NL8201763A - PROCESS FOR PREPARING FRUCTOSE FROM INSULIN. - Google Patents

Description

V * '* / / N.0. 30993 1

Process for the preparation of fructose from inulin.

The present invention relates to a. new microbial inulinase preparation suitable for an industrial process for the production of fructose and for an inulin sugar-making process.

Fructose is the sweetest, naturally nutritious sugar with a sweetness level approximately 1.4 times that of sucrose. Fructose is easily soluble in water and has a greater solubility than sucrose. It could find wide application in the food industry, for example in non-alcoholic drinks, if production costs could be significantly reduced.

10 Fructose is present in a large number of fruits and in honey.

Fructose is 50% of sucrose and can be prepared from sucrose by acidic or enzymatic hydrolysis followed by separation. However, the separation of equal amounts of glucose and fructose, which can be performed by special crystallization techniques or by chromatographic separations, is expensive and is one of the reasons why. costs of fructose production so far have been considerably greater than for sucrose.

Fructose is also prepared in large amounts by enzymatic isomerization of glucose syrups to prepare syrups similar in sweetness to sucrose. However, in isomerized syrups, the fructose content is limited to about 42% due to the isomerization equilibrium between glucose and fructose. Syrups with higher levels of fructose, for example 55% or 90%, are prepared by performing chromatographic separation techniques on isomerized syrups.

Another way to increase the fructose content of isomerized syrups would be to mix them with pure or nearly pure fructose syrups.

A potential source of fructose is inulin. Inulin is an unbranched fructose polymer, which in its natural form consists of 35 beta-fructofuranose residues with a terminal glucose molecule bound to fructose by an alpha-1, beta-2 bond as in sucrose.

Inulin is found as an energy reserve in several plants, in particular in the Compositae family, with good sources of earth limbs, dahlia tubers, chicory and dandelion roots. Inulin is practically insoluble in cold water, but can be dissolved in warm water (see Yanousuy et al. J.A.C.S. 55, (1933) 3658-3633).

8201763 sZ i 2

Inulin can be extracted with warm water from the plant material by known methods, such as countercurrent diffusion of plant parts at elevated temperature or by squeezing the juice. The crude juice is purified by filtration through activated carbon or otherwise treated with lime, as with sugar beet processing and decolorized by coal.

In addition to inulin, the extract may contain polymers of shorter chain length, for example fructose polymers and fructose polymers with terminal glucose. The average chain length of such polymers can vary with the plant source and growing, harvesting and storage conditions, but will often be between DP5 to DP15 (DP * polymerization degree).

Inulin can be hydrolyzed by acid under relatively mild conditions, pH 1-2, 1 to 2 hours at 80-90 ° C. However, acid hydrolysis gives rise to color and by-product formation, for example, formation of about 5% difructose dianhydrides, which reduces the yield accordingly. (Whistler i.s. "Polysaccharide Chemistry" Academy Press, Inc., N.Y. 1953, p. 287).

Since fructose in solution appears to be most stable in the pH range of 4-6, a process for hydrolyzing inulin performed in this range is believed to be advantageous to avoid by-product formation and isomerization.

Enzymes capable of hydrolyzing inulin have been described in the literature as available from plant material sources (roots and tubers containing inulin) and from microorganisms. The microbial enzymes can be obtained from yeasts such as Kluyveromyces fragilis, Candida pseudotrophlcalis or kefyr as well as from fungi such as Aspergillus, Fusarium and Penicillium species.

The microbial enzymes are described with pH optima in the range of 3.5-5.5 and temperature optima between 45 ° C and 55 ° C. The enzymes are reported to rapidly lose activity at 60 ° C.

The microbial inulinases are prepared both intra- and extracellularly. The ability of the enzymes to hydrolyze both inulin and sucrose has in some cases been attributed (Kluyveromyces fragilis) to a dual activity enzyme. (Nham et al. "Purification and Characterization of Inulase from Kluyveromyces fragilis", Korean Biochem, J. 10 (2), (1977) 95-108).

