DE2404101A1 - PROCESS FOR THE MANUFACTURING OF FRUCTOSE - Google Patents

Description

PATENTANWÄLTE DlPL-ING. DIETER JANDER ββ, -JNG. MANFRED BONING

I EEiTJN 15

KURFÜRSTENDAMM 66 TELEPHONE 883 50 71

97/15 139 DE

January 25, 1974-

Patent application

of the company
NAARDEN INTERNATIONAL NV

Huizerstraatweg 28, Naarden, The Netherlands

"Process for the production of fructose"

The invention relates to an enzymatic method for Production of fructose, in particular based on an enzymatic process for the production of fructose containing Carbohydrate syrups made from starch or containing starch Fabrics and raw materials.

Invert syrup is a solution of equimolecular amounts

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Glucose (dextrose) and fructose (laevulose) and residual amounts of sucrose. It's a very popular product for the production of food and beverages. Invert syrup has more sweetness than an equimolecular one Amount of sucrose, it shows less tendency to crystallize and possesses because of its higher osmotic pressure stronger preservative properties.

The technical production of invert syrup has so far been limited to the so-called inversion of sucrose with acids or with the enzyme invertase. Invert syrups are also known which are produced by alkaline isomerization made from glucose. However, due to their strong color and bad smell, which are caused by side reactions do not prevail.

The discovery of enzymes that can convert glucose into fructose opened up theoretical possibilities for technical production of invert syrup from glucose syrups, which in turn consists of raw materials containing starch, such as Corn or potatoes. Such a process would bring considerable benefits if it were would be technically feasible.

Sucrose, especially that from sugar beet, can only be produced at a certain time of the year, which is based on the Losses due to long storage of the beet are due. As a result, the manufacturing capacities remain during the unused for a larger part of a year. Since sucrose can only be stored in crystalline form for a long time

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can, must sucrose for the production of invert syrup brought back into solution, which is a disadvantage. If the inversion is performed during sucrose production, it should be done before evaporation will. This requires a large capacity for storing liquid. If you put invert syrup off Starch-containing materials, especially from grain, v / ie wheat, shark, etc., have much fewer storage problems, since the raw material can easily be stored in silos Can be stored at low cost until the time the invert syrup is made. In countries that relying on imports to meet their sucrose needs, foreign currency could be saved if Invert syrup made from local grains.

Glucose isomerizing enzymes are only recently in technical quantities and in suitable quality have become available (GB-I-S 1 103 394-, owner: Agency of Industrial Science and Technology; Dutch laid-open patent application 72 034-91, applicant: Miles Laboratories Inc.).

Technical processes for the production of fructose-containing carbohydrate syrups which contain fructose with glucose isoiuerase have already been proposed (ER Kooi, RJ Smith, Food Technology 1972, 57-59, FR-PS 2 086 752, FR-PS 2 086 926, FR- PS 2 086 927, FR-PS 2 086 928; CH-PS 4-73 221; brochures from enzyme manufacturers: Miles Kalichemie GmbH, Nienburg, Weser, Product Information Glucose Isomerase). All of these technical processes that have been proposed start either from pure glucose solutions or from highly converted (96 DE) cereal

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syrups or of "sweet liquid" made up of the mother liquor in the production of crystalline glucose or dextrose.

Such a method, according to Kooi and Smith (loc. Cit.) the following steps: 1. Preparation of the enzyme; 2. Production of a dextrose raw material; 3. Conversion of the Dextrose from contact with the enzyme; 4. Separating the syrup from the enzyme; 5 · Refining and concentrating the Syrups.

The production of a dextrose raw material either from purified starch or from substances containing starch like grain, especially maize, is technically possible and consists of the following stages:

1. Gelatinization and liquefaction of the starch or des Starting grain with acid or enzyme (pC-amylase) or a combination of both (A.J. Wieg, Die Starke 20, 324, 1968).

2. Saccharification of the liquefied starch material using glucamylase (amyloglucosidase) under specific conditions of temperature, pH and substrate concentration.

The material thus obtained can be used as such for isomerization. One can get the product too concentrate, isolate the crystalline glucose and use the purified glucose to convert it into fructose.

The technical saccharification of liquefied starch or grain sludge is currently most common at pH 4.0-4.5, 60C, a concentration of $ 30-35 Solids and an enzyme concentration of 0.2-0.3 AGU

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per gram of starch. Until it reaches a value of 96 DE takes about 48 hours.

