KR800001395B1 - Isomerization method of glucose - Google Patents

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Description

Isomerization method of glucose

The present invention relates to a method for the continuous isomerization of glucose at low processing costs.

Known glucose isomerization agents are virtually all activated cobalt, which significantly adds to the cost of syrup conversion since any cobalt added to the injected glucose syrup must be removed from the glucose-fructose product by ion exchange methods, which are relatively expensive. It becomes an argument.

The fact that cobalt maximal activity against enzyme is observed when cobalt ion concentration in glucose syrup is about 10 ^-3 M. A study on glucose isomerase derived from B. coagulans cells (see Danno et al, Agriculture Biologic Chem. Vol. 31, No. 3,1967, pp. 284-292) was reported. The concentrations for the formulated enzyme preparations have been shown to be somewhat higher. It is believed that most of the patented technologies published after this paper were adopted to use the cobalt concentration required for enzymes as described above regardless of the microbial source. Since cobalt ions added to the glucose injection syrup should be thoroughly removed (by ion exchange) from the glucose-fructose product syrup, substantially reducing the amount of cobalt added to the injection syrup can significantly reduce the cost of purification of the glucose-fructose product syrup. have.

Also rain. It is also known that the activity of cogurans is affected by the magnesium-ion content of the syrup, and by conventional methods, the magnesium in the syrup is usually contained in a high content, that is, at a concentration of 10 ^-2 M, regardless of the enzyme source. Was used. According to a conventional method glucose syrup receiving the enzymatic isomerization reaction typically contains CO ⁺⁺ to 10 ^-3 M and contains a Mg ⁺⁺ as a 10 ^-2 M. Typically, aftertreatment formulations of syrup are designed to remove not only cobalt but also magnesium. However, magnesium is a common non-toxic ingredient in foods, so it is not necessary to actually remove magnesium.

In other words, a significant reduction in the amount of cobalt used can be handled at a practically low cost. In addition, even if the amount of magnesium used is reduced, it can be processed at low cost.

According to the present invention, Co ⁺⁺ is not used at all, and furthermore, according to a suitable method of the present invention, the additional use amount of magnesium is considerably reduced. When the present invention can be described in more detail as follows.

The method of the present invention comprises glucose syrup containing less than or equal to Ca ⁺⁺ and about 10 ⁻³ M and less than or equal to about 10 ⁻² M of Mg ⁺⁺ , preferably not more than 5 × 10 ⁻⁴ M but not containing Co ⁺⁺ . It consists of treating with glucose having a concentration of 30-55% by weight to perform enzymatic isomerization. The inversion reaction is carried out at pH 7.8-8.6 by treating the total contact time of the enzyme and syrup to about 3.5 hours or less, in particular 2 hours or less. Processes suitable for the present invention are intended to be treated with the addition of a small amount of magnesium to avoid post treatment isomerized ion exchange. Temperatures convenient for the isomerization reaction can be varied in the range 60-85 ° C., but particularly suitable temperatures are in the range 60-70 ° C.

ratio. Glucose isomerase derived from coogurans has been studied in a variety of ways to obtain enzyme preparations with long half-life and high unit activity that are long enough for industrial combustion treatment. Current rain. Highly active glucose isomerases with long half-lives derived from coogurans cells are used in the art. Specific enzyme preparations suitable for the practice of the present invention can be exemplified by the enzyme products described in US Patent Application No. 501,292 (filed August 28, 1974) and Belgian Patent No. 832,852.

Enzyme formulations described in this patent are glutaraldehyde cross-linked b. Coogurans microorganisms. In particular, the agonist is preferably prepared from homogenized cells prior to reaction with glutaraldehyde. ratio. Coogullans is a fungus that is somewhat different from many known variants. Variant productivity of an excellent glucose isomerase is one of its characteristics and has the ability to uniquely proliferate in inorganic nitrogen sources as nitrogen nutrition. Examples of references describing such enzymes and detailed descriptions of their microbial sources include U.S. Patent Application No. 428,682 (Dec. 27, 1973) and Belgian Patent No. 809,546.

