CA1184519A - Fructose production from inulin with a novel inulinase preparation - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Fructose is produced from inulin by subjecting a solution of inulin to enzymatic hydrolysis at pH 3.5-7, 60°-65°C with an inulinase preparation containing exo-inulinase and endo-inulinase in a ratio of 1:10 to 10:1, the inulinase preparation further exhibiting a high degree of thermal stability.

Description

"~ 11~34519 This invention relates ~o a novel microbial inulinase prepara~ion sui~ed to an industrial process for ~ruc~ose produc~ion, and to an inulin saccharification process.

Fruc~ose is the sweetest, naturally nutrltious su~ar having a swee~ness of approximately l.~ tlmes that of sucrose. It is easily soluble in water and has a higher .so1.ubili~y than sucrose. It could find a wide application in ~he ~ood industry, e.g., in soft drinks, if production costs can be reduced substantiall~.
Fructose is present in a large number o ~rults and ~n hone~. ~'ructose amounts to 50~ o~ sucrose and can be pxo~uc~c:l from sucros~ by acid or enzyma~ic hydrolyis followed by ~epar~tioll. However, separation of equal àmounts of glucose and ~ructose, ~/hi.ch can be performed by special crystallization technlques or by chromatographic separations, is expensive, and is one reason why the costs of fructose production till nowhave been considerably higher than for sucrose.

. . . ..-1- ~

`` 111~151~ ~

~ ruc~ose is also produced in large quantities by enz~ma~ic .isomerization of ~lucose syxups to produce syrups comparable in sweetness with sucrose. However, in isomerized syr~ps the fructose content is limited to appro~lmately 42 due to the isor,leriza~ioll equilibrium between glucose and fruc~ose. Syrups ~ith higher frùctose contents, e.g., 55~
or 90%, are produced by conduct of chromato~raphic separation techni~ues on isomerized syxups.
An ~lternative way of increasing the fructose content of isomerized syrups would be by hlending them with pure or al~os~ pure fructose syrups.
~ poten~ial source o~ fructose is inulin. Inulin i~ an un~ranched fructose polymer ~hich, in native form, consists of about 35 Beta-fructofuranose residues te~ninated by a glucose molecule linked to fructose ~y an alpha-l, b~t~ linka~e as in sucrose.
Inulirl occurs as energy reserve in various plants, ~3 par~icularly in those of the Co~ ltae amily, good sources beiny Jeruscllem artichoke tubers, Dahlia tubers, Chicory and D~ndelion roots. Inulin is almost insoluble in cold water, ~ut can be dissolved in hot water. (See Yanousuy et al.
.A.C.S. 55, (1933) 3658-3633)~
~ nuli~ can be hot water extracted from the plant material by well known methods such as countercurrent diffusion of plant slices at elevated temperature or by pressing out the juice. The raw ~uice is purified by filtration through activated carbon or alternatively treated with lime, as in sugar beet processing, and decolorizea by carbon.

~ ~18'1'~
~J Besides inulin, ~he e~tract l~ay contaill lower chain lencJ~h polymers, e.g./ fructose poly~ers and fructose polymers terminated by ylucose. The average chain length o such polymers may vary ~ith the plant source and gro~ing~
harvestin~ and storing conditions, ~ut will often be bet~een DP5 to DP15 (DP -- degree of polymerization).
Inulin can be hydrolyzed by acid under relatively mild conditions, pH 1-2t 1-2 hours at ~0-90C. However, acid hyd~olysis gives rise to color and byproduct formation, e.~., formation of approxir.lat:ely S~ difruc~osediallhydrides hich reduces the yield coxrespondingly. ~Whistler et al , "~olysaccharide Chemistry" Academic Press, Inc., N.Y. 1953, page 287).
Since fructose in solution seems to be most stable in the p~ range of 4-6, a process for hydrolyzing inulin carried out in this ran~e is believed to be advantageous in oxder to avoid byproduct formation and isomerization.
Enzyrnes capable of h~drolyzing inulin have been de~cri.bed ln ~he literature as being available from plant material sources (inulin containing roots and tubers) and from microorganisms. ~he microbial enzymes may be obtained from yeasts, such as ~luyveromyces fragilis, Candida pseudotrophicalis or kefyr as well as from fungi such as Aspergillus, Fusarium and Penicillium s~ecies.
__ The m;.crobial enzymes are described to have pH
optima in the range from pH 3,5-5.5 and temperature optima bet~een 45C. and 55C. The enzymes are reported to lose activity rapidly at 60C~
~3 ~ 16~ ~