The reaction conditions for the isomerization of the dextrose raw material into fructose-containing syrup, which are mentioned in a number of publications are shown in Table 1.

Table 1 I. II III IV V Glucose content 30-50 55.5 67.5 45 36/50 of raw material, % 6.2-6.5 6.5 6.5 6.5 6.8 / 7.2 pH 65 70 70 ■ 70 65 Temperature, ⁰ C 80 92 92 92 92 Response time, hours 19.5 2.3 4.6 20th 8.5 / 6.15 GIU / g glucose 50 17.5 32.5 50 54.2 / 52 % Isomerization

I: Glucose isomerase brochure, Miles Kalichemie

II: Dutch patent application 70 04989, table A,

Sample 3
III: Dutch patent application 70 04989, table A, sample 4

IT: Dutch patent application 70 04989, table E. V: GB-PS 1 103 394, Table 6

From this table it can be seen that a sufficient Conversion of glucose into fructose requires a reaction time of at least 80 hours, provided that the enzyme is added in sufficient quantity and of suitable quality. It follows that for Manufacture of a fructose-glucose mixture from starch

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a total reaction time of between 128 and 14-0 hours is required.

It has now been found that the reaction time can be shortened considerably if the saccharification of the liquefied Starch using glucamylase and the conversion of glucose to fructose using glucose isomerase at the same time is carried out. This can be done by adding glucamylase and glucose isomerase at the same time a liquefied starch paste and there reaction conditions which are favorable for the action of glucose isomerase, e.g. what the pH and the Temperature concerns. One can expect that under these conditions the effect of glucamylase will be considerable is decreased. Surprisingly, it has been found that saccharification and isomerization work together and at the same time can be carried out in the same time that is optimal for the saccharification step alone Process conditions for the glucamylase activity is required, or in even less time.

In the context of this invention, liquefied starch is to be understood as meaning starch or material containing starch, which has been gelled by heating, converted by acid or modified by enzymes. Come as enzymes those from microorganisms - e.g. Amylase from bacteria or fungi - or from malt in question. Liquefied starches of this type, DE values (DE = dextrose equivalent) of practically 0 to 67 have. They can contain glucose in concentrations up to 4-5 # (Tables 2 and 3), but themselves these highly converted products contain substantial amounts of di- and higher saccharides produced by glucose isomerase can only be changed if glucamylase acts at the same time.

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Table 2

Composition of corn starch hydrolysates converted with acid, determined by chromatographic analysis (Carbohydrate base)

Dex mono- Di- percent Saccharides 3.0 Hexa- Hepta- higher trose 2.3 2.8 Tri- Tetra penta 4.3 2.2 2.1 81.7 Equi
especially 3.7 4.4 2.9 5.0 5.5 5.5 3.0 72.4 lente 5.5 5.9 4.4 4.5 6.5 4.5 5.9 63.3 10 7.7 7.5 5.8 5.8 7.2 5.2 4.6 54.1 15th 10.4 9.3 7.2 7.2 7.8 6.0 5.2 45.1 20th 15.4 11.5 8.6 8.2 8.3 6.5 5.5 36.4 25th 16.9 15.2 10.0 9.1 8.4 6.7 5.7 28.3 50 21.0 14.9 11.2 9.7 7.9 6.5 5.6 21.3 55 25.8 16.6 12.2 10.1 7.2 5.9 5.0 15.9 40 50.8 18.1 12.9 10.0 6.3 5.1 4.2 11.9 4-5 56.2 19.5 15.2 9.6 5.1 4.4 5.2 8.5 50 42.5 20.9 15.2 8.7 4.6 3.6 2.2 5.5 55 4-5.1 21.4 12.7 7.5 3.2 1.8 4.5 60 12.5 6.9 65 67

"The data is based on recent chromatographic analyzes of 91 samples from all manufacturers in the USA. The data is accurate to within - Λ% . (Source: Technical Advisory Committee, Corn Industries Research Foundation, Inc.)

Table 5

Some data on brewery syrups

(Source: Process Biochemistry 5, 59, 1970)

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Code no. A. Έ J source Corn wheat barley % Total solids 79.4 80.4 75.2 % Carbohydrates (the total solids) 98.0 97.0 91.1 Sugar (# based on Total GHO) Hexosis 40.7 41.0 27.0 Maltose 31.0 16.4 60.2 Maltotriose 8.4 13.1 7.7 Dextrins 19.9 29.5 5.1

It is known that glucamylase has its greatest activity in the pH range from 3 to 5.7, the maximum being between 4.0 and 4.5. At the pH optimum, the activity is strongest at 50 ° C. In practice, however, 60 G is used in order to reduce microbial contamination to a minimum. At 70 ° C, the glucamylase has only 7 % of its activity at 50 ° C. At pH 6.2, the activity is only 40 % of the maximum value.