The present invention provides a fixed ratio. It has been found that the biochemical requirement of cobalt glucose isomerase for cobalt can be kept fully satisfied during the isomerization without cobalt supplementation. In fact, it was found that omitting Co ⁺⁺ from glucose syrup does not impair the productivity and enzyme stability during isomerization and in some cases improves it.

As mentioned earlier, it was also found that the biochemical demand for glucose isomerase on magnesium was much less than previously thought. In fact, the addition of Mg ⁺⁺ can be avoided in all cases where the concentration of Ca ⁺⁺ is low. Active requirement for Mg ⁺⁺ of glucose isomerase is required more is sufficiently satisfied or Mg ⁺⁺ concentration of 10 ^-3 M or less at pH 7.8. However, it is assumed that the calcirium ions in the syrup are much more restrained than previously thought. It is clear that Mg ⁺⁺ in syrup acts to suppress Ca ⁺⁺ . If the syrup is low, the magnesium content may be lower. The Mg ⁺⁺ / Ca ⁺⁺ ratio of the syrup should exceed 5: 1 on a molar basis, especially this ratio should exceed 10: 1 but above 500: 1 when Mg ⁺⁺ > 10 ⁺³ M Should not.

However, according to the present invention, the enzyme glucose isomerization reaction can be carried out above 8.0 according to the present invention, in contrast to the conventional method that the isomerization reaction is performed at pH 8.0 or lower to reduce pigmentation. To remove the pigment from the syrup product as in conventional methods, treatment with activated carbon, for example, adds to the processing cost. It is highly desirable to minimize pigmentation, but unfortunately the pigmentation rate of glucose syrup increases with increasing pH.

Examination of the pigmentation trend revealed that the pigmentation rate varies slightly over time at all pH levels. In summary, the higher the pigmentation ratio at pH 8.0+, the faster the isomerization reaction can be performed.

Limiting the residence time of glucose syrup in the enzymatic conversion reactor, that is, the contact time to about 3.5 hours or less, yields an acceptable syrup product of the pigment. If the contact time is limited to 1 hour or less, then no pigmentation treatment of the syrup product is needed, and the presence of psicose and other by-products cannot be measured by thin layer chromatography. (Sven Åge Hansen, TLC method for the identification of monosaccharides, disaccharides and trisaccharides, chromatography).

According to the present invention has the following advantages.

(One). No addition of Co ⁺⁺ in connection with isomerization.

(2). No need to add Mg ⁺⁺ in connection with isomerization.

(3). No need for ion exchange treatment to remove harmful ions after isomerization.

(4). Enzyme shows excellent thermal stability.

(5). Process productivity is improved.

(6). The process is well suited for continuous column operation and has a small pressure drop per unit length of the column.

Referring to the isomerization method of the present invention in more detail as follows.

In the isomerization of glucose syrup, the isomerization of glucose syrup is performed as a continuous operation in a contact time of about 3.5 hours or less, especially 2 hours or less, and a syrup injection pH of about 7.8-8.6. In the continuous process, 30-35% w / w glucose syrup is converted to a syrup product containing a desired fructose concentration of at least 40%, usually about 45%. The inversion temperature is in the range of 60-85 ° C., in particular in the range of 60-70 ° C. The pH range of the feedstock syrup is adjusted to alkali (NaoH or Na ₂ CO ₃ ) to attract 7.8-8.6 and, if necessary, readjusted at the point of choice of the reactor.

The pH of the isomerized discharge syrup is lower than the pH of the injected syrup. Typically, the difference between the infusion syrup pH and the discharge serum pH is in the range of 0.2-0.6.

Control is important in the practice of the present invention. If the pH is too high, that is, about 8.6 or more, unwanted pigmentation (when the contact time is long) occurs. If the pH is too low, i.e. below 7.6, the addition of CO ⁺⁺ is necessary to maintain enzymatic activity. Because of these two effects, changes in enzyme activity with pH should be considered. Rain The optimal pH for coogurans is about 8.5. Industrial batch processes are usually carried out with a long contact time since they use relatively low enzyme concentrations.