The microbial inulinases are produced both intra-and cxtracellularly. The ability of the enzymes to hy~ro~yze both inulin and sucrose is in some cases (Kluyveromyces fragil s) ascribed ~o one enzyme having two activities. (Nahm et al., "Purification and Characterization of Inulase from ~luyveromvces ~rac~ilis", Korean Biochem. J., Vol. 10 (2), p. 95-108, 1977.) _~_ Both an endo- and an exo-lnulinase have been puri~ied from ~spergillus ni~er. The endo component has its maximum ac~ivity ak pH 5.3 and ~5C.; the exo co~ponent at pH 5.0 and 55C. (Nakamura et al., "S~udies on Microbial Inulase part IV," Nippon No~eikagaku I~aishi, Vol. 52 (~), p. 159-16G, 1978 and 71a~amura et al., "Studies on ~icrobial Xnulcl~e part V"/ N.N.K., Vol. 52 (12), p. 581-587, 1978.) Due to the relatively limited solubility of inulin, th~ hydrolysi.s of inulin will have to be per~ormed in relatively dilu~e 301utions, and, as has already been pointed outl preferably within the p~I range of about 4-6. A 20~ w/w solution o~ pure inulin from D~hLia rooks can be prepared at 60C.
~ut such solut~on is supersaturated and precipitates on extended standing. ~he rnaximum amount of inulin soluble in ater at 50-60C. will depend naturally on the degree of polymerization o the fructose polymer. Of course, the 10~1er the processing ~emperature, ~he lower the solubility o~
inulin, and the more dilute -the inulin solution will need be~
To avoid microbial infections in the relatively dilute inulin solutions industrial process opera~in~ temperature should be at least about 60C. and preferably 60-~5~C. This tem~erature level is ordinary practice in the starch industry wl~ere e,g., the sacchari~ication process is usually carried out at 60C. and 30% w/w DS ~or 2-5 days.
Unfortun2tely, the enzymes reported on in the prior art are poorly suited to commercial usage at 60C.;
~hey deactivate too rapidly. ~et, as a practical matter, -60C. is the mlnimum reasonable hydrolysis kemperature level ~O~ or xeason of inulin sol~lbility and for avoidance of micro~ial con~amination.

~ herefore, there exists a need ~or an effective inulJnase prepclralion which is sufficiently thermostable to be ~mployed at 60~65C. for extended periods of time such as l-S days, to allow hydrolysis of the inulin in an economical way~ Insofar as the inventors hereof are aware, up till now no inulinase preparation having these properties has been reported upon.
Since khe present invention concerns itsel~ with commercial practi~ality, it must be appreciated that an 1~84~

~ enzy~natically produced hydrolysate product superior in cost I and quallty to the acid hydrolysis product is required since high quality fructose eit~ler from inversion of sucrose or fxom fractional separation of isosyrups is available. Thus, the inulin hydrolysa~e should exceed 98% monosaccharide w/w of khe (dry) solids in solution. The term "full hydrolysis"
as h~reinafter employed refers to production of an lnulin hydrolysate that exceeds 98% fructose plus glucose on a dry solids baQis.
. The invention resides in the discovery that inulin i8 ~ost effectively hydrolyzed when both an endo inulinase activ.ity and an exo-inulinase activity are present, and when these inulinase components are present in a certain ratio, suitably a weight xati,o. A suitable endo-inulinase (.inulase) is that designatecl 3.2.1.7, accordi.ng to Enzyme Nomenclature ¦ (1978)~ published or the International Union of Biochemistry ¦ by Aca~emic Press Incu A suitable exo~inullnase (invertase) is th~t ~es:i.~ ted 3.2.1.26, ~cording to the ~ N ~ nclature sys~em.
Accorclis~y to l.ts first aspect, the present invention provide~ a pxocess fox hydrolyzing inulin ~ith a microbial inullnase preparation which process comprises hydrolyzing inulin in an aqueous medium at a pH in the range of 3.5~7, at a temperature in ~he range of 60-65C. in the presence of an effec:tive dosage of an inulinase preparation having a ratio ~f endo-inul~nase to exo~inulinase in the range of 1:10 to 10~1 on a weight basis, whereby an enzymatically full hydrolysls of inulin is obtained.
In a preferred embodiment of the present invention the endo- and e~o-component of the inulinase are present in a weight ratio of 1:7 to 7:1, more preferably 1:2 to ~:1.

