CN1283804C - Trehalose-releasing enzyme, and its preparation and use - Google Patents

Abstract

Disclosed is a trehalose-releasing enzyme which specifically hydrolyzes the linkage between a trehalose moiety and the remaining glycosyl moiety in a non-reducing saccharide having a trehalose structure as an end unit and having a degree of glucose polymerization of 3 or higher. The molecular weight of the enzyme is about 57,000 to 68,000 daltons on SDS-PAGE, and the isoelectric point is about 3.3 to 4.6 on isoelectrophoresis. The enzyme is useful in an industrial-scale preparation of trehalose, and the trehalose prepared therewith can be readily incorporated into food products, as well as cosmetic- and pharmaceutical-compositions.

Description

Trehalose-release enzyme and preparation and purposes

The present invention relates to a kind of trehalose-releasing enzyme, and preparation and purposes, particularly, relate to a kind of new trehalose-release enzyme, it can specificity hydrolysis irreducibility polysaccharide in key between trehalose part and all the other glycosyl parts, it is 3 or higher that described irreducibility polysaccharide contains as the trehalose structure of a terminal unit and glucose polymerization degree, and relates to the preparation of this enzyme.The invention still further relates to the microorganism that can produce described enzyme, the trehalose that obtains with described enzyme, and contain the composition of described trehalose.

Known trehalose or α, α-trehalose are a kind of irreducibility polysaccharide of being made up of glucose unit.As at Advances in Carbohydrate Chemistry (Vol.18, pp.201-225 (1963), Academic Press, USA publishes) and Appliedand Environmental Microbiology (Vol.56, pp.3,213-3,215 (1990)) as described in, trehalose extensively is present among microorganism, mushroom, the insect etc., although its content is low relatively.Trehalose is a kind of non-reduced polysaccharide, it neither with contain amino material such as amino acid and proteins react, induce amino-carbonyl reaction, can not make that to contain amino acid whose material rotten yet.Therefore, can relieved use trehalose and needn't fear to cause coking dissatisfactory and rotten.Owing to these reasons, an urgent demand can large-scale industrialization prepare trehalose.

The traditional preparation process method of trehalose is, for example, at the open No.154 of Japanese Patent, the disclosed method of microorganism that utilizes in 485/75, with the open No.216 of Japanese Patent, reported method in 695/83, wherein with maltose-and trehalose-Starch phosphorylase combine use, maltose is become trehalose.Yet, the former, because in (calculating) microorganism as parent material with dried solid (d.s.b), the content of trehalose is usually less than 15w/w% (unless stated otherwise, term in this specification sheets " w/w% " is abbreviated as " % "), and therefore extraction and purification step complexity are unsuitable for suitability for industrialized production.The latter has following shortcoming: because trehalose forms through Cori ester, can not adjust to satisfactory high level as the concentration of the maltose of substrate; (ii) the enzymatic reaction system of Phosphoric acid esterase is to pass reaction, and actual trehalose productivity ratio is lower, and (iii) is difficult to basically obtain stable reactive system and successfully enzyme reaction is carried out continuously.Therefore, in fact, these traditional preparation process methods can not be as industrialized preparing process.

About Preparation methods of trehalose, at " Food Chemicals " (No.88, pp.67-72 (1992,8)) be entitled as in the special column of " Oligosaccharides ", title is for pointing out in the article of " Current Status of Starch Application Developmentand Related Problems ": " although being widely used of trehalose; reported in this field; through the direct enzyme preparation method of polysaccharide-shift reaction or hydrolysis reaction, almost be impossible theoretically." therefore, consider that think and make material with starch, it is very difficult preparing trehalose by enzyme reaction from professional angle.

Know, from the starch partial hydrolysate of raw starch such as liquefying starch, dextrin and maltose oligosaccharides (maltooligosaccharides) preparation, because of its reduction end group shows reducing power.Usually represent the reducing power of these reductibility starch partial hydrolysates with " dextrose equivalent (DE) value " (with dry weight basis).Understand, in these reductibility starch partial hydrolysates, it has high relatively DE value those, usually molecule is heavy relative with viscosity low, and sugariness and level of reactivity are high relatively, and easily with the material such as amino acid and the proteins react that have amino, thereby cause tedious coking, smell and damage its quality.