For this reason, the pH should be kept low to avoid pigmentation, and at this pH the activity of the enzyme is significantly lower than at optimum pH. Therefore, the addition of CO ⁺⁺ is required. The continuous method according to the present invention can keep the contact time small and keep the pH high. Isomerization is carried out at the optimum pH of the enzyme or at very close proximity to the optimum pH of the enzyme. Therefore, the addition of CO ⁺⁺ can be avoided.

The continuous process according to the invention thus has several advantages over the batch process. The amount of Mg ⁺⁺ added to the raw syrup is about 10 ⁻² M or less, preferably 5 × 10 ⁻⁴ M or less. However, to lower the Mg ⁺⁺ concentration, the syrup of raw syrup may be prepared from low water or the glucose syrup may be appropriately pretreated so that the calcium is limited to 2.5 × 10 ⁻⁵ M or less. In either case, when calcium is present, a sufficient amount of magnesium is added to the syrup to increase the calcium content of Mg ⁺⁺ / ca ⁺⁺ , especially Mg ⁺⁺ / ca ^++, by more than 10: 1. It causes you to lose your balance.

The total content of Mg ⁺⁺ and Ca ⁺⁺ is determined by Ca ⁺⁺ to ion exchange removal from glucose serum to avoid Ca ⁺⁺ of 10 ^-3 M or more and Mg ⁺⁺ of 10 ^-2 M or more, if necessary. By controlling and matching. If glucose syrup if the case of containing a low concentration of Ca ⁺⁺ to Mg addition amount of Mg ^{++ ++:} Ca ⁺⁺ is staying in the 5-500 range when the ^{Mg ++> 10 -3 M Mg ++} ≤10 - ^At 3M the ratio is reduced to greater than 5 In this way, in a suitable method of the present invention, the syrup contains Ca ⁺⁺ at 2.5 × 10 ⁻⁵ M or less and Mg ⁺⁺ at 5 × 10 ⁻⁴ M or less.

Glucose syrup used in the present invention is derived from starch hydrolyzate. Syrup preparations sometimes have some Ca ⁺⁺ because they start by suspending starch in slightly mild tap water. Starch hydrolysis and saccharification of starch hydrolysates are enzymatically performed using Ca ⁺⁺ activated enzymes. In summary, Ca ⁺⁺ is not contained in any glucose syrup at all (except as prepared directly in the laboratory from crystalline glucose and deionized water) and care should be taken not to exceed the Ca ⁺⁺ content of 10 ^-3 MCa ⁺⁺ . .

In the continuous isomerization method according to the present invention, the particle size of the enzyme preparation is determined according to the syrup treatment conditions (and facilities). In the column operation, which is a suitable method of the present invention, the column tends to agglomerate when small particles are used, so the particle size is preferably about 10 microns or more. In the case of a shallow bed method (ie, isomerization in a pressurized reactor), any particle size can be used. However, the particle size used in the isomerization method may be a factor that governs the efficiency of a particular method (or facility), since it depends somewhat on the size of the unit activating particle of the enzyme preparation. In either case, the enzymatic formulations disclosed in pending U.S. Patent Application No. 501,292 (filed August 28, 1974) and Belgian Patent No. 832,852 can be formulated to a desired particle size.

In the following examples specifically describing the method of the present invention, the activity of the immobilized enzyme is measured in IGIC units (immobilized glucose isomerase column method). “1IGIC” means 40w / w% glucose, infusion pH 8.5,65 ℃ and 4 × 10 ^-3 M Mg ⁺⁺ , without Ca ⁺⁺ : continuous packed bed column: column size: 25cm × 40cm etc. This is the amount of enzyme required to convert glucose to fructose at an initial rate of 1 μ / mol for standard conditions.

In each of the following examples, the productivity of the enzyme within a given time is expressed as the amount of glucose (kg) that can be converted into a mixture of fructose and glucose, with a conversion of 0.45 per kg of enzyme with an initial activity of 100 IGIC / g. Enzyme preparations with 150250IGIC activity per gram were used. In order to compare the results obtained with different enzyme preparations, the productivity values were all redefined with reference to 100IGIC / gdmnl enzyme activity. In each example, the contact time refers to an enzyme preparation having an activity of 1001 GIC / g. For example, a 1 hour contact time with an enzyme formulation having 200IGIC / g activity corresponds to a 2 hour contact time with a standard formulation having 100IGIC / g activity.