The inu].ixlase enzyme preparation of this invention ha~ its temperature optimum at 60Co I but can be employed at 65C. a~ pH values between 3.5-7, preferably 4-6, more pxeferably p~ 4.5 5Ø It retains approximately 50% of its original ac~ivity a~ 60C. in 20~ inulin w/w after 48 hoursO
Preferably~ 75% and more pre~erably, 90% o the activity should be retained.
According to a further aspect of the present inven~ion there is provided an inulinase preparation well suited to perform the process accordiny to the invention said preparation being characterized in that it exhibits a r.atio of endo-inulinase ~o exo-inulinase in the range of 1:10 to 10:1 on a weight basis.

The inulin hydrolysis process according to the presen~ invention may be performed as a batch process in conventional (dextrin) saccharification equipmen-t. Full hydrolysis of the inulin (i.e., ~8~ or more, fructose plus gluco~e) is obtained in the process. The reaction time depends on enzyme dosage a~d can be extended to 2-4 days for more efficient use of ~h~ enzyme without byproduct or color forrnation~ The total inulinase (exo and endo combined) dosage employed is in the range of 0.25-10 Somogyi units per gram of inulin, preferably 1-5 Somogyi units.

In an industrial process, the substrate for hydrolysis will be either an extract of an inulin containing plant, e.g., chicory, Jerusalem artichoke, dahlia, etc. or a suspension of a sliced plant material in water. Such an extrac~ is prepared by well-known techniques and i~ may be purified by liming, carbon-treatment or ion-exchange, etc.
be~ore hydrolysis is carried out.
In both laboratory scale and industrial scale, the inulin extracts will often be relatively dilute, e.g., 5-25%
dry ~ubstance. The inulin extract may be concentrated to the substance conten~ limit of the solubility of inulin or other fructo~e polymers therein at the processing temperature.
As has already been pointed out, the inulin extract will be su~ceptible to microbial infection unless the hydrolysis reaction temperatuxe is made suffici.ently high to prevent microbial growth. Preservatives may, of course, be added, bu~ doing so would add to processing costs throuyh the cost o preservatives and for their removal after processing.
It has been found that microbial inulinase enzyme pr~parations with a sufficiently high thermostability to be applied in i~dustrial hydrolysis processes at 60-65C. can be produced from Aspergilli belonging to the Asper~illus ni~er group.
In a preferred embodiment of the present invention the novel inulinase preparations are produced by cultivation of As~ergillus ficuum ~trains. These novel inulinases are sufficiently thermally stable for practice of this invention.
Their p~ optimum is suited to practice of this invention.
The above microorganisms produce both endo- and exo-inulinase.
However, the ratio of endo- and exo-inulinase activity of the inulinase elaboxated by the Aspergillus ficuum has been found to be ~train speciic.

The inventors hereof have found that the inulinase enzyme produced by means of Aspergillus ficuum, strain CBS
55565 which is identical to ATCC 16882 which ls identical to IMI 91881 (our designation A 524) contains the endo- and exo-inulinase component within ~he above weight ratio.~.~
Accordingly, the use of said specific mi.cxoorganism for producing the inulinase is preerred. However, it is re-coqnized that other worker in the art ma~ prefer to mix se-parately produced ~ndo- and exo-inulinases, and, therefore, such is contemplated expressly as also falling within practice of this invention albe~t as a less preferred mode of this :.

invention.
Re~erring now to the drawings, Figures 1, 2, 3 and ~ are ch~omatoc~xaF.,s il].ustrating the different enzymatic action o~ th~ endo and exo inulinase components from A. ficuum A-324.
The endo and exo enzyme can be separate~ by ion-exchange on CM-sepharose CL-6B~ which is a cationic ion-~xchan~er, delivered as a gel, ~ide Ion~-Exchanye Chromatography, Pxinciples and ~e~hods, edited by Pharmacia Fine Chemicals, pa~e 18. The endo activity moves at pH 4.1 in 0.01 2~ acet~te ~ 9 bu~er, whereas ~he exo activlty is ixed to the ion-exchanger, ~hereafter the exo activity can ~e eluated by means o~ a salt gradient in the }~uffer (~aCl O M - 0.3 M). By means of subsequent gel filtration on Sephacryl S-200~(prepared by covalently cross-linkillg of allyl dex~ran with N,~ methylene-bisacrylamide, v e "Gel Fil~ratlon, Theory and Practice,"
edited by Pharmacia Fine Chemicals, page 12, practically pure exo activity is produced~ The endo activity front fraction was ultrafiltered and the buffer was changed to a 0.01 M phosphate buffer of pH 7Ø This endo enzyme solution ~as ion-exchanged on the anionic ion-exchanger DE~E Sepharose cL-6s, vide Ion-Exchange Chromatvgraphy, ~rinciples an-l Methods," edited by Pharmacia Fine Chemicals, page 18. Under these changed conditions~ the endo.enzyme i.s fixed on the ion-exchanger, and subsequently it is eluated by means of a salt gradient in the buffer (NaCl, 0 M - 0.3 M~.
T~le ~ndo ~nzyme was further purified by adsorption chromatograph~
on a hydroxyapatite agarose gel (~HA-Ultrogel). In this way, very pure endo and exo comE)onents were produced.
By m~ans of the pure endo and exo activities specific an~isera were produced by immunization of rabbits.
On the basis of these antisera, quantitative analyses on the endo and exo components could be carriea out by means of rocket immunoelectrophoresis, as described in ~ . Axelsen et al., "A Manual of ~uantitative Immunoelectrophoresis, n 1977t page 37. By means of rockf~t immunoelectrophoresis of solutions of endo and exo.components with known concentration (dilution series) it has been found tha~ an almost linear relationship exists between the logarithm of the concentration and the area below the rocket. On the basis of corresponding t~ ks 10 ' 14L519:

