to the invention is at least 90%. However, glucose concentrations above 99% can easily be achieved by appropriately adjusting the process variables.

Another advantage of the process is that it can be adapted for continuous production of high purity glucose, whereby glucose purity above 99% can be achieved.

Yet another advantage of the process according to the invention is that it allows for virtually complete conversion of starch to glucose.

From the large number of advantages mentioned above, which the method according to the invention brings, it is clear that the invention represents a significant advance in the production of glucose with a concentration of about 94% and higher by enzymatically catalyzed hydrolysis of digested liquefied starch.

According to one embodiment of the invention, the invention provides a method for selectively recovering sugar, which is either glucose or maltose, from digested liquefied starch. The digested liquefied starch is hydrolyzed by the action of an enzyme producing glucose or maltose, resulting in a product stream containing 50 to 92% sugar. This stream is separated into a sugar-enriched product and a sugar-depleted stream. The sugar-enriched stream is isolated and the sugar-depleted stream is recycled to the hydrolysis stage.

In a more specific embodiment of the invention, the stream leaving the hydrolysis stage contains 70 to 80% sugar, and in an even more specific embodiment, the sugar-enriched product stream contains at least 90% sugar.

The process for obtaining glucose or maltose from digested liquefied starch according to the invention represents a radical departure from the methods known up to now. One characteristic feature of this departure is the partial hydrolysis of the digested liquefied starch.

While according to known methods the digested liquefied starch is hydrolyzed until maximum glucose formation is reached, in the method according to the invention the hydrolysis is continued for a considerably shorter time, thereby obtaining a product stream containing a lower glucose concentration than the maximum concentration.

Another characteristic feature of this diversion is the separation of the hydrolysis product into a glucose-enriched product stream and a glucose-depleted stream, the latter of which is recycled to the hydrolysis stage. The process according to the invention is illustrated in the block diagram in Fig. 2.

The digested liquefied starch, as defined above, containing from about 30 to about 45% solids serving as a raw material, is passed through line 11 to a saccharification reaction zone 12. Although only one reaction zone is shown for saccharification in the figure, this variant represents only one possible embodiment.

In other embodiments, a plurality of reaction zones connected in series may be used, and all such embodiments are within the scope of the invention. A stream 13 containing about 50 to about 92% glucose on a dry matter basis leaves the saccharification zone and is used as feed to separation stage 15. The separation is shown in the figure as a single-stage process, but multi-stage separation variants are also within the scope of the invention.

Two streams emerge from the separation zone. One is a glucose-enriched product stream 16, which contains at least 90% glucose on a dry matter basis, and the other is a glucose-depleted stream 14. This second stream is recycled to the saccharification reaction zone 12.

In the event that the glucose-enriched product stream 16 is to serve as a feedstock for an isomerization reactor in the final stage, it is directed to an evaporation zone 17, from which a stream 18 containing about 35 to 50% dry matter is obtained. Stream 18 is then used as a feedstock for an isomerization reaction zone 19, in which the glucose is converted to fructose. However, it should be understood that the glucose-enriched product stream may serve as a source of glucose for purposes other than as a feedstock for isomerization to fructose.

Other uses for which glucose can be used include hydrogenation to sorbitol, fermentation to ethanol, and as a sweetener in food.

If the product is maltose, the isomerization reaction zone 19 is not included in Figure 2. The maltose-enriched product (stream 16) is either used as is or sent to the evaporation zone 17 to obtain more concentrated maltose solutions or even dry maltose.

In the initial step of the process, the digested liquefied starch is selectively enzymatically hydrolyzed by a glucose-producing enzyme, particularly AG, to produce a product stream containing from about 50 to about 92% glucose. This selective hydrolysis step is often referred to as the saccharification step. The AG used may be soluble, in which case it is recycled with the glucose-depleted stream, or it may be immobilized AG.

In either case, pullulanase or alpha-amylase, or both, may be present in the digested liquefied starch feed to facilitate hydrolysis. The temperature at which the enzymatic hydrolysis is carried out depends on the thermal stability of the enzyme used, but is typically in the range of about 40 to about 80°C, with 60°C being most common. However, it is known that AG from at least one microorganism is sufficiently thermally stable to permit the process to be carried out at temperatures up to about 100°C.