Glucose isomerizing enzymes show little activity below pH 5.5. Most of these enzymes possess one pH optimum of 7 or above.

According to the method according to the invention, a paste of liquefied starch which contains between 15 and 50 %, preferably 30-40%, solids is heated to 40-80.degree. C., preferably 60-70.degree. The pH is adjusted to 5.0-7.5, preferably 6.0-6.5. The glucamylase and the glucose isomerase are used simultaneously in amounts of 50-500 AGU, preferably 75-150 AGU and 2,000-20,000 GIU, preferably 3,5OO-12,000 GIU, per kg of carbohydrate

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added in the paste ..

1 AGU is the amount of glueamylase you can use in one Hour at pH 4.2 and at 60 ° 0 1g of soluble dextrose Maintains Strength (Miles Product Information).

1 GIU is the amount of glucose isomerase with which 1 mg of fructose is obtained from glucose in one hour at? Ο ° G and at pH 7.2 (Y. Takasaki, Igr. Biol. Chem. _T 30, 1247-1253, 1966) .

For the conversion of the starch molecules by the enzymes it is necessary that the starch granules are gelatinized. Gelatinization takes place under the influence of water and heat, in some technically used devices shear forces are applied at the same time. To reduce the viscosity of the gelatinized starch paste, you can before, during or immediately after gelatinization, enzymes with o <-amylase activity are added. Many oC-amylases develop their activity under the conditions given above for the interaction of glucamylase and glucose isomerase.

Therefore you can use the viscosity-reducing effect of «X-amylase on gelatinized starch with the invention Connect procedures. For this purpose, an o <-amylase activity of 400,000-800,000 MWU (modified Wohlgemuth units) required per kg of starch.

Commercially available enzymes are - in terms of their activity standardized by mixing the enzymes with suitable solvents or inert fillers. It's on the Hand that using separate enzymes as they are for

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the method of the present invention can be cumbersome. The invention therefore continues Enzyme mixtures for the production of fructose from starch or raw materials containing starch, which Mixtures of a combined glucamylase-glucose isomerase activity or a combined oC-amylase-glucamylase-glucose isomerase activity exhibit. These mixtures are standardized with regard to their individual activities.

Combined enzyme preparations which are used for the method according to the invention should be so composed be that the activities of the individual enzymes therein lie in the ranges given above for the enzymes are.

The resulting carbohydrate solutions containing fructose can be purified as is known to others Carbohydrate solutions is described, e.g. by treatment with activated carbon, ion exchangers or active clays Electrodialysis etc. sulfur dioxide, which is used in some starch conversion processes (A.J. Wieg, The strength 20, 324, 1968), has no negative influence on the glucamylase or glucose isomerase activity.

Example 1 Production of a 96 DE glucose raw material from potato starch in a manner known per se.

1000 g potato starch (moisture content: 18 #) was mixed with 1600 g v / water and 1.07 g Tenase (R) (Miles Amylase from bacteria) (0.13 % based on dry starch).

The mixture was stirred at pH 6.5 while the temperature

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was increased from 50 to 70 ° C in 20 minutes. The temperature was held at 70 ° C for 10 minutes, then increased to 80 ° C over 10 minutes and the same time held at 80 ° G, after which it was over the course of 5 Set at 85 ° C for minutes and at 85 ° C for 20 minutes held. After this time, the DE value was 18.

It was cooled to 60 ° C and the pH with dilute Hydrochloric acid adjusted to 4.2.

1.83 nil glucamylase (Miles amyloglucosidase L 100) with an activity of 100 AGU / ml was added, and the mixture was then ^{stirred continuously for 48 hours at the temperature v} P ⁿ 60 C. After this time, the DE value was 96, 7 increased.

Example 2 Production of a 96 DE glucose raw material from corn starch in a manner known per se

1000 g corn starch (14 % moisture) was mixed with 1720 g water and 1.12 g Tenase (E) (bacterial amylase from Miles Laboratories Inc. Elkhart, Ind. USA) (0.13 % based on dry starch). The process was carried out as indicated in Example 1, with the difference that the final temperature was kept at 85 ° C. for 35 minutes. The DE value was then 12.