standard curves the above indicated ratio between endo and exo component can be calculated for an inulinase preparation.
It has been found that ~he maximum obtainable inulin conversion (about99%) is reached in a relatively short time when inulina~e preparations with the above indicated preferred range ratios be~ween endo and exo activity components are 115 ed.
However~ due to the fact that the rocket immuno-electrophoresis analysis does not ne-~e~sarily disti,nguish between active and inactive inulinase, the inulinase activity and the invertase activity of the inulinase preparations, according to the invention is additionally determined by mean~ o the below indicated enzymatic activity determinations~
The inulinase ac~ivity ~covering both endo and exo activity) is d~termined 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., S0C.
Stop reagent: 4 ml 0.5 N NaOH
The released reducing sugars (fructose and a small amount of glucose) are determined quantitatively b~ means of the Somogyi-Nel~on method, vide Journal of Biological Chemistry, Vol. 153, page 375-380,1944.
One inulinase activity unit is defined as the amount of enzy~e which is able to form one micromole reducing sugar per minute under,the above indicated conditions.
The invertase activity (mainly covering exo activit~) is detexmined in the following manner:

~L~L8'1S19 Substrate: 10% sucrose in acetate bu~fer - ~0.1 ~) p~ 4.7 Incubation: 1 ml substrate ~ 1 ml enzy~
~0 min., 50C.
Stop reagent: 1 ml 0.1 N NaOH
The r~leased glucose is de~ermined quantitatively by means of ~he GOD-P~RID methocl, vide ~nstruction Sheets for Manual As~ays '77, Boehringer Mannheim GmbH Diagnostica, Sheet 277.439.6 (1). ' Although separation of the inulinase into pur~e exo and endo components has been discussed at length above and the 90 purified enzyme products were employed in the test work from which some o~ the hereinafter provided examples are drawn, large scale practice o~ this invention would normally be carri.ed out with industrial grade en7,ymes (i.e., less pure enzyme produ`cts) produced by cul~ivation of a preferred microorganism strain. In addition, should a ~e,~ire exi~t to tailor the endo- and exo~inulinase proportions o~ the lndustrlal yrade enzyme preparation to match the particular inulin source substrate available to a large scale acility, fractionation of a mixed inulinase enzyme as above described would not be the usual choice of those skilled in the art. Preferable would be the addition of some predominantly endo- or exo-inulinase, whichevèr is appropriata (made by cultivation of a different strain) into the enzyme prepara~ion~
In summary then~ inulin is hydrolyzed at 50-65C., and the hydrolysis is most efficient when the ratio between endo and exo components in the inulinase is in the preferred 1-7 to 7-1 weight range described above. Almost no byproducts are formed during the hydrolysis process. The resulting fructose syrup can be concentrated and purified by con-ventional methods, ~

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for blendinc3 purposes. ~lternatively~ ~ructose is crys~allized, possibly ater separation from glucose, to yield crystalline fructose.
The present invention is further illustrated in the following examples ~hich are not to be considered to limit the invention.