The present invention will be described in detail by way of examples.

Example 1

Isomerization with and without Co

The isomerization was all done as a continuous packed bed plug flow reaction.

ratio. Coogland enzyme was prepared according to the method of Example 2 described in US Patent Application No. 501,292, particle size ranged from 150 μ-2800 μm.

This enzyme preparation was pre-soaked in 40% by weight glucose syrup for 1 hour at room temperature and the thus-prepared glucose isomerase was charged to different water-cooled columns. A stream of glucose syrup with data of concentration, pH and other properties according to the following table (mono) was passed upward through the enzyme preparation.

The flow rate was adjusted so that the syrup conversion yielded to 45%. The linear flow velocity always exceeded 10 cm / hour (film diffusion limit), and in this example the Ca ⁺⁺ concentration was 2.5 × 10 ⁻⁵ M or less.

[Table 1]

Columns were used in different sizes, such as size (1.5 cm × 35 cm) and 5.8 cm × 45 cm) columns. In the size indication above, the first value is the diameter of the column and the second value is the height of the column. For convenience, the columns will be referred to as 60 ml columns and 1 l columns. A 60 ml column was charged with 9 g of enzyme and 1 L column with 225 g of enzyme.

All of the isomerization reactions were performed after 450 hours, and the residual activity was measured, and the productivity per 100IGIC / g and the contact time per 100IGIC / g were calculated. The results are shown in the following table (1 b).

[Table (1)]

Given one or more values for contact time in the table (and similar tables below), the first value corresponds to the beginning of the high activity, and the last value corresponds to the end of the rather low activity.

As can be seen from the table above,

(1) The productivity per 100 IGIC / g of residual activity is further improved when Co is not added than when Co is added when it is maintained at the recommended level.

(2) When the pH is below the recommended level, the productivity per 100IGIC / g of residual activity is more improved when Co is added than without Co, but the value when Co is added is the pH at the recommended level without Co. It is smaller than the value obtained when.

Example 2

Isomerization in the presence and absence of Mg

Isomerization was carried out as described in the method of Example 1.

1. During the test, the following isomerization conditions were kept constant.

After 450 hours of isomerization, the test was stopped and residual activity was measured to calculate the fish per 100 IGIC / g and contact time per 100 IGIC / g. The results are shown in Table 2 below.

TABLE 2

As can be seen from Table 2, the addition of Mg ⁺⁺ does not affect the productivity and residual activity per 100 IGIC / g.

Example 3

Isomerization according to the ratio change between Mg ⁺⁺ and Ca ⁺⁺

Isomerization was carried out according to the method described in Example 1. 60ml column was used for the five different from the ratio between the injection, but syrup constant Mg ⁺⁺ is added to the Ca ⁺⁺ was not added as glucose syrup of Mg ⁺⁺ and Ca ⁺⁺ hangeoteuro.

The isomerization conditions used are as follows.

The activity of the control column (without Ca addition) was measured using the values of the parameters indicated in () above the isomerization conditions. After 800 hours of operation, the control columnase was reduced to 25% of its initial activity.

The numerical values shown in the following Table 3 indicate the activity rate for the enzymatic action of the control column after the same time operation.

TABLE 3

The results shown in Table 3 above indicate that the Mg ⁺⁺ / C ⁺⁺ ratio increases in enzyme life, and the Mg ⁺⁺ / Ca ⁺⁺ ratio should exceed 5, in particular this ratio should exceed 10 .

Example 4

Isomerization of Glucose Concentration in Injected Syrup

Isomerization was carried out according to the method described in Example 1.

The isomerization conditions used are as follows.

In Table 4, productivity, residual activity and contact time were obtained after 450 hours of operation on a 60 ml column.