Enzymatic hydrolysis is carried out at a pressure of 6.87 kPa to 6.87 MPa. The residence time for which the starch is in contact with the immobilized enzyme is relatively short, i.e., in the range of 4 minutes to 1.6 hours, and is correlated with the hourly space velocity of the liquid phase, which may be in the range of about 2 to about 50 ^h_1 .

Space velocity and residence time are correlated such that, for example, the degree of conversion is maintained within the desired range by a short residence time and a high space velocity (which lie within the range specified above). Conversely, long residence times and low space velocities (which lie within the range specified above) can also be used to achieve the desired result.

The residence time for which the starch is in contact with the soluble enzyme is relatively short compared to conventional processes, i.e., it ranges from 2 to 48 hours.

A desirable consequence of the hydrolysis of digested liquefied starch to a product stream containing 50 to 92% glucose on a dry matter basis is a substantial reduction in reaction time. Hydrolysis to a product containing 50% glucose may take less than one-quarter of the time required to obtain 94% glucose, hydrolysis to 90% glucose may take only half that time, and even hydrolysis to 92% glucose may take only three-fifths of that time.

Another desirable consequence is a decisive improvement in organoleptic properties, which is achieved by a substantial reduction in the content of the bitter component, isomaltose. Hydrolysis to 50% glucose may be accompanied by the formation of only one-fourth of the amount of isomaltose that accompanies the production of 94% glucose.

Digested liquefied starch containing about 30 to about 45% dry matter is common in industry.

10 as the raw material, but the process of the invention is not limited to this raw material. However, a dry matter content in the range of about 35 to about 45% is preferred in the practice of the invention, as this allows full utilization of the beneficial effect of higher dry matter content on enzyme stability and glucose production rate.

In the processes known up to now, the hydrolysis is continued until the maximum glucose formation, which corresponds to a glucose concentration in the product of about 94%. A characteristic feature of the process according to the invention, on the other hand, is that the hydrolysis is continued until the glucose content in the stream is in the range of from 50 to 92%. Usually, the process is not carried out to a glucose content higher than 90 percent. It is desirable that the glucose content in the product leaving the saccharification zone is in the range of from about 60 to about 85% and the most preferred content is about 70 to 80%.

The stream leaving the hydrolysis (saccharification) zone is then divided in a separation step into a glucose-enriched product stream and a glucose-depleted stream. If the glucose-enriched product stream is to be used as feedstock for isomerization to fructose, this stream must contain at least about 94% glucose, since current processes for producing fructose from glucose require a feedstock containing glucose of at least this purity.

In those cases where a feedstock with a glucose content lower than 94% is acceptable, the separation does not have to be carried out under such stringent conditions and glucose of lower purity is obtained. From a practical point of view, the product stream enriched with glucose contains at least about 90% glucose. It is also clear that the separation can be carried out ⁱⁿ such a way as to obtain glucose of even higher purity, i.e. 94% glucose. According to the invention, it is also possible to continuously produce glucose with a purity higher than 99%, if such a high purity is required.

The separation stage usually consists of a single stage. However, in some circumstances, a multi-stage separation may be advantageous and such an embodiment is also within the scope of the invention. In any case, the glucose-enriched product stream from the separation stage is isolated for subsequent use and processing.

Any method that can selectively separate glucose from disaccharides and higher polysaccharides is suitable for performing the separation. In the case where maltose, i.e. a disaccharide, is formed, the separation must be selective only for higher polysaccharides. For example, membrane separation can be effectively used.

Another example is separation based on the use of solid adsorbents. Examples of adsorbents include substances such as alumina, silica, various clays, zeolites, etc. Yet another separation method is the selective crystallization of glucose from a carbohydrate mixture. At least two other separation methods that can be used in the method according to the invention are solvent extraction and supercritical e-t^ction.

An integral part of the invention is the recycling of the glucose-depleted stream to the hydrolysis stage. If multiple hydrolysis zones are used in the hydrolysis (saccharification) stage connected in series, the choice of the hydrolysis zone to which the recycled stream is returned depends on operating parameters such as:

— reactor configuration;

— activity of the relevant enzyme;

— concentration of reactants and/or products in the recycle stream;

— the circumstance, whether the enzyme appears dissolved or immobilized;

— enzyme concentration, if the enzyme is soluble;

— required product purity;

— specifically the separation method used, etc.