EXA~5PLES
Example 1 _ . .
Gro~ing of an Inulinase producing Mlcroorganism ~spergillus icuum (A 524) was grown in the ~ollowing way:

Growth of inoculum Irhe seed culture was ~rown on an agar slant at 37C for 7 days on agar having ~he following composition:
Yeast e~tract (Difco)............ ;

K2HPa, ~ -- --- ~ -~............... ... 1 ,~ MgSO~,7H20.......................... .. 0.5 Glucose.. ~.... ~............................................. 15 ~gar (Di~co).. ~..... l..... ~.~............................... 15 H20 up to..... ~.......................................... l,OOO ml Seedi~ of shake flask . . _ _ Growth of the inulinase producing microorganisms is performed in 500 ml Erlenmeyer flasks equipped with a notch and inoculation rubber port.

Inoculum from one agar slant tube was prepared by pouring 9 ml steril.e water containing 0.1% ~Jeen 80 over the agar slant with subsequent shaking on a ~hirling mixer. The total suspension was used for seeding of one shake flask~

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- ~ 5~

Growth _ _ Growth in shake flas~s is performed at 37 GC for 7 days using lO0 ml of a substrate wi~h the following composition:
. ~. .
Corn steep liquor........ .. 20 NH~H2P04.... o....~........ 12 7 ~Cl.~.................... 0.7 M~S04,7H20.. ,............ 0.5 ~ K2HPOI,, 11:) . O
.. ... .. _ ... .
FeS0~,7H20............... 0.1 Sucrose.~................. 25 cac~3---.... ~.~.......... 0.05 Pluronic.... ~........... 0.1 Prior to autoclaving, pH of the substrate is adjusted to 4 0 5.
Th~ ollowing yi~lds were obtair.ed:
Inulinase activity Invertase activity units/ml A. ficuum A $~4 3 l9 _ ___ Example 2 ~__ Mode of action of the two_inulinase components 90 grams of inulin from Dahlia roots Sigma No. I-3754 hereafter referred to as Sigma inulin was dissolved in hot water to a total weight of 450 g. pH was adjusted at 60C to . ~

p~I 4.5, t~o ~lasks were fi].led with each 150 ~ of the solution, The flasks were equipped with magnetic stirrer and placed in a water bath at 60 C . To the first ~lask was aclded pure ~:~;o inulinase component 0.1i5 ml correspondiny to 2.2 inulinase units per gram inu~in. To the other flask was added pure endo co~ponent 0.7 ml corresponding to 1.~ uni~s~g inulin.
The exo and endo enzymes ~ere prepared from ficuum A 52~.as described in Example 1 and separated as has been described herein.
Samples were withd.rawn at set intervals of time, and heated in a boiling water bath for 10 minutes to inacti.vate the enæymes. A~ter cooling the samples were filtered and then hea-ted with a mixed bed ion exchange resin (Bio-Rad ~G 501-X8 [D]) to remove ash and soluble N be~ore being analysed by ~elchra~togra~hy.

The chromatograms after 4 and 48 hours reaction are cleplc~d on Figures 1, 2, 3, and ~. It is seen that th~ modes o~ action o~ the two enzymes are quite different.
~ he exo enzyme forms mainly DPl and only minor pea~s between DP2 and DPl~ are seen. This indicates a mode of action where the inulin molecule is degraded stepwise to fructo~e and glucose from the fructose end. As opposite the endo ~nzyme gives rise to fo~nation of mainly DP3 to DP5 and only minor peaks of DPl and DP2. This is a typical endo attack pattern.
~owever, with this endo inulinase apparently also DPl and DP2 groups can be split off, but at a much slower rate than seen with the exo enzyme.
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~ k 3 . ~,~,f~

e 3 Mixin~ of exo and endo comPOnents -- ~
160 g. o~ Sigma inulin were dissolved in hot water to a total weigl~t of 800 g. p~ was adjus~ed at approximately 60C to pH 4.5. 7 flasks were each ~illed with 100 g. of solution and equipped wi~h magnetic stirrers and then placed i.n water hath at 60Co Endo and exo-components were added to the flasks keeping the total enzyme amount on protein basis constant but varying the propor~ion between endo- and exo-en~yme according to the following scheme:
. _ . _ ~ Endo/exo ~1r~l ~otal actlvity/g~inulin ratio Endo Exo_Somogyi units 1 oo 0.192 0 0.54 ~ 7 0~168 0.024 0 d ~ 2 3 3 0.1~ 0.048 1.3 1 0.096 0.096 2.1.
1/3 0.0~8 0.1~4 2~8 6 .1/7 0.024 0.168 3.2 7 0 0 0.192 3.6 _ _ ~ _ _ 1 ml of endo enzyme and 1 ml Gf exo enzyme had the same amount of enzyme on a protein basis.
At set intervals of time samples were withdrawn and heated in a boiling water bath ~or 10 minutes to inactivate enzyme acti~ity. After cooling ~he samples were filtered and then treated with a mixed bed ion exchange resin (Bio-Rad AG
501-X8 [D~) to remove ash and soluble 27 before ~eing analyzed by HPLC. Results are tabulated below.