Both an endo and an exo-inulase have been purified from Aspergillus niger. The endo component has its maximum activity at pH 5.3 and 45 ° C; the exo component at pH 5.0 and 55 ° C (Nakamura et al., "Studies on 8201763 * z 3

Microbial Inulase part IV, "Nippon Nogeikagaku Kaishi, 52 (4), (1978) 159-166 and Nagamura et al.," Studies on Microbial Inulase part V, "N.N.K., 52 (12), (1978) 581-587).

Due to the relatively limited solubility of inulin, the hydrolysis of inulin will be carried out in relatively dilute solutions and, as already explained, preferably within the pH range of about 4-6. A 20 wt% solution of pure inulin from dahlia roots can be prepared at 60 ° C, but such a solution is supersaturated and precipitates on long storage. The maximum amount of inulin which is soluble in water at 50-60 ° C will, of course, depend on the degree of polymerization of the fructose polymer. Obviously, the lower the processing temperature, the lower the solubility of inulin and the more dilute the inulin solution will have to be.

To avoid microbial infections in the relatively dilute inulin solutions, the industrial process operating temperature should be at least about 60 ° C and preferably 60-65 ° C. This temperature level is common practice in the starch industry, where, for example, the purification process is usually carried out at 60 ° C and 30 wt% DS for 2-5 days.

Unfortunately, the enzymes reported in the prior art are poorly suited for commercial use at 60 ° C; they deactivate too quickly. Still, as a practical matter, 60 ° C is the minimum temperature level for reasonable hydrolysis both because of the inulin solubility and to avoid microbial contamination.

There is therefore a need for an effective inulinase preparation that is sufficiently thermostable to be used at 60-65 ° G for extended periods of time, such as 1-5 days, to allow the hydrolysis of the inulin to be economical. As far as the applicant is aware, no inulinase preparation with these properties has been reported so far.

Since the present invention is concerned with commercial applicability, it is to be understood that an enzymatically produced hydrolyzate product is much better in cost and quality than the acid hydrolysis product, since high quality fructose is obtained both from the inversion of sucrose and from the fractional separation of isosiropes is available. Therefore, the inulin hydrolyzate should exceed 98 wt% monosaccharide of the (dry) solids in solution. The term "complete hydrolysis" as used hereinafter refers to the preparation of an inulin 40 hydrolyzate which exceeds 98% fructose plus glutose based on dry solids 8201763 / * 4.

The invention lies in the surprising finding that inulin becomes most yd when both an endo-inulase actives inulinase activity are present and when these 5 inulinase components are present in a certain ratio, suitably a weight ratio.

In a first aspect, the present invention provides a method of hydrolyzing inulin with a microbial inulin seperate, which method comprises hydrolyzing inulin in an aqueous medium at a pH in the range of 3.5-7, at a temperature in the range of 60-65 ° C, in the presence of an effective dose of an inulinase preparation having a weight ratio of endo-inulinase to exo-inulinase in the range of 1:10 to 10: 1, with an enzymatically complete hydrolysis of inulin is obtained.

In a preferred embodiment of the present invention, the endo and exo component of the inulinase are present in a weight ratio of 1: 7 to 7: 1 and more preferably 1: 2 to 4: 1.

The inulinase enzyme preparation of the present invention has its optimum temperature at 60 ° C, but can be used at 65 ° C at pH values between 3.5-7, preferably 4-6 and more preferably 4.5-5, 0. It retains about 50% of its original activity at 60 ° C in 20 wt% inulin after 48 hours. Preferably 75% and more preferably 90% of the activity should be retained.

According to another aspect of the present invention there is provided an inulinase preparation which is excellent for carrying out the method of the invention, which preparation is characterized in that it has a ratio of endo-inulinase to exo-inulinase in the range of 1: 10 to 10: 1 by weight.