Under many operating conditions, the location of the recycle inlet can vary considerably without significant effect. For example, where the enzyme is immobilized, a portion of the glucose-depleted stream can be recycled by combining with the feed prior to its entry into the hydrolysis zone. In all circumstances, however, it should be understood that the determination of the appropriate inlet location will depend on the specific parameters employed in any particular process, and such determination is within the skill of the person skilled in the art.

As already mentioned, the enzymes used in the process of the invention most often include amyloglucosidase or beta-amylase, which can be supported on a solid support. By using enzymes immobilized on a support, it is possible to relatively stabilize the enzymes, ensure their efficient utilization and prevent their loss in the reaction medium.

Such mobilized or insolubilized enzymes can then be used in various reactor systems, such as packed columns, stirred tanks, etc., depending on the type of substrate used. By allowing the reuse of enzymes that are in a relatively stable state and can therefore be used for a relatively long period of time, the entire process becomes economical and attractive from an industrial point of view.

Enzymes can be immobilized on a solid support by any method known in the art. One method of immobilization that may be cited for illustration is the enzyme immobilization method disclosed in U.S. Patent No. 4,141,857.

As previously mentioned, ultrafiltration membranes can be used to separate glucose or maltose from the partially hydrolyzed reaction mixture resulting from the action of the enzyme on liquefied starch. Any membrane having a suitable molecular weight and pore size is suitable, which is capable of providing a high glucose or maltose content in the permeate and which is capable of retaining other substances containing unhydrolyzed oligosaccharides. Ultrafiltration membranes typically have a molecular weight cutoff of from 100 to 5,000 and typically have pores of from about 0.5 to about 12.5 nm in size.

Specific examples of materials from which these membranes can be made include cellulose acetates such as:

cellulose diacetate, cellulose triacetate and mixtures thereof, polyethyleneimine crosslinked with dialdehyde, dicarboxylic acid chloride or diisocyanate, polyacrolein, chitosan crosslinked with dialdehyde, such as glutaraldehyde, polystyrene sulfonates, etc.

The above ultrafiltration membranes are only representative examples illustrating individual classes of materials and the invention is not limited to the use of these particular membranes.

The two-stage process of the invention can be carried out in any suitable manner and can be carried out either batchwise or continuously. For example, when operating in batches and a high glucose syrup is to be obtained, a predetermined amount of feed containing liquid starch, which has been previously treated with alpha-amylase to increase the dextrose equivalent, is contacted with immobilized amyloglucosidase for a predetermined time under reaction conditions comprising a temperature of from about 45 to about 70°C, and a pressure in the range of from about 6.87 kPa to about 6.87 MPa.

In addition, the residence time for the feedstock to be in contact with the immobilized enzyme is correlated with the hourly space velocity of the liquid phase fed into the system to achieve a conversion of the liquid starch to glucose within the desired range. After passing through the immobilized enzyme, the effluent is collected and the partially hydrolyzed starch is subjected to ultrafiltration. In this step, the reaction mixture is passed through an ultrafiltration membrane of the type mentioned above, obtaining a product stream with a high glucose (or maltose) content as the permeate.

Products that do not pass through the membrane are recycled. A portion of the recycled stream may be mixed with the stream leaving the enzyme treatment, while another portion of the recycled stream may be mixed with the feed stream entering the zone containing the immobilized enzyme. If desired, a third portion may be recycled back to the zone where the feedstock is pretreated with alpha-amylase.

The process of the invention can also be carried out continuously. When operating in this manner, a feed containing the processed liquefied starch is continuously fed into a reaction vessel, such as a column containing the desired immobilized enzyme, the column being maintained under suitable operating conditions of temperature and pressure. After passing through the enzyme, which proceeds at a predetermined hourly space velocity which is sufficiently high to cause only partial hydrolysis of the feed with little or no formation of reverse products such as isomaltose, the reaction mixture stream is continuously withdrawn and fed to an ultrafiltration device containing a membrane of the type mentioned above.

After passing through this device, which is also maintained under suitable operating conditions of temperature and pressure, the permeate containing a syrup with a high glucose or maltose content (i.e. above 90 percent) is obtained as a product. The retentate, containing unhydrolyzed oligosaccharides, such as saccharides with a degree of polymerization (DPJ DPz, DPe, DP9 _+, is also collected.

A portion of this product is recycled back to the immobilized enzyme column where it is reused as feedstock, another portion is mixed with the effluent from the immobilized enzyme treatment stage, and, if desired, still another portion is fed to the alpha-amylase pretreatment stage.