Because the character of reductibility starch partial hydrolysate changes with its DE value, therefore, the relation between reductibility starch partial hydrolysate and its DE value is remarkable.In this field, even can not believe and to break away from this relation.

Yet, the inventor has changed this prevailing paradigm really, and successfully founded a kind of the sort of method for preparing trehalose that resembles described in the Japanese patent application No.362131/92, wherein, with glucoamylase with can form non-reduced polysaccharide (have one as the trehalose of a terminal unit and have 3 or higher glucose polymerization degree) non-reduced polysaccharide-formation enzyme use, so that to what prepare by raw starch, glucose polymerization degree be 3 or higher reductibility starch partial hydrolysate have an effect, from irreducibility starch partial hydrolysate direct production trehalose.Although with this trehalose preparation method, the productive rate of producing trehalose from irreducibility starch partial hydrolysate is about 30%, and is a kind of feasible industrialized process for preparing, from the trehalose productive rate, still exists the misgivings that cause production cost high.Therefore, urgently need to found a kind of new trehalose preparation method, it can improve the productive rate of being produced trehalose by irreducibility starch partial hydrolysate.

The objective of the invention is with low relatively cost with from stablizing the starch of supply, provide Preparation methods of trehalose with high relatively productive rate.

In order to achieve the above object. the inventor has extensively screened the microorganism that can produce a kind of new enzyme, described enzyme can have trehalose structure as a terminal unit and glucose polymerization degree be 3 or higher irreducibility starch partial hydrolysate in discharge trehalose.As a result, we find, and are disclosed in 131/92 as at Japanese patent application No.362, can form the microorganism of non-reduced polysaccharide in the isolating rhizobium from soil, i.e. root nodule bacterium (Rhizobium sp.) M-11 (CCTCC M94031); With as at Japanese patent application No.265, disclosed in 416/93, from soil, can form the microorganism of non-reduced polysaccharide in the isolating genus arthrobacter; Be that Arthrobacter (Arthrobacter sp.) Q36 (CCTCC M94030) produces a kind of new trehalose-release enzyme.We also find, when with non-reduced polysaccharide-when the formation enzyme is used in combination, this new trehalose-release endonuclease capable promotes to form with gratifying high yield the reaction of trehalose, and by allowing this new trehalose-release enzyme and a kind of non-reduced polysaccharide-formation enzyme that reductibility starch partial hydrolysate is worked, and reclaim the reaction mixture that contains relative high purity trehalose, prepare trehalose easily.We have screened the microorganism that produces above-mentioned trehalose-release enzyme widely from known microorganism.The result, we find, the microorganisms that tyrothricin (Brevibacterium) and micrococci (Micrococcus) belong to can form the trehalose-release enzyme of trehalose by (having trehalose structure is 3 or higher as a terminal unit and glucose polymerization degree) non-reduced polysaccharide, similar to trehalose-release enzyme from the microorganism of root nodule bacterium and genus arthrobacter, and finished the present invention.We have also developed the composition that contains by the trehalose of above-mentioned preparation method's preparation, as food, makeup and medicine, and have finished the present invention.

Fig. 1 represents from the wash-out collection of illustrative plates with wash-out trehalose of the present invention-release enzyme and non-reduced polysaccharide-formation enzyme on the post of " DEAE-Toyopearl " gel dress.

Fig. 2 represents the influence of temperature to trehalose-releasing enzyme activity of the present invention of obtaining from root nodule bacterium M-11 microorganism.

Fig. 3 represents the influence of pH to trehalose-releasing enzyme activity of the present invention of obtaining from root nodule bacterium M-11 microorganism.

Fig. 4 represents the influence of temperature to trehalose of the present invention-release enzyme stability of obtaining from root nodule bacterium M-11 microorganism.