The inulin hydrolyzing process of the present invention can be carried out as a discontinuous process in conventional (dextrin) confectionery equipment. Complete hydrolysis of the inulin (i.e., 98% or more fructose plus glucose) is obtained in the process. The reaction time depends on the enzyme dose and can extend up to 2-4 days for more efficient use of the enzyme without forming by-product or color. The total inulinase (exo and endo combined) dose used is in the range of 0.25-10 Somo-gyi units per gram of inulin, preferably 1-5 Somogyi units. "

In an industrial process, the substrate for the hydrolysis will be either an extract of an inulin-containing plant, for example, ci-40 chorei, Jerusalem artichoke, dahlia, etc., or a suspension of pieces of plant material in water. Such an extract is prepared according to known techniques and can be purified by liming, treatment with coal or ion exchange, etc. before the hydrolysis is carried out.

In both laboratory scale and industrial scale, inulin 5ne extracts will often be relatively dilute, for example 5-25% dry matter.

The inulin extract can be concentrated to the limit of the solubility of inulin or the other fructose polymers contained therein at the processing temperature. As already explained, the inulin extract will be susceptible to microbial infection unless the hydrolysis reaction temperature is made high enough to prevent microbial growth. Of course, preservatives can be added, but this will add to the processing costs due to the cost of the preservatives and because of their removal after preparation.

It has been found that microbial inulase enzyme preparations with sufficiently high thermostability to be used in industrial hydrolysis processes at 60-65 ° C can be prepared from Aspergilli belonging to the Aspergillus niger group.

In a preferred embodiment of the present invention, the new inulase preparations are prepared by cultivation of Aspergillus ficuum strains. These new inulases are sufficiently thermally stable for the practice of the present invention. Their pH optimum is suitable for the practice of the invention. The aforementioned micro-organisms produce both endo and exo-inulase. However, the ratio of endo and exo-inulinase activity of the inulinase generated by the Aspergillus ficuum strain has been found to be specific.

Applicant has found that the inulinase enzyme produced by Aspergillus ficuum, strain CBS 55565, which is identical with ATCC 16882, which is identical with IMI 91881 (our designation A 524), the endo-inulinase component within the above weight ratio contains.

Accordingly, the use of this particular microorganism for the preparation of the inulinase is preferred. However, it is recognized that other skilled artisans may prefer to mix separately generated endo- and exo-inulinases, and therefore this method is also within the scope of the present invention, although it is a less preferred method.

The attached Figures 1, 2, 3 and 4 are chromatograms illustrating the different enzymatic activity of the endo and exo-inulinase compo-40 buds of A. ficuum A-524.

8201763 '* · «- 6

The endo and exo enzyme can be separated by ion exchange on CM-Sepharose CL-6B, which is a cationic ion exchanger, supplied as a gel, see Ion-Exchange Chromatography, Principles and Methods, published by Pharmacia Fine Chemicals, biz . 18.

The endo activity moves at pH 4.1 in 0.01 molar acetate buffer, while the exo activity is fixed on the ion exchanger, after which the exo activity can be eluted by means of a salt gradient in the buffer (NaCl 0 M - 0.3 M). By subsequent gel filtration on Sephracryl S-200 (prepared by covalent cross-linking of al-10-lyldextran with Ν, η'-methylenebisacrylamide, see "Gel Filtration, THeory and Practice," published by Pharmacia Fine Chemicals, page 12 , practically a pure exo-activity is generated The front fraction with endo-activity was ultrafiltrated and the buffer was changed to a 0.01 M phosphate buffer with a pH of 7.0 This endoenzyme solution was subjected to an ion exchange treatment subject to the anionic ion exchanger DEAE-Sepharose CL-6B, see Ion-Exchange Chromatography, Principles and Methods, "published by Pharmacia Fine Chemicals, page 18. Under these changed conditions, the endo enzyme is fixed on the ion exchanger and then 20 this was eluted by a salt gradient in the buffer (NaCl, 0 M - 0.3 M) The endoenzyme was further purified by adsorption chromatography on a hydroxyapatite agarose gel (HA-Ultrogel). Highly pure endo and exo components were produced in this way.