Instead of any of the above membrane separations, separation by means of adsorbents may also be used. One suitable adsorbent is described in British Patent No. 1,585,369 and comprises zeolites X or Y containing one or more selected cations at the exchangeable cation sites. A preferred adsorbent exhibiting selectivity for glucose, in the case of its mixture with disaccharides, higher oligosaccharides and polysaccharides, is the potassium form of zeolite X.

The following eleven examples serve to further illustrate the invention. The examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Percentages in the examples are by weight, as in the preceding description, unless otherwise stated.

Example I

The starch feed is treated with alpha-amylase to adjust its dextrose equivalent to 15 DE. In this case, the enzyme was immobilized and contained amylglucosidase supported on a solid alumina support. The treated starch feed is passed through a column containing 40 cm ³ of immobilized

24952S enzyme. The processed starch feed used contains 0.1% benzoate, SO ppm sodium omadine and has a pH of 4.2. The starch is processed at a temperature of 45 ^J C and a pressure higher than atmospheric pressure with a liquid phase space hourly velocity of 3.21 h S Current off,+ ?Pe DP7 DPe dps

19.1 0.2 0.1 —

The product obtained from this column (200 ml) is then passed through a cellulose acetate membrane. From the original 200 ml, 130.7 ml of product is obtained as permeate. Analysis of the permeate passed through the membrane at a temperature of 22 °C and a pressure of 0.02 MPa gives the following composition:

94.2% glucose,

3.6% maltose and only

1.7% oligosaccharides having a value

DP„ ₊ .

The material not passed through the cellulose acetate membrane (retentate) can be recycled and used as part of the feed, which is introduced into a zone containing the immobilized enzyme amyloglucosidase supported on a modified alumina support.

Example II

We proceed as described in Example I. The modified liquefied starch forming the feed is again passed through a column of mobilized amyloglucosidase under similar conditions as in Example I. The column effluent containing 77.2% glucose, a smaller ^amount of maltose, DP3, DP7, DPs and a significant amount of DP ₊ oligosaccharides is passed through a polystyrene sulfonate membrane sold under the trade name Amicon UM2. A total of 122.8 ml of permeate passes through the membrane. Liquid chromatography analysis shows that the permeate contains 97.6% glucose, 2.3% maltose and only 0.1% DP-x oligosaccharides.

When the above experiment is repeated under HPLC analysis: (area %)

DP ₉₊ DPs DP7 DPfi DPs

36.9 0.9 0.3 0.3 0.1

A 100 ml stream from this column is passed through a cellulose acetate membrane, while the temperature in the membrane filter is maintained at 22 °C and the pressure at 0.62 MPa. Liquid chromatography analysis of the permeate shows the presence of 79.0% maltose, 16.3% DP3 product and 2.4% DP ₉₊ oligosaccharides.

When the above experiment is repeated using an ultrafiltration membrane sold under the name Amicon UM2, a permeate containing 90.1% maltose is obtained, 9.7% of which is eluted from the column and analyzed by liquid chromatography. The analysis gives the following percentage composition, where DP stands for the degree of polymerization (for example, DPz stands for an oligosaccharide containing 7 monomeric glucose units.

DPd DPs DPz Glucose — 0.3 3.1 77.2 using a cellulose acetate membrane with the trade name Nucleipore with the product from the column containing immobilized amyloglucosidase, is passed through this membrane at a temperature of 22 ^: C and a pressure of 0.62 MPa, a permeate containing 97.5% glucose, 2.3% maltose and 0.2% oligosaccharides DP ₉₊ is obtained.

The retentate that has not passed through the cellulose acetate membrane can be collected and recycled. A portion of the retentate is mixed with the product from the immobilized enzyme treatment stage and the remaining portion is recycled as part of the feed entering the immobilized enzyme treatment zone.

Example ¹¹ This example illustrates the ability of the process of the invention to produce a product with a high maltose concentration. A liquefied starch feed, which has been pretreated with alpha-amylase to adjust the dextrose equivalent to the desired level, is passed through a column containing 100 ^cm3 of beta-amylase immobilized on a solid matrix similar to that used in Example 1. The feed at pH 5.0 contains 0.1 moles of acetate and 0.2 moles of benzoate, which serve as buffers. The feed is passed through the enzyme at a temperature of 55°C under a pressure greater than atmospheric pressure with a liquid phase hourly space velocity of 5 h- ¹ . The product passed through the column is analyzed by liquid chromatography, the following results being obtained:

np.i DPs DP? glucose

0.5 11.1 50.2 0.5 gosaccharide DP3 and only 0.2% oligosaccharide. DP ₉₊ .