Fig. 5 represents the influence of pH to trehalose of the present invention-release enzyme stability of obtaining from root nodule bacterium M-11 microorganism.

Fig. 6 represents the influence of temperature to trehalose-releasing enzyme activity of the present invention of obtaining from Arthrobacter Q36 microorganism.

Fig. 7 represents the influence of pH to trehalose-releasing enzyme activity of the present invention of obtaining from Arthrobacter Q36 microorganism.

Fig. 8 represents the influence of temperature to trehalose of the present invention-release enzyme stability of obtaining from Arthrobacter Q36 microorganism.

Fig. 9 represents the influence of pH to trehalose of the present invention-release enzyme stability of obtaining from Arthrobacter Q36 microorganism.

According to the present invention, a kind of microorganism of rhizobium, namely the discrimination test of rhizobium M-11 draws following result. (edited by Takeji Hasegawk by " Biseibutsu-no-Bunrui-to-Dote i " (classification of microorganism and evaluation); Japan Scinticfic Socities Press, Tokyo, Japan, publish (1985)) the middle test that the side finishes of describing:

A. morphology:

At 27 ℃, when in nutrient agar medium, cultivating, the feature of cell:

Usually the bar-shaped form with 0.6-0.8 * 1.0-1.5 μ m exists;

Single existence, but rare paired or continuous in order form;

There is not polymorphism;

Have motility, do not produce spore and amphitrichous;

Nonacidfast;

Gramstaining: feminine gender;

Pod membrane: feminine gender;

Volutin granules: the positive; With

The accumulation Poly-.

B. cultural characters:

(1) at 27 ℃, the colony characteristics that forms when cultivating on the nutrient agar medium plate:

Form: cultivate after 24 hours, obtain the circular bacterium colony of diameter for about 1.5mm;

The edge: the edge is smooth;

Projection: flat or semisphere;

Gloss: the positive;

Surface: smooth;

Color: form the butteriness translucent colony that does not have the pink colour element.

(2) 27 ℃ of colony characteristicses that form when on agar plate, cultivating with dextrose and tryptone:

Myxoid butteriness translucent colony.

(3) 27 ℃, the colony characteristics that forms when on agar plate, cultivating with yeast extract and N.F,USP MANNITOL

Form: cultivate after 5 days, form the round property bacterium colony of the about 3mm of diameter;

Color: myxoid butteriness translucent colony.

(4) 27 ℃, the colony characteristics that on agar plate, forms with yeast extract, N.F,USP MANNITOL and Congo red cultivation

Neither it is Congo red that pale pink does not absorb basically yet;

(5) 27 ℃, on agar plate, with yeast extract, N.F,USP MANNITOL and 2%NaCl grow together;

(6) 27 ℃, the colony characteristics that forms when on the nutrient agar medium inclined-plane, cultivating

Growth: satisfactory;

Form: wire; With

(7) 27 ℃, in nutrient gelatin during stab culture, can not liquefy gelatin.

C. physiological characteristic

(1) reduction of nitrate: the positive;

(2) denitrification reaction: feminine gender;

(3) methyl red test: feminine gender;

(4) VP-test: feminine gender;

(5) formation of indoles: feminine gender;

(6) formation of hydrogen sulfide: the positive;

(7) starch hydrolysis: feminine gender;

(8) utilization of citric acid: the positive;

(9) inorganic nitrogen-sourced utilization: utilize ammonium salt and nitrate;

(10) formation of pigment: do not have;

(11) urase: the positive;

(12) oxydase: feminine gender;

(13) catalase: the positive;

(14) growth conditions: in the temperature range of the pH of 5.5-9.0 and 4-35 ℃, grow;

(15) oxygen requires: aerobic

(16) utilization of carbon source and acid form

Utilization of carbon source acid forms

D-glucose++

The D-semi-lactosi++

D-fructose++

L-arabinose++

The D-wood sugar++

The L-rhamnosyl++

Maltose+-

Sucrose++

Lactose+-

Trehalose+-

Raffinose++

Seminose+-

Dextrin+-

Galactitol+-

(17) to amino acid whose decarboxylase test

To L-Lys, L-Arg and L-ornithine feminine gender;

(18) amino acid whose utilization

Utilize the L-Sodium Glutamate, L-Sodium L-aspartate, L-Histidine and L-proline(Pro);

(19) DNase: feminine gender

(20) formation of 3-ketone lactose (3-ketolactose): feminine gender; With

(21) the mole %:61% of DNA guanine (G)+cytosine(Cyt) (C).