Specific antisera were prepared by immunization of rabbits by means of the pure endo and exo activities. Based on this antisera, quantitative analyzes of the endo and exo components could be performed by "rocket" immunoelectrophoresis, as described in NH Axelsen, "A Manual of Quantitative Immunoelectrophoresis," 1977, p. 37. Rocket immunoelectrophoresis of solutions of endo and exo components of known concentration (dilution series) has shown that there is an almost linear relationship between the logarithm of the concentration and the surface below the rocket. On the basis of corresponding standard curves, the aforementioned ratio between endo and exo component can be calculated for an inulinase preparation. It has been found that the maximum obtainable inulin conversion (about 99%) is achieved in a relatively short time when using inulinase preparations having the aforementioned preferred ratios between endo and exo activity components.

However, due to the fact that the rocket immunoelectrophoresis 8201763 7 analysis does not necessarily distinguish between active and inactive inulinase, the inulinase activity and the invertase activity of the inulinase preparations according to the present invention are also determined by the enzymatic activity assays indicated below. The inulinase activity (which includes both the endo and exo activity) is determined in the following manner: substrate: 5% inulin in acetate buffer (0.1 M) pH 4.7

incubation: 1 ml substrate + - 1 ml enzyme 20 min, 50 ° C

stop reagent: 4 ml 0.5 N NaOB.

The released reducing sugars (fructose and a small amount of glucose) are quantitated by the (Somo-gyi-Nelson method, see Journal of Biological Chemistry, 153 (1944) 375-380.

An inulinase activity unit is defined as the amount of enzyme available to form a micromole of reducing sugar per minute under the conditions indicated above.

The invertase activity (which mainly includes the exo activity) is determined in the following manner: substrate 10Z sucrose in acetate buffer (0.1 molar) pH 4.7

incubation: 1 ml substrate + 1 ml enzyme 20 min, 50 ° C

stop reagent: 1 ml 0.1 N NaOH

The released glucose is quantitated by the (G0D-PERID method, see Instruction Shewets for Manual Assays '77, Boehringer Mannheim GmbH Diagnistica, Sheet 277.439.6 (1).

Although separation of the inulinase into pure exo and endo components has been discussed in detail above and the thus purified enzyme products have been used in the research work, from which some of the examples provided below are derived, the practice of the present invention widely carried out with industrial grade enzymes (ie less pure enzyme products) produced by processing a preferred microorganism strain. In addition, there would be a desire to tailor the endo and exo-inuli-35 industrial grade enzyme preparation ratios to conform the available particular inulin source substrate to a large scale facility, fractionation of a mixed inulinase enzyme as previously described. will not be the expert's usual choice. Preferred would be the addition of any predominantly endo- or exo-inulinase, which is suitable 8201763 ** (8 (prepared by culturing a different strain) into the enzyme preparation.

Summarizing; inulin is hydrolyzed at 60-65 ° C and the hydrolysis is most effective when the ratio of the endo and 5 exo components in the inulinase is in the above-described preferred weight range of 1-7 and 7-1. Virtually no by-products are formed during the hydrolysis process. The obtained fructose syrup can be concentrated and purified by conventional mixing methods. Fructose is also crystallized, if possible after separation of glucose, to yield crystalline fructose. Examples Example I

Culturing an inulinase producing microorganism.

Aspergillus ficuum (A 524) was grown in the following manner: Inoculum culture

The seed culture was grown on agar at 37 ° C for 7 days on agar of the following composition: 1/1 yeast extract (Difco) ................ 4 K2HPO4. .......................... 1

MgS04.7H20 ....................... 0.5 glucose. ....................... 15 agar (Difco) ..................... 15 H20 to ....................... 1000 ml 25 Inoculate the shaking bottle

The culture of the inulinase-producing microorganisms is carried out in 500 ml Erlenmeyer flasks, which are provided with an incision and an inoculation opening of rubber.