r 1.1~ Ibl9 On ~he chromatogram glucose cannot in all cases be separatecl ~rom ano~her peak, presumab~.y difruc~ose. Greater amOUlltS of this compound are reflec~ed in a little changed reten~ion ~ime for glucose. ~P~ includes saccharides of chain length of 4 or higher.
DPl-DP3 are saccharides with chain length from 1 (except glllcose and fxuctose) to 4 The ~.xample illustrates that the most eficien~
hydrolysis is pexformed when the Endo/Exo propor~ion is approxim~tely 1.
_ ; , _ ..
,Endo/Exo 2~ hours 48 hours 72 hours Ratio - Fructose14.3 29.1 47.3 Glucose 9.3 12.5 12.6 . DP~ 31.9 19.8 1108 , DPl-DP3 .44.6 3B.5 2~.~
. . _ _ _ . 7 Fructose62.2 83.6 89~3 Glucose10.1 7.0 5.2 DP4~ 13~8 3.2 1~2 . DP~DP314 J 0 6.2 4.~
. . ~ ~
3 ~'ructose 84.8 90.4 91 2 Glucose 4.8 5~5 5 8 DP~ 6.2 1~5 0.9 DPl--DP34 a 2 2 ~ 6 2~ 1 _ ___ _ __ . .

1 Fruc~ose93.3 n4.3 93.7 Glucose 3.8 4~7 5.4 ~P4~ 1.9 0.6 0.~
DPl-~P3 1.0 0O4 0.4 CO~T.

. 1~

.~ ~3~5~ ~
. . . . ___ ~ndo/Exo 24 hours 48 hours 72 hours Ratio . .__ _ . _ 1~3 ~ructose91.1 ~3. ~ 940 3 Glucose3.9 4.8 4.8 DP4~ ~.1 1.0 0.5 . 3 0.9 0.5 0 4 ..
1/7 Fxuctose90.0 93.5 94-4 Glucose3.9 4~6 4.3 DP~ 5.3 1.5 0.8 .
DPl-~P3 0.8 0.4 0~4 .. . . _ _ 0 Fructose~4.8 89,6 90.4 Glucose3.4 ~.1 5~0 DP~ 3 5~7 3.4 1 3 1.3 . _ , _ .

Exampl.e 4 __ ~ydro~sis of Inulin Extract using Various Endo Exo Component Ratios Preparati.on of substrate 22.8 kg of dried chicory root slîces are extracted with ~27 kg water at 7$C for approximately 1 hour at natural pH 5.3. The mixture is filtered and th~. ~et roots extracted again with 150 kg water at 75C for 30 minutes.
The ~wo extracts yield a total of 295 1 extract with a dry substance content measured ~o be 3.5 Brix.
~ he extract is purified by liming, Two por~ions o 687 g Ca(OH)2 each are added successively at a te~pera~ure of 35-40C, Filtration is performed at 40-45C~ C02 is added to the filtrate thus decreasing pH to approximately 7, no precipitate i~ observed. The solution is treated with activated carbon ~2% carhon on the basis o~ dried roots) at 75C fol- 30 minutes. The car~on is removed by filtration, and the solu~ion evaporated under vacuum to a dry matter content of approximately 24 Brix. The resulting inulin solu~ion had a brownish colour.
llyclro ~ s of inulin extract ~ pproximately 800 ml of the above i.nulin solutio., is diluted to a Brix value of 20, p~l is adjusted to 4.$, and the solution divided in 7 flasks with 100 ~ solution in each. The fl~s~s were equipped with magnetic stirrers and pl~ced in water bath at 60.
Enclo and exo enz~nes ~Jere added to the 1asks keeping the to~l enzyme amount Oll protei.n basis constant, but varying ~he enclo-exo component ratio according ~o the followi.ng Table:
Tot.act./
Endo/Exo Ml. ~11 Brix DS
ratio on protein basis exo end~ Somogy;. ~ni.ts _~
:~ - 0 0.096 0.27 -ci 7 0.012 0.084 0.46 3 0.024 0.072 0.65 1 0.~48 0~048 1.05 1/3 0.072 0.024 1.4 ./7 ~.084 0.012 1.6 ~ 0~0~6 ~ 108 _. :___~_~-.... ___.____ ._- 7 _ _ - -. ' _ _._ __ _._ _ __.
1 ml of endo enzyme and 1 ml of exo enz~me had on protein weight basis, a~Droxl~.ately -~e same am3unt of enzy~e cc~onent.
. ' ' '-' At set in~.ervals of time, samples were wit~dra~n and heated in a boiling water bath for 10 minutes to inactivate the enzylr~e activity. After cooling, khe samples, i~cluding a sample of substrate ~ithout enzyme added, were filtered and tl~en treated with a mixed bed ion-exchange resin (Bio-Rad AG
501~X8 [D]) to remove ash and soluhle N before HPLC~analysis.
u~s are given in Tables III and IV.
"C,lucose" is the combined amounts of glucose and another component, possibly difructose, which are not separated on ~he chromatograms. DP~ includes saccharides having a cll.~in len~th of ~ or higher plus impuriti~s in this sample, c. g . salt and protein.
DPl-DP3 are saccharides having a chain length from ~o ~" ~xcept ~lucose, fructose.
The ~inal glucose yield in this experiment is x~ther hi~h indicating a low average chain length of the .ruct.ose .
l polymcrs.
I ~owever~ here too i~ is seen that the mos~ eicient ! hydrolysis is caxried ou~ whe.n the endo/exo enzyme ratio is close to 1.