The retentate obtained from the ultrafiltration membrane separation described in the previous paragraph can be recycled. Part of it can be mixed with the effluent from the immobilized beta-amylase treatment stage and another part can be mixed with the liquefied starch feed before it is brought into contact with the immobilized enzyme.

Example IV

In this example, a liquefied starch feed, optionally pretreated with alpha-amylase to adjust the dextrose equivalent of the starch to a predetermined level, is passed through a column of immobilized amyloglucosidase at a temperature of 45 degrees Celsius and a pressure greater than atmospheric pressure. The reaction mixture is left in contact with the immobilized enzyme for a predetermined time to achieve a conversion to glucose of about 75%, and the column stream is then passed to an ultrafiltration device containing a polyacrolein membrane. Membrane filtration is performed at ambient temperature and a pressure of about 172 kPa. The permeate is collected while the retentate is recycled and mixed with the stream entering the membrane filter.

In a similar manner, a liquefied starch feedstock having a dextrose equivalent of about 15 can be passed over a mobilized enzyme comprising beta-amylase supported on a modified alumina support under reaction conditions comprising a temperature of about 50°C and a pressure greater than atmospheric pressure. The reaction mixture is passed over the enzyme at a predetermined hourly space velocity for a residence time sufficient to achieve a degree of starch to glucose conversion of about 75 percent. A continuous product stream is withdrawn from the hydrolysis zone and passed to a membrane filter comprising a polyethyleneimine dialdehyde membrane. Membrane filtration is carried out at a temperature of about 25°C and a pressure of about 687 kPa. The permeate containing more than 90% glucose is collected while the retentate is recycled.

One portion of the retentate is mixed with the stream leaving the enzyme treatment zone, a second portion is recycled and mixed with the liquefied starch feed introduced into the mobilized enzyme zone, and a third portion is recycled to the pretreatment zone, in which the starch feed is contacted with alpha-amylase, serving to increase the dextrose-equivalent of the starch to a predetermined level.

The stream continuously withdrawn from the immobilized enzyme treatment can also be passed through a chitosan dialdehyde ultrafiltration membrane under reaction conditions comprising a temperature of about 22°C and a pressure of about 687 kPa. The permeate contains a major portion of glucose and is isolated, while the retentate is recycled by mixing with the stream leaving the immobilized enzyme treatment zone.

The following seven examples are illustrative of the invention and are not intended to be limiting in any way.

The product Maltrin-150 is used as the digested liquefied starch that serves as the starting material, and has the following typical composition:

% glucose, % DP2, % DP3, % DP4, % DPs, «/o DPe, % DP7 and % DPa and higher polysaccharides.

Glucoamylase activity is determined as follows:

To 4 ml of starch solution containing 30% dry matter, 25 μΐ of enzyme solution is added and the mixture is incubated for 30 minutes at 60 °C. The hydrolysis is stopped by adding 1 ml of 0.2N sodium hydroxide and the mixture is cooled. The amount of glucose produced is determined using a glucose analyser. The number of grams of glucose produced per hour represents the AG activity expressed in Miles units.

Example V

AG solutions (77 units/liter) at pH 4.2 in glucose solutions of various dry matter contents are maintained at 60 °C. Aliquots are periodically taken from the solutions and AG activity is determined. The half-life of the enzyme at dry matter contents of 30; 44 and 55% is 4.8; 12.3 and 21.9 days, respectively. The stability of the enzyme, measured by the half-life at 60 °C, increases 4.5-fold over a range of dry matter contents from 30 to 55%.

Example VI

A batch of Maltrin 150 starch with varying dry matter (ds) content at pH 4.2 containing 77 units/litre AG is hydrolysed at 60°C. The glucose concentration is monitored over time, measured by high-performance liquid chromatography (HPLC). The results are summarised in the following table:

Table 1

Dependence of glucose concentration on hydrolysis time

Time Glucose concentration in g/1

(h) Dry 30% Dry 44% Dry 55% 5 260 290 305 10 320 405 425 20 330 500 540 40 340 525 600 80 340 525 600

The results clearly show that the rate of glucose formation increases with increasing dry matter content in the crop.