The feature of known microorganisms among bacterial characteristics and the Bergeys Manual of Systematic Bacteriology (the 1st edition (1984)) is compared.As a result, show with this microorganism identification to be the microorganism of rhizobium.Those of this microorganism and alfalfa Phylloxera bacterium kind are similar, but itself and they can be differentiated, reason is, this microorganism utilizes maltose, lactose and N.F,USP MANNITOL but do not form acid, and it had both produced when acting on reductibility starch partial hydrolysate, can form the enzyme of the non-reduced polysaccharide with trehalose structure, but also produce the key between the trehalose part and all the other glycosyl parts in the non-reduced polysaccharide of specificity hydrolysis, thereby discharge the novel alga sugar-release enzyme of trehalose part.Also do not report the microorganism of these features.

The inventor is this microorganism called after " Rhizobium sp M-11 ", and in 1992,12,24 are deposited in Fermsntation Reasearch Institute, Agency of Industrial Science and Technology, Ibaraki, Japan.The same day, this microbial preservation was accepted, and gave preservation by this mechanism with the preserving number of FERM BP-4130.

Except that the microorganism of above-mentioned evaluation, other bacterial strain of rhizobium and its mutant just are applicable to the present invention as long as can produce trehalose of the present invention-release enzyme.

According to the present invention, a kind of genus arthrobacter microorganism, promptly the qualification test of Arthrobacter Q36 draws following result.Finish test by the description in " Biseibutsu-no-Bunrui-to-Dotei " (classification of microorganism and evaluation) (compiling Japan Scientific Societies Press, Tokyo, Japan (1985) publication by Takeji Hasegawa).The result is as follows:

A. morphology

(1) 27 ℃ of cell characteristic when in nutrient agar medium, cultivating

Be generally the bar-shaped form of 0.5-0.7 * 0.8-1.6 μ m; Single existence;

The performance polymorphism;

Do not have motility, flagellum and do not produce spore;

Nonacidfast;

Gramstaining: the positive;

Pod membrane: feminine gender; With

(2) 27 ℃ of cell characteristics when in EYG agar, cultivating

Present a baseball ring.

B. cultural characteristic

(1) 27 ℃ of colony characteristics that forms when in the nutrient agar medium plate, cultivating

Form: cultivate after 3 days, form the circular bacterium colony of the about 2-2.5mm of diameter;

The edge: the edge is smooth;

Projection: semisphere;

Gloss: the gloss that secretory product is arranged;

Surface: smooth;

Color: translucent and white or faint yellow.

(2) 27 ℃, the colony characteristics that forms when in the nutrient agar medium plate, cultivating

Growth rate: satisfactory; With

Shape: wire

(3) 27 ℃, in containing the agar plate of yeast extract and peptone, the cell characteristic during slant culture

Growth rate: satisfactory;

Form: wire; With

(4) 27 ℃ of cell characteristics during stab culture in meat soup and gelatin

Liquefaction meat soup and gelatin.