Table III

! omposition of Inulin Substrate !~xuctose ~ 0 7%
Glucose............. ~...... 0.2%
5ucrose....... ~..... ~...... 5.5%
DP~ ;.~.90.4%
DPl~DP3 ----.. 3.2%

2~

,,. ,_ .. ,_. ,., .,.~, ,., .. ,.. , . .

l ~

Tdble IV
. . _ _ Endo/~xo 2~ 4 9 Rat:io Hours Hcurs , , ,, __ _ _ _ _ Fructose12. 7 21. 5 "Glucose"7 .1 g D ;2 DP,l~42.731.8 DP -DP37. 637.5 .__ . ~, , ,. ~_ _~.,.,.. _ _ .
. 7 Fxuctose 43. 6 71. 2 'Glucose'' 10 . 9 11. 0 18.4 ~,~
l DP3 21.1 12.9 __ .. _. . . . .... . ~ . ~ _ . .. ... ..

3 Fructos~ 68~ 9 82. 2 "Glucose" 11. 4 12~ 3 DP,~ 8.2 2.4 D 1 D 3 11.6 3.
...... _ . , l;'~ ct:oxc 31 . 5 ~ 8~
"G~uco~i~ " . .12. ~ 13 . O
. nJ?~. . 401 ; 1.8.
. - ' L~ vr3 _ 2. 2 ,, ,... . , ~,,, . . ~ ~,___ 1,~3 Fructose 79. 5 83~ 7 "Glucose"12. 8. 13 o l DP,~+ 4.S 2.0 - DPl--DP33. 2 1.1 1~7 Fxuctose 79~ 0 83. 6 nGlucose"12~ 512. 8 DP4~ 5~ 6 ~. 5 ~Pl-~P32 . 9 1. 1 . ... , . _ .. . _., O Fructose 79. 4 83 . 4 "Glucose" 12~ 5 12. 2 DP~ 6;5 300 . DPl-DP3 - lo 5 1~ 4 _ . . ~ -- _. -- ~ . ., _ . 29. .

i9 ~ I
Example 5 ~Iydrolysis of Inulin using Various Endo/Exo Com~onent Ratio 6 flasks, each containing 100 g of a 20% Sigma inulin solution at pH 4.~, wer~ prepared~ The flasks were equipped wi~h magnetic stirrers and pla.ced in water bath at 6QC.
. Endo and exo inulinase enzymes were added to the ~lasks keeping the enzyme activity wi~hin a more narrow range than in Example 3, but again varying the proportion between endo and exo enzymes gravimetrically de~ermined according to the following Table:

Endo/Exo Rat.ioEndo/~xo Ratio Sum of ~ctivities on Protein ~asison Act~ Basis per Gram Inulin ~ 0.67 .1 '0.6 ~.90 207 0.4 0.~
. 1.35 0.~ 0.55 0,~5 0.~7 1.00 o ' ' o ' 1.09 At set intervals of time samples were withdra~m and heated in a boiling water bath for 10 minutes to inactivate the enzyme activity. After cooling, the samples were filtered and then treated with a mixed bed ion-exchange resin (Bio-Rad AG
501-X8 [D3) to remove ash and soluble N before being analyzed by HPLC~ P~esults are shown in Table V.