Example V I I The approach Maltrinu-150 0 dry matter content 30 percent and pH 4.2, containing 77 units of AG per liter is hydrolyzed at 6 0°C. The product is analyzes for glucose, total content disa- charides (DP2) and isomaltose. Results from two different attempts with different AG are compo- summarized in the following table: Table 2 Production of glucose, disaccharides and isomaltose Attempt No. 1 Time Content (% by weight) (h) glucose breath- isomal- rids tose 2 28 8.5 0 4 58 9.0 0.6 6 72 4.5 0.9 8 88 4.0 0.9 10 90 4.5 1.3 12 92 4.8 1.3 20 94 5.8 2.2 30 94 7.0 3.4 Attempt No. 2 Time Contents (% by weight) (h) glucose breath- isomal- rids tosis 1.8 45.1 17.3 0 4.7 69.0 10.8 0 5.6 74.4 7.5 0 7.5 80.9 4.5 0 13.0 91.1 3.2 0.0 20.0 93.5 3.4 0.8 48.0 94.8 4.1 1.6

It is clear from the results that a product containing 90% glucose is obtained in about half and a product containing about 92% glucose in about 60% of the total time required to obtain a product containing 94% glucose.

The results also clearly show the accumulation of isomaltose during the later stages of conversion.

Example VIII

This example compares the results obtained in a single-pass reactor (i.e. conventional operation) in which the digested liquefied starch is hydrolyzed to a glucose content of 94% or more, with the results obtained in a closed-loop system in which the product from the saccharification zone is fed to membrane ultrafiltration, the glucose-enriched product is withdrawn, and the glucose-depleted product is recycled to the reactor inlet. The hydrolysis is always carried out at 60°C at a pH of 4.5 to 5.5.

Experiment A represents an experiment in a conventional reactor with a single pass of the reaction mixture, experiments B and G are experiments with a system comprising a closed technological loop.

System B uses a cellulose acetate membrane with a molecular weight cutoff of 10,000, operating at 60°C and 1.03 MPa, and system C uses a polyelectrolyte membrane with a molecular weight cutoff of .500, operating at 60°C and 2.06 MPa.

Both membranes are manufactured by Amicon and are available under the designations YM 10 and UM 05. The results are shown in Table 3, where DPi represents the total monosaccharide content, which is practically exclusively glucose, DPs represents disaccharides, DP ₄₊ represents polysaccharides with 4 or more units and Dry. represents the dry matter content. The analytical data relating to the composition of the reactor effluent, product stream and recycle stream in runs B and C are equilibrium data.

T a b l e 1 a n d 3

Comparison of single-pass operation with closed-loop system operation

Experiment B

Experiment C

Experiment A

Enzyme dose (AGj 77.0 34.0 58.0 (units/1 j residence time (h) 48.0 14.0 20.0 Handle composition (%/o) Dry. 30.0 26.1 24.7 DPI 1.0 1.2 1.6 DP2 4.5 4.3 3.9 DP., ₊ 87.5 87.8 88.3 Composition of the stream leaving the reactor Dry. 33.0 27.0 30.4 DPt 95.7 90.5 83.4 DP2 4.1 2.9 4.4 DPt-i 0.2 6.3 11.6 Product flow Dry. 26.1 24.7 DPI 93.6 98.0 DP2 3.1 2.0 DP,+ 2.9 0.0 Recycled electricity Dry. 27.4 34.2 DPi 89.9 82.5 DP? 3.5 3.8 DP, ₊ 6 3 13.1

Example IX

A kinetic model was developed from the experimental data, which not only closely reproduced the existing experimental data, but could also be used relatively reliably to predict the course, the following factors were used as variables (experimental parameters) in the model: composition of the feed (liquefied digested starch), enzyme dosage, residence time in the reactor, membrane type and operating conditions. The glucose and isomaltose concentrations in the product stream were obtained as outputs. The liquefied digested starch feed contained 30.6 percent by mass. dry matter with a content of:

1.2% DPI,

4.8% DP2 and

87.0% DP _{4 +} .

In experiment A, a single-pass reactor was used. In experiments P and C, closed loop (recycle) systems were used using the polyelectrolyte membrane described in the previous example. The glucose-depleted stream was recycled to the top of the reactor. The compositions of the reactor effluent and product streams represent equilibrium values.