Inoculum of an angled agar tube was prepared by pouring 9 ml of sterile water containing 0.1% Tween 80 over the angled agar with subsequent shaking on a Whirling mixer. The total suspension was used to inoculate one shake flask.

Grow

The culture in shaking flasks is carried out at 37 ° C for 7 days using 100 ml of a substrate of the following composition: g / 1 soaked tender liquid ............... 20 NH4H2PO4. ......................... 12 40 KC1 ...................... .......... 0.7 82.017 63 9 Λ ♦ τ

MgS04,7H20 ......................... 0.5 κ2ηρο4 .................. .......... 10.0

FeS04,7H20 .................. .0.1 sucrose ........................ ... 25 5 CaC03 .............................. 0.05

Pluronic ........................... 0.1

The substrate pH is adjusted to 4.5 prior to autoclaving.

The following yields were obtained: 10 inulinase activity invertase activity units / ml units / ml A.ficuum A 524 3 19

Example II

Mode of action of the two inulinase components 90 grams of inulin from dahlia roots Sigma No. 1-3754, hereinafter referred to as Sigma inulin, were dissolved in warm water to a total weight of 450 g. The pH was adjusted to 4.5 at 60 ° C and two flasks were charged with 150 g of the solution each. The flasks were equipped with magnetic stirrers and placed in a 60 ° C water bath. 0.175 ml of pure exo-inulinase component, corresponding to: 2.2 inulinase units per gram of inulin, was added to the first flask. 0.7 ml of pure endocomponent, corresponding to 1.4 units / g inulin, was added to the other flask.

The exo and endo enzymes were prepared from Aspergillus fieuum A 524 as described in Example 1 and separated as described therein.

Samples were taken at set time intervals and heated in a boiling water bath for 10 minutes to inactivate the enzymes. After cooling, the samples were filtered and then heated as a mixed bed with an ion exchange resin (Bio-Rad AG 501-X8 [D]) to remove ash and soluble nitrogen before analysis by gel chromatography.

The chromatograms after a reaction of 4 and 48 hours are shown in Figures 1, 2, 3 and 4. It appears that the modes of action of the two enzymes are completely different.

The exoenzyme mainly forms DP ^ and only minor peaks between DP2 and DP ^ 4 are seen. This indicates a mode of action in which the inulin molecule is gradually broken down to fructose and glucose from the fructose end. In addition, the endoenzyme gives rise to the formation of mainly DP3 to DPg Λ- ψ ίο and only minor peaks of DP] and DP2 * This is a typical endo attack pattern. However, it is apparently also possible to cleave DP and DP groups with this endo-inulinase, but at a much slower rate than is seen with the exo-enzyme.

Example III

Mix of exo and endo components 160 g of Sigma inulin were dissolved in warm water to a total weight of 800 g. The pH was adjusted to about 4.5 at 60 ° C. Seven flasks were each filled with 100 g of solution and provided with magnetic stirrers and then placed in a 60 ° C water bath.

Endo and exo components were added to the flasks, keeping the total protein amount on a protein basis constant, but the ratio between endo and exo enzyme was varied according to the following scheme: 15 endo / exo ml ml total activity / g inulin ratio endo exo Somogyi units 1 00 0.192 0 0.54 2 7 0.168 0.024 0.92 3 3 0.144 0.048 1.3 20 4 1 0.096 0.096 2.1 5 1/3 0.048 0.144 2.8 6 1/7 0.024 0.168 3.2 ml-0.192-3J-1 ml endo-enzyme and 1 ml exo-enzyme had the same amount of enzyme on a protein basis.

Samples were taken at set time intervals and heated in a boiling water bath for 10 minutes to inactivate enzyme activity. After cooling, the samples were filtered and then treated with an ion exchange resin (Bio-RAD AG 501-X8 [D]) as a mixed bed to remove ash and insoluble nitrogen before analysis by HPLC. Results are shown in the table below.