The saccharification was carried out as indicated in Example 1 with 1.91 ml of amyloglucosidase L 100. The final DE value was 96.1.

Example 5 Conversion of the reaction products from Examples 1 and 2 into fructose-glucose syrup in a manner known per se

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The 96 DE-glucose raw material of Examples 1 and 2 was used converted with glucose isomerase. The reaction conditions and results are shown in Table 4.

Tabel

Initial DE value

% Glucose based on dry solids

Added stabilizers, percent by weight of the total solution:

CoGl ₂ • 6H ₂ O
Temperature ⁰ C

pH (continuously adjusted with 0.1N NaOH

represents)

Glucose isomerase concentration, GIU / g glucose (Hollo U) Reaction time (hours)

% Conversion of the glucose used

! Fructose, % based on weight of dry solids

Glucose, % based on weight of dry solids

96 DE-glucose raw material from Example 1 Example 2 96.7 96.1

91.2 91.0

0.2 0.2 0.02 0.02 65 65

6.2

6.2

7.5 7.3 90 90 49.2 48.8 44.8 44.4 46.2 46.6

From Examples 1-3 it can be seen that for the production of a crude fructose-glucose syrup, which is still must be cleaned, it takes 138 hours to ground when using liquefied starch according to the prior art • goes out.

Example 4 Preparation of a fructose-glucose syrup by the simultaneous action of glucamylase and glucose isomerase

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Liquefied potato and corn starches according to Examples 1 and 2 were made with a combined enzyme preparation treats both glucamylase and glucose isomerase activity exhibited. The enzyme complex was standardized to contain 50 AGU and 1,000 GIU per g.

7.0 g of the enzyme preparation were used to make 1000 g to convert liquefied starch. The remaining conditions were as follows:

liquefied starch

Initial DE value

Added stabilizers,
Weight percent of the total solution:

potato Grain off the end example 1 Example 2 18 ■ 12th

MgSO _z CoCl.

7H ₂ O

0.2 0.2 0.02 0.02 65 65 6.2 6.2 72 72 46.1 45.9 4-3.9 44.1

Temperature C

pH (continuously with 0.1N NaOH
set)

Response time, hours

Fructose, % based on weight of dry solids

Glucose, % based on weight
of dry solids

The total reaction time for the production of fructose-glucose syrup from liquefied starch was only 72 hours using the combined enzyme preparation.

Example 5

530 g of wheat starch (14 % moisture) was mixed with 1500 g

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Water and 0.6 g Tenase ( ^R ^ mixed and reacted analogously to Example 1. After liquefaction, the DE value was 18. As in Example 1, the mixture was cooled to 65 ° C., the pH value was corrected and the amount of activators 3.5 g of an enzyme mixture (50 AGU / g glucamylase and 1000 GIU / g glucose isomerase) were added. The reaction mixture was kept at 65 ° C. and pH 6.2 for 60 hours. After this time, the fructose content was 47 , 9 #, glucose 46.1 % and di- and oligosaccharides 6 % based on dry matter.

Example 6 Production of a fructose-glucore syrup from corn meal by the simultaneous action of α-amylase, glucamylase and glucose isorrterase

1000 kg of sterilized Mc.is meal and 3000 kg of worms were in a Henze kettle at 6 atm. Section. Heated for 1 hour. It also took 1 hour to reach these conditions. The contents were under expansion into a mash tun transferred and immediately cooled to 65 ° C. The pH was adjusted to 6.2 and so was the temperature kept constant during the further procedure. -

Stabilizers were added in the same concentrations as in Examples 3 and 4.

6.5 kg of an enzyme preparation was added which had an activity in α-amylase of 100,000 MWU / g, in amyloglucosidesis of 50 AGÜ / g and glucose isomerase of 1000 GIU / g. the Liquefaction, saccharification and isomerization ran simultaneously for 50 hours. After that the solution was through filtered through a filter press with Hyflo Supersil. The thus obtained clear solution had a solids content of 755 kg with a total weight of 3551 kg. The fructose content was 316 kg, glucose 344 kg and di- and oligosaccharides 75 kg. The solution was made with activated charcoal and

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Treated ion exchange resins to remove color, ash and nitrogenous material, as is common in the manufacture of grain syrups. Finally, it was concentrated to a solids content of 75% in a thin-film evaporator (type Sambay ' ^R '). The syrup obtained was crystal clear and contained 18 percent by weight fructose, 35 percent by weight glucose, 7-7 percent by weight di- and oligosaccharides and no D-psicose.