Table 4

Isomaltose accumulation

Experiment A

Experiment B Experiment C

AG concentration (units/l) 77.0 residence time (h) 48.0

Current leaving the reactor:

glucose content (%) 95.7 isomaltose content 1.68

Product flow:

glucose content (%) isomaltose content (%)

9.0 18.0 10.0 10.0 74.0 87.3 0.7 1.85 95.5 97.2 0.34 0.73

The data clearly show that the closed loop, i.e., recycle system provides glucose at the same high concentration as the process performed in a conventional single-pass reactor, however, in the recycle system the enzyme dosage can be significantly reduced and the product contains Ϋ4 of the isomaltose content.

Experiment C shows that the glucose content in the product can be increased to over 97% (using Ϋ4 enzyme concentration) and still the isomaltose content in the product is less than half that in the product obtained by the conventional method.

Example X

In this example, the kinetic model described above is used to determine the effect of recycle location on the glucose and isomaltose content of the product stream and the reactor effluent stream under steady state conditions. The data are summarized in Table 5. “Reactor volume” is the percentage of the reactor volume that precedes the recycle inlet; “0” represents the top of the reactor, “100” represents the bottom of the reactor. The liquefied digested starch feed has the same composition as in the previous example, the reaction time is 10 h, and the polyelectrolyte membranes from Example 4 are used, operating under the same conditions.

Table 5

The influence of the location of the recycling input on the product composition

30 50

90

AG concentration: 18 μ/\

Current leaving the reactor

glucose 87.4 88.0 87.0 85.0 73.0 57.0 isomaltose 1.85 1.8 1.7 1.3 0.6 0.25 Product glucose 97.2 97.2 97.2 96.9 96.9 90.7 isomaltose 0.73 0.71 0.66 0.55 0.27 0.13 AG concentration: 77 μ/1 Current leaving the reactor glucose 91.5 91.5 91.5 91.5 92.3 91.0 isomaltose 4.75 4.8 4.7 4.4 3.35 2.5 Product ‘ glucose 96.9 96.9 96.9 97.0 97.3 97.4 isomaltose 1.73 1.74 1.70 1.59 1.22 0.93

The results presented in the table reflect the fact that at high AG concentration the product composition is relatively insensitive to the location of the recycle inlet and at lower enzyme concentration no significant change in product composition is evident when the recycle inlet is located between 0 and 90% of the reactor volume. The overall conclusion is that the recycle location is an optional variable dependent on other operating parameters such as enzyme concentration, residence time, separation method used, desired product composition, etc.

Example XI

The data provided by the kinetic model show the advantages of using a high solids feed in a closed loop, i.e. recycle, system that cannot be utilized in a conventional single-pass reactor. The feed has the composition shown in Example 5, with only the dry matter content (%wt) varying. The recycle reactor uses the polyelectrolyte membrane from Example 4 operating under the conditions shown in Example 4.

Table 6

Effect of dry matter content

Single-pass reactor

Dry. [ %) 30 Residence time (h) 48 AG concentration, (units/l) 77 Product, glucose content (% by weight) 95.7 isomaltose content (% wt.} 1.7

Closed loop recycle reactor

40 40 71 10 77 18 93.6 95.6 2.2 0.7

In a single-pass reactor, the residence time is unacceptably long and the isomaltose concentration is unacceptably high when using a feed with a dry matter content of 49%, while when operating in a closed loop recycle reactor, the residence time is reduced by 7X, the isomaltose concentration by 3X, and the enzyme concentration by 4X.

Claims (5)

A method for selectively obtaining sugar, either glucose or maltose, from cleaved liquefied starch, characterized in that the cleaved liquefied starch feed is hydrolyzed by treatment with a soluble or mobilized glucose-providing or maltose-producing enzyme to produce a stream leaving the reaction zone containing 50 to 50 92 wt. of the aforementioned sugar, said stream being divided into a sugar-enriched product stream containing at least 90% by weight of sugar; and a sugar-depleted stream, the sugar-enriched stream is recovered, and the sugar-depleted stream is recycled to the hydrolysis stage. VYNALEZU 2. A process according to claim 1 wherein the stream leaving the reaction zone comprises 70 to 80 wt. of sugar. 3. The method of claim 1, wherein the enzyme is amyloglucosidase. 4. The method of claim 1, wherein the enzyme is beta-amylase. 5. The process of claim 1, wherein the separation of the reactor effluent is performed using a membrane or solid adsorbents.