On the chromatogram it is not always possible to separate glucose from another peak, probably difructose. Large amounts of this compound are reflected in a little changed glucose retention time. DP4 includes saccharides with a chain length of 4 or higher.

DP1-DP3 are saccharides with a chain length from 1 (except glucose and fructose) to 4.

The example illustrates that the most effective hydrolysis is performed when the endo / exo ratio is about 1.

8201763 -Λ 9% t 11 endo / exo 24 hours 48 hours 72 hours ratio ___ fructose 14.3 29.1 47.3 glucose 9.3 12.5 12.6 5 DP4 + 31.9 19.8 11.8 _DPt- DP ^ 44.6_39.5_28.4 7 fructuse 62.2 83.6 89.3 glucose 10.1 7.0 5.2 DP4 + 13.8 3.2 1.2 10 _DPt-DPt 14.0_6j2_4.4 3 fructose 84.8 90.4 91.2 glucose 4.8 5.5 5.8 DP4 + 6.2 1.5 0.9 _DPt-DP ^ 4.2_2J_2jl 15 1 fructose 93.3 94.3 93.7 glucose 3.8 4.7 5.4 DP4 + 1.9 0.6 0.4 _DP-ι -PP7 1.0_0 ^ 4_0Λ 1/3 fructose 91.1 93.8 94.3 20 glucose 3.9 4.8 4.8 DP4-h 1.4 1.0 0.5 _DP-i-DP ^ 0.9_0 ^ 5_0 ^ 4 1/7 fructose 90.0 93.5 94.4 glucose 3.9 4.6 4, 3 25 DP4 + 5.3 '1.5 0.8 _DPt-DP ^ 0.8 __ <^ 4 0 fructose 84.8 89.6 90.4 glucose 3.4 4.1 5.0 DP4 + 11.3 5, 7 3.4 30 _DPt-DPs 0.5_0 ^ 5_1.3

Example IV

Hydrolysis of inulin extraet using different endo-exo component ratios.

Preparation of substrate. 22.8 g pieces of dried chicory root are extracted with 227 kg of water at 75 ° C for 1 hour at the natural pH 5.3. The mixture is filtered and the wet roots are extracted again with 150 kg of water at 75 ° C for 30 minutes.

40 The two extracts gave a total of 295 1 extract with a content of 8201763 t 12 dry matter measured as 3.5 ° Brix.

The extract is purified by liming. Two portions of 687 g Ca (0H) 2 each are successively added at a temperature of 35-40 ° C. The filtration is performed at 40-45 ° G. CO2 is added to the filtrate to lower the pH to about 7; no precipitation is observed. The solution is treated with activated charcoal (2% charcoal based on dried roots) at 75 ° C for 30 minutes. The carbon is removed by filtration and the solution is evaporated under reduced pressure. a dry matter content of about 24 ° Brix. The inulin solution obtained had a brownish color.

Hydrolysis of inulin extract.

About 800 ml of the previous inulin solution is diluted to a Brix value of 20 °, the pH is adjusted to 4.5 and the solution is divided into 7 flasks with 800 g of solution in each. The flasks were equipped with magnetic stirrers and placed in a 50 ° C water bath.

Sndo and exo enzymes were added to the flasks, keeping the total protein amount on a protein basis constant, but the endo-exo component ratio was varied according to the following table:

endo / exo volume ml to. act./$Brix DS

thing protein base_exo_endo Somogyi units 0 0.096 0.27 25 7 0.012 0.084 0.46 3 0.024 0.072 0.65 1 0.048 0.048 1.05 1/3 0.072 0.024 1.4 1/7 0.084 0.012 1.6 30 _0_096 0_ 1 1.8 ml of endo-enzyme and 1 ml of exo-enzyme had about the same amount of enzyme component on a protein weight basis.