C. physiological characteristic:

(1) reduction of nitrate: the positive;

(2) denitrification reaction: feminine gender;

(3) methyl red test: feminine gender;

(4) VP-test: the positive;

(5) formation of indoles: feminine gender;

(6) formation of hydrogen sulfide: the positive;

(7) starch hydrolysis: feminine gender;

(8) cellulose hydrolysis: feminine gender;

(9) utilization of citric acid: the positive;

(10) inorganic nitrogen-sourced utilization;

Utilize ammonium salt and nitrate;

(11) formation of pigment: feminine gender;

(12) urase: the positive;

(13) oxydase: feminine gender;

(14) catalase: the positive;

(15) growth conditions: in the temperature range of the pH of 5-10 and 4-37 ℃, grow;

(16) oxygen demand: aerobic;

(17) utilization of carbon source and acid form

Utilization of carbon source acid forms

D-glucose+-

The D-semi-lactosi+-

D-fructose+-

L-arabinose+-

The D-wood sugar+-

The L-rhamnosyl+-

Maltose+-

Sucrose+-

Lactose+-

Raffinose+-

Seminose+-

Dextrin+-

Galactitol+-

(18) amino acid whose utilization

Utilize the L-Sodium Glutamate, L-Sodium L-aspartate, L-Histidine and L-proline(Pro);

(19) DNase: the positive

(20) formation of 3-ketone lactose (3-ketolatose): feminine gender;

(21) the main diamino acid of cell walls: Methionin; With

(22) the mole %:63% of DNA guanine (G)+cytosine(Cyt) (C).

The feature of known microorganisms among bacterial characteristics and the Bergeys Manual of Systematic BacterioCogy (Vol.2 (1986)) is compared.As a result, showing, is a kind of microorganism of genus arthrobacter with this microorganism identification.This microorganism is characterised in that, it produces a kind of when acting on reductibility starch partial hydrolysate, can form the non-reduced polysaccharide-formation enzyme of (having trehalose structure) non-reduced polysaccharide, and the key between trehalose part and all the other glycosyl parts in the non-reduced polysaccharide of a specific specificity hydrolysis, thereby discharge the trehalose-release enzyme of trehalose part.Unexposed report is crossed the mentioned microorganism of these features.

The inventor is with this microorganism called after " Arthrobacter Q36 ", and in 1993,6,3 are preserved in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, Ibaraki, Japan.The same day, the preservation of microorganism was accepted, and gave preservation by this mechanism with the preserving number of FERMBP-4316.

Except that mentioned microorganism, other bacterial strain of genus arthrobacter and its mutant; As long as can produce trehalose of the present invention-release enzyme, just be applicable to the present invention, the key in described trehalose-release endonuclease capable specificity hydrolysis (having trehalose structure is 3 or higher as terminal unit and glucose polymerization degree) the non-reducing sugar starch partial hydrolysate between trehalose part and all the other glycosyl parts.

Other microorganism is as long as produce enzyme of the present invention, and is just in the present invention available.For example, except that above-mentioned root nodule bacterium M-11 (FERM BP-4130) and Arthrobacter Q36 (FERMBP-4316), up to now, other known microbial strains such as little yellow tyrothricin (Brevibacterium Helvolum) (ATCC11822) and micrococcus roseus (Micrococcus roseus) (ATCC186) also be applicable to very much the present invention.

Any nutritional medium needs only mentioned microorganism and can grow thereon, and can produce trehalose of the present invention-release enzyme, just can be used for the present invention.For example, can arbitrarily use synthetic and natural nutrition substratum.Any carbonaceous material just can be used as carbon source and is used for the present invention as long as it can be utilized by described microorganism.The example of described carbon source is a polysaccharide, as glucose, and fructose, lactose, sucrose, N.F,USP MANNITOL, sorbyl alcohol, molasses and starch partial hydrolysate; With organic acid such as citric acid and succsinic acid.Suitably select the concentration of these carbon sources in nutritional medium.For example, when using starch partial hydrolysate, from the angle of described microorganism growth and breeding, preferred concentration normally 20% or lower, preferably 5% or lower (d.s.b.).Can be used for nitrogenous source of the present invention is, for example, inorganic nitrogen compound is as ammonium salt and nitrate; The material that contains organonitrogen, as urea, corn steep liquor, casein, peptone, yeast extract and beef extract.Can be used for inorganic components of the present invention is, calcium salt for example, magnesium salts, sylvite, sodium salt, phosphoric acid salt and magnesium, zinc, iron, copper, other salt of molybdenum and cobalt.If desired, can use amino acid and VITAMIN.