TABLE 4

Example 5

Effect of pH on Activity

The effect of pH on the activity of immobilized glucose isomerase prepared according to the method of Example 2 described in US Patent Application No. 501,292 and Example 2 described in Belgian Patent No. 832,852 is 2.5 × 35 cm. Measured on a continuous plug flow column with specifications. The isomerization reaction was carried out according to the method described in Example 1, and the activity was measured in the following manner, respectively. The column was left to equilibrate for 5 hours before measuring activity.

After changing the pH of the feedstock to the value indicated in the following isomerization conditions, the column was further operated for 5 hours. The isomerization conditions used are as follows.

Relative activity rates are shown in Table 5 below, and the maximum activity is expressed as 100 at PH 8.5.

TABLE 5

Example 6

Relationship of temperature to activity and stability

The isomerization reaction was carried out as a glucose isomerase prepared by the method of Example 2 described in US Patent Application No. 501,292 and the equivalent Bilgi Patent 832,852 according to the method described in Example 1. It was. Seven columns were operated isothermally at different temperatures in the range of 60-90 ° C. and initial activity was measured at the same time as the half life (half life is indicated by the time required for the activity to be reduced to 50% of the initial activity).

The isomerization conditions used are as follows.

The results obtained in this example are as follows.

Example 7

Effect of Particle Size on Activity

The isomerization reaction was carried out according to the method described in Example 1 except for the enzyme preparation. In this example, two enzyme preparations were used, the last of which is denoted by X and Y in US Patent Application No. 501,292, respectively, as described in Examples 2 and 3, and in the corresponding Bilchi patents 832,852. The immobilized glucose isomerase formulation described in the corresponding examples was used.

Enzyme preparations were sorted both before filling the column. The isomerization conditions used are as follows.

The effect of particle size on the activity of enzymes X and Y is shown in Table 7 below.

TABLE 7

It was found that the stability does not depend greatly on the particle size. As can be seen from the table, the homogeneity of some enzyme preparations varies greatly depending on the particle size than others. It is not known why the activity of the X enzyme is much smaller depending on the particle size than the activity of the Y agent, but this phenomenon is believed to be related to particle formation. It was found that the X formulation is mainly in the slab form while the Y formulation is mainly in the spherical form.

At a given particle size, the average pore length of the slab is much smaller than the average pore length at the same particle size. This fact generally means that the activity is less with agent Y than with agent X.

Example 8

Pigment formation

Isomerization was carried out as described in Example 1. Six columns were packed with different amounts of enzyme to maintain constant conversion and contact time at 45% and 1 hour. The isomerization conditions used are as follows.

It was found that after about 100 hours of isomerization, the Icumsa pigment index was increased as shown in Table 8 below.

TABLE 8

Where

a is OD420 (light density)

b is DS in g / ml

c is the cell length in cm

Example 9

Upscaling

Isomerization was carried out as described in Example 1 except for the enzyme preparation. The enzyme was prepared according to the method of Example 3 described in US Patent No. 501,292 and corresponding Belgian Patent 832,852. The particle size ranged from 150-500μ and the isomerization conditions used are shown in Table 9 below.

TABLE 9

The results obtained in this example are as follows.

As can be seen from the above table, upscaling of the isomerization method from 60 ml columns can be performed without difficulty, and the values for productivity and residual activity can be performed essentially the same.

Claims (1)

As described above in the text and 10 ^-3 M or less Ca ⁺⁺ and Mg ⁺⁺ containing ^10-2 M or less and a, Mg ^++: Ca ⁺⁺ mole ratio of Mg ^{++ 10-3} M when the 5- 30-35% by weight Staphylococcus syrup, which is in the range 500 and no less than 5 when Mg ⁺⁺ ≤ 10 ^-3 M, at a temperature in the range of 60-85 ° C. for a total contact time of 3.5 hours or less in the pH 7.8-8.6 range. At least 40% by weight of fructose in the glucose-fructose content by passing syrup through a bed of gurutar aldehyde crosslinked immobilized glucose isomerase having a size of at least about 100 microns derived from B. coagulans Continuous isomerization of a glucose syrup to a glucose-fructose mixture, characterized in that it is isomerized.