Samples were taken at set time intervals and heated in a boiling water bath for 10 minutes to inactivate enzyme activity. After cooling, the samples, including a sample substrate with no added enzyme, were filtered and then treated with an ion exchange resin (Bio-RAD AG 501-X8 [D]) as a mixed bed to remove ash and insoluble nitrogen before HPLC analysis. . The results are shown in Tables C and D.

40 "Glucose" is the combined amount of glucose and other components, possibly difructose, that are not separated on the chromatograms. DP4 + includes saccharides with a chain length of 4 or more plus impurities in this example, for example, salt and protein.

5 DP ^ -DPg are saccharides with a chain length of 1 to 4, except glucose, fructose.

The final yield of glucose in this experiment is quite high, indicating a low mean chain length of the fructose polymers.

However, it can be seen here that the most effective hydrolysis is performed when the endo / exo enzyme ratio is close to 1.

Table C

Composition inulin substrate fructose 0.7% 15 glucose 0.2% sucrose 5.5% DP4 + 90.4% DPL-DP3 3.2%

Table D

endo / exo 24 hours 49 hours ratio __ fructose 12.7 21.5 '' glucose "7.1 9.2 25 DP4 + 42.7 31.8 _DPl -PP 3_ 37.6_37.5 7 fructose 49.6 71, 2 "glucose" 10.9 11.0 DP4 + 18.4 4.9 30 _DPi-DPq_.21.1_12.9 3 fructose 68.9 82.2 "glucose" 11.4 12.3 DP4 + 8.2 2, 4 _DPl -PP? _1M_3.2 35 1 fructose 81-, 5 84.0 "glucose" 12.2 13.0 DP4 + 4.1 1.8 _DPl -PP? _2j2_1.2 8201763 * 14 1/3 fructose 79, 5 83.7 •• glucose "12.8 13.1 DP4 + 4.5 2.0 _DPt -PP 7_3 ^ 2_1.1 5 1/7 fructose 79.0 83.6" glucose "12.5 12.8 DP4 + - 5.6 2.5 _DPi -DP ^ _ 2J | _1.1 10 0 fructose 79.4 83.4 "glucose" 12.5 12.2 DP4 + 6.5 3.0 _DPt -DPrt_ lj5_1.4

Example V

Hydrolysis of inulin using different endo / exocomponent ratios.

6 flasks, each. 100 g of a 20% 's Sigma, inulin solution at a pH of 4.5, were formulated. The flasks were equipped with magnetic stirrers and placed in a 60 ° C water bath.

Endo and exo-inulase enzymes were added to the flasks, keeping the enzyme activity within a narrower range than in Example III, however again varying the ratio between the endo and exo enzymes determined gravimetrically according to the following 25 the table: endo / exo ratio endo / exo ratio soar of activities · on protein basis_on act, basis_per gram inulin_ CO OO 0.67 30 4.1 0.6 0.90 2.7 0.4 0.88 1.35 0.2 0.95 0.45 0.07 1.00 0_0_1 ^ 09_ 35

Samples were taken at set time intervals and heated in a boiling water bath for 10 minutes to inactivate enzyme activity. After cooling, the samples were filtered and then treated with an ion exchange resin (Bio-RAD AG 501-X8 [D]) as a mixed bed to remove ash and soluble nitrogen before analysis according to HPLC 8201763 ¢ 5. The results are shown in Table E.

Glucose is the combined amount of glucose and another component, possibly difructose, that cannot be separated on the chromatogram. DP4 + includes saccharides with a chain length 5 of 4 or more. DP ^ -DPj are saccharides with chain lengths from 1 to 4, except glucose and fructose.

In this experiment, the most effective hydrolysis for a dosage of about one unit appears to be obtained when the ratio, determined gravimetrically, is 4.1.