- ' ~ , ,i Glucose is the combined ar.loun~s of glucose and a~oth~r c~mponel~t, possibly difructose, which canno~ be separated on the chromatogram. DP4~ includes saccharides havillg a chaill length of 4 or higher. DPl-DP3 are saccharides having chain lengths rom 1 to 4, e~cept glucose and fructose.
In this experiment ~he mos~ efficient hydrolysis for a dosage of approxima~ely 1 unit is found when the ratio, .
~ravimetrically determined, i5 4 o ~

Table V
Hours Endo/Exo Componen~.s 24 ~8 72 .. . . . . _ . _ Fructose 3.7 5.6 33.8 "~lucose" 5.1 6.8 13~1 DO DP4~ 37.1 17.1 15.3 1 .3 54.2 70.5 37.9 .. . . . _ _ _ . ___ .1 Fructose 77~4 91.5 9~.7 "Glucose" 5, 5 3. 8 30 3 DP~ . 10.0 2.9 1.4 DP~-DP3 7.1 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 DRl~DP3 5.~ 1.2 0~5 , ~
1~35 Fructose 75.9 90.0 92.8 . "Glucose" 3.8 3.5 303 DP4~ 16.8 5.1 3.1 DPl-DP3 3.5 1.5 008 . . _ _ . _ _ .
. 0~45 Fructose 71.0 88.1 92.5 "Glucose" 3.1 3.7 3.4 : DP~ 23~8 7~3 3.5 DPl-DP3 2.1 - 0.9 007 . 0 Fxuctose 64.8 75.5 82.8 . '1Glucose" 2.3 2.9 3.2 DP4+ 31.9 20.8 13.2 DPl-DP3 1.0 008 0.8 _~

. 23 Exarrple 6 2 flasks each containing 100 g o 20% w/w Sigma inulin solution at pH 4.5 were preparedO The flasks ~ere equipped with magnetic stirrers and placed in a water bath at 60 and 65C, respectively.
Inulinase enzymes ~rom A. ~icuum A 52~ was adde~ at a dosage of 2.8 units/g inulin. The inulinases from A ficuum had an endo/exo ratio of l.
At set in~ervals of time samples were withdrawn and heated in a boiling water ba~ch for lO minutes to inacti~ate ~he enzyme. After cooling, the samples were filtered and treat~d ~lith a mixed b~d ion-exchange resin (Bio Rad AG
SOl~X8 [D]) to remove ash and soluble N before being analysed by ~IPLC.
The follo~ing results were obtained:
Temp. DSiage HOURS
inulin Comp5. 24 4B 72 96 6P 2.8 Fructose34.0 95.5 95.3 95.1 Glucose 4.9 4.3 4.3 4.4 DP~-~ 0.8 0~2 0.2 0.1 DPl DP3 0.3 0.1 0.2 0.3 _ __ 65. 2.8 Fructose93.5 95.0 . g5.4 95.3 ~lucose 4.7 ~.2 4~0 4.2 DP4~ 1.2 0.5 002 0.1 DPl~D~3 0.6 0.3 0.3 0~4 .~
It is seen thata full hydrolysis fructose + "glucose" content of 99~ is obtained at both ~emperatures using this enzyme prepar~tion and dosage.

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for hydrolyzing inulin with a microbial inulinase preparation, wherein inulin is subjected to hydrolysis in an aqueous medium at a pH in the range of 3.5 - 7, at a temperature in the range of 60 - 65°C and in the presence of an effective dosage of an inulinase preparation having a ratio of endo-inulinase to exo-inulinase in the range of 1:10 to 10:1 on a weight basis whereby an enzymatically full hydrolysis of inulin is obtained.

2. The inulin hydrolyzing process of claim 1, wherein said ratio is in the range of 7:1 to 1:7.

3. The process of claim 1, wherein said ratio is in the range of 1:2 to 4:1.

4. The process of claim 1, wherein the inulinase preparation comprises an inulinase obtained by cultivating an inulinase mixture producing strain of Aspergillus ficuum.

5. The process of claim 1, wherein the dosage of the inulinase preparation is in the range of 0.25 - 10 Somogyi units per gram of inulin.

6. An inulinase preparation suitable for performing the process according to claim 1, and comprising a ratio of endo-inulinase to exo-inulinase in the range of 1:10 to 10:1 on a weight basis.

7. Inulinase preparation according to claim 6, obtained by cultivating an inulinase producing strain of Aspergillus ficuum.

8. Inulinase preparation according to claim 7, obtained by cultivating the Aspergillus ficuum strain CBS
55565.