Example 7

A granular starch sludge with a solids content of 44 percent by weight was in a Kroyer (- ^) converter with Hydrochloric acid liquefied up to the DB value 17 »5. 10 kg des products thus obtained were used for conversion into fructose. The pH was adjusted with dilute sodium hydroxide solution set at 6.2 and held at that value. As in Example 4, stabilizers were added and the temperature was kept at 65 ° C. Glucamylase (0.25 AGU per g solids) and glucose isomerase (8 GIU per g solids) were added. After 40 hours, the following concentrations were found: 21.2 percent by weight fructose, 20.6 percent by weight glucose, no D-psicose and the remainder di- and oligosaccharides.

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Claims (13)

Patent claims: Method of preparation: of fructose-containing Dropping carbohydrates directly from starch or starch containing Substances that consist in gelatinizing the starch, liquefying the gelatinized starch, the liquefied starch is saccharified to glucose with glucoamylase and the glucose to fructose with glucose isomerase isomerized, characterized in that the conversion of a carbohydrate-containing Solution in glucose and the isomerization of glucose into fructose at the same time with the help of an enzyme complex, which has glucamylase and glucose isomerase activities, using the complex in an amount that is sufficient for the desired degree of conversion. 2. The method according to claim 1, characterized in that the simultaneous conversion into Glucose and fructose takes place at a temperature of about 4-0 to 80 ° C and a pH of about 5.0 to 7.5. 3. The method according to claims 1 or 2, characterized in that the simultaneous conversion takes place in glucose and fructose at a temperature of 60 to 70 C and a pH of 6.0 to 6.5. 4-, method according to any one of claims 1-3, characterized characterized in that an amount of enzyme is used per 1 kg of carbohydrate in the raw material which 50-500 glucamylase units and 2000-20,000 glucose isoraerase units amounts to. 5. The method according to any one of claims 1-4, characterized - 17 409831 / 0873 characterized in that an amount of enzyme is used per 1 kg of carbohydrate in the raw material, the 75-350 glucamylase units and 3.5ΟΟ-12.ΟΟΟ Glucose isomerase units. 6. Process for the preparation of fructose-containing carbohydrate solutions directly from starch or starch-containing Substances that consist in gelatinizing the starch, liquefying the gelatinized starch, the liquefied starch is saccharified to glucose with glucoaraylase and the glucose to fructose with glucose isomerase isomerized, characterized in that that the liquefaction of the gelatinized starch with the help of cC-amylase and the conversion of the carbohydrate in the solution in glucose and the isomerization of glucose into fructose with an enzyme complex, the oi-amylase, Glucamylase and glucose isomerase activity, is carried out simultaneously, said Enzyme complex is used in an amount sufficient for the desired degree of conversion. 7. The method according to claim 6, characterized in that the liquefaction of the gelatinized Starch and the conversion of the carbohydrate in the 'solution in glucose and the isomerization of glucose into fructose at a 'temperature of about 40 to 80 ° 0 and a pH value from about 5.0 to 7 »5 takes place. 8. The method according to claim 6 or 7, characterized in that the liquefaction of the gelatinized Starch and the conversion of the carbohydrate in the solution into glucose und.die isomerization of glucose into fructose at a Temperature from 60 to 70 ° C and a pH value from 6.0 to 6.5 takes place. 409831/0873 9. The method according to any one of claims 6-8, characterized in that nan per 1 kg of carbohydrate an amount of enzyme used in the raw material that contains 100,000 - 1,000,000 modified Wohlgemuth- oi-amylase units, 50-500 glucamylase units and?:. 000-20,000 Glucose isomerase units. 10. The method according to any one of claims 6 - 9j thereby characterized that one per 1 kg of carbohydrate an amount of enzyme used in the raw material that is 400,000 - 1,000,000 modified tfohlgemuth- oo-aylase units, 75-350 glucamylase units and 0.500-12.OOO Glucose isomerase units. 11. Carbohydrate syrups containing fructose according to one or more of claims 1-10. 12. Carbohydrate syrups containing fructose according to one or more of claims 1-10, characterized in that they contain glucose and Contain maltooligosaccharides. 13. Enzyme mixture consisting of glucamylase and glucose isomerase. 14-. Enzyme mixture according to Claim 13, characterized in that it additionally contains c <-amylase. 409831/0873