10 Table E

hours endo / exo components_ 24_48_72_ fructose 3.7 5.6 33.8 "glucose" 5.1 6.8 13.1 15 DP4 + 37.1 17.1 15.3 DPI-DPI_54.2_70.5_37.9 4, 1 fructose 77.4 91.5 94.7 "glucose" * 5.5 3.8 3.3 DP4-K 10.0 2.9 1.4 20 _DPi-DP ^ _Tjl_1 * 8_0.6 2.7 fructose 80.0 92.9 94.5 "glucose" 5.4 3.3 3.4 DP4 + - 9.1 2.6 1.7 _DP1 -DP ^ _5 * 5_1 * 2_0.5 25 1.35 fructose 75, 9 90.0 92.8 "glucose" 3.8 3.5 3.3 DP4j- 16.8 * 5.1 3.1 _DPT-DPg_3 * 5_1.5 0.8 0.45 fructose 71.0 88 , 1 92.5 30 "glucose" 3.1 3.7 3.4 DP4 + 23.8 7.3 3.5 _DP1-DP ^ _2 * 1_0 * 9_0.7 0 fructose 64.8 75.5 82.8 "glucose" 2.3 2.9 3.2 35 DP4 + 31.9 20.8 13.2 _DPI -DP ^ _1 * 0_0.8 0.8

Example VI

2 Flasks, each containing 100 g of a 20 wt% Sigma inulin solution at a pH of 4.5, were made up. The flasks were fitted with magnetic stirrers and placed in a water bath at 60 and 65 ° C, respectively.

Inulase enzymes from A. ficuum A.524 were added at a dose of 2.8 units per g of inulin. The inulase of A. ficuum had an endo / exo ratio of 1.

Samples were taken at set time intervals and heated in a boiling water bath for 10 minutes to inactivate the enzyme. After. cooling, the samples were filtered and treated with an ion exchange resin (Bio-RAD AG 501-X8 [D]) as a mixed bed to remove ash and soluble nitrogen before analysis by IPLC.

The following results were obtained: temp.eC dosage units components hours _inulin 24_48_72 96 15 60 2.8 fructose 94.0 95.5 95.3 95.1 "glucose" 4.9 4.3 4.3 4.4 DP4 + · 0.8 0.2 0.2 0.1 ___ ΡΡΊ -DFq_0.3 0.1 0.2 0.3 65 2.8 fructose 93.5 95.0 95.4 95.3 20 "glucose" 4, 7 4.2 4.0 4.2 DP4 + 1.2 0.5 0.2 0.1 _DP-ι -DP ^ _0.6 Q, 3 0.3 0.4

It. it appears that a complete hydrolysis fructose + "glucose" content of 99% at both temperatures is obtained using this enzyme preparation and this dosage.

8201763

Claims (9)

Process for hydrolyzing inulin with a microbial inulinase preparation, characterized in that inulin is hydrolyzed in an aqueous medium at a pH in the range of 3.5-7, at a temperature in the range of 60-65 ° C, in the presence of an effective dose of an inulinase preparation having an endo-inulinase to exo-inulinase ratio in the range 1:10 to 10: 1 by weight, whereby an enzymatically complete hydrolysis of inulin is obtained 2. Process according to claim 1, characterized in that a ratio is applied in the range from 7: 1 to 1: 7. Process according to claim 1 or 2, characterized in that a ratio is applied in the range from 1: 2 to 4: 1. 4. Process according to claims 1 to 3, characterized in that an inulinase preparation is used, which contains an inulinase obtained by cultivation of an inulinase mixture producing strain of Aspergillus ficuum. Method according to claims 1 to 4, characterized in that a dosage of the inulinase preparation is used in the range of 0.25-10 Somogyi units per gram of inulin. Inulinase preparation for carrying out the method according to claim 1, characterized in that the preparation has a ratio of en-inulinase to exo-inulinase activities in the range of 1:10 to 10: 1 by weight. Inulinase preparation according to claim 6, characterized in that the preparation is obtained by cultivation of an inulinase producing strain: Aspergillus ficuum. Inulinase preparation according to claim 7, characterized in that the preparation is obtained by cultivation of the Aspergillus ficuum strain 30 CBS 55565. ************** 8201763