Will be in the present invention the available microorganism under aerobic condition, usually 4-40 ℃ scope, preferred 20-35 ℃ scope; And the pH of 4-10; The pH of preferred 5-9 cultivates down.The incubation time that is suitable in the present invention is adjusted to begin the required time than described microorganism growth longer, preferably, 10-100 hour.Be not particularly limited the concentration of dissolved oxygen in the nutritional medium (DO), usually, it is gratifying using the DO of 0.5-20ppm scope.By the control ventilation rate, stir nutritional medium, ventilation delivery of supplemental oxygen, and the interior pressure of increase fermenting container can remain on DO concentration in this scope.

After microorganism culturing is finished, reclaim enzyme of the present invention.In cell and acellular supernatant liquor, all find to have active enzyme of the present invention, can be with its recovery and as thick enzyme.The culture of gained intactly can be used as thick enzyme.In the present invention, can use traditional liquid-solid separation method, so that from culture, remove cell.For example, can use, and the precoating filter is with the method for its filtration or the membrane filtration isolated cell by using dull and stereotyped filter or hollow fiber the direct centrifugation method of the culture of gained.Therefore the cell-free filtrate that obtains intactly can be used as enzyme solution or before use that it is concentrated.The available concentration method is among the present invention, for example, uses saltouing of ammonium sulfate, uses the precipitator method of acetone and alcohol and the method for enrichment of use film such as dull and stereotyped filter and hollow fiber.

Cell-free filtrate and its enriched material can be used for traditional fixing means.The example of traditional method is to use the combined techniques of ion-exchanger, uses the covalent linkage and the absorption process of resin and film, and the entrapping method that uses high molecular weight material.Isolated cells from the gained nutrient solution can not needed any further processing or is fixed before use as thick enzyme.For example, cell is mixed with alginate, in calcium chloride solution, drip the mixture of gained, the droplet gelling is become particle, thus fixed cell.Can be with the fixing particle of gained of polyethylene imine based or glutaraldehyde.Enzyme preparation by cell extraction in the present invention, can be used as thick enzyme solution.By from cell, extracting enzyme of the present invention, comprise and use ultrasonic wave, use the Mechanical Crushing of granulated glass sphere and aluminum oxide, France pressure (freneh-press) fragmentation etc., make the extract that obtains through centrifugal or membrane filtration, thereby obtain containing the thick enzyme settled solution of enzyme of the present invention.

Can intactly use the thick enzyme solution that obtains thus or before use, be purified with traditional method.For example, the pure enzyme preparation that presents a band on electrophoresis can prepare by the thick enzyme preparation of dialysis, by concentrating with the thick enzyme culture of ammonium sulfate precipitation and with products therefrom, then, is using " DEAE-Toyopearl ^" chromatography on the anion-exchange column of (a kind of anionite); With " Butyl-Toyopearl ^" hydrophobic chromatography of (a kind of hydrophobic resin); And use " Toyopearl ^HW-55 " gel permeation chromatography (all products all are TosohCorpration, Tokyo, the product of Japan) of (a kind of gel-filtration resin), the solution of purifying dialysis successfully, thus prepare described thick enzyme preparation.

Trehalose of the present invention-release the enzyme that obtains thus has following physics-chem characteristic:

(1) effect

The specificity hydrolysis have trehalose structure be a terminal unit and glucose polymerization degree be 3 or higher non-reduced polysaccharide in, the key between trehalose part and all the other glycosyl parts;

(2) molecular weight:

Go up at sodium laurylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) about 57,000-68,000 dalton;

(3) iso-electric point (pI)

With amphotericeledrolyte be about 3.3-4.6 on the iso-electric point electrophoresis;

(4) optimum temps:

When pH7.0 cultivates 30 minutes, about 35-45 ℃;

(5) best pH:

When cultivating 30 minutes for 40 ℃, about 6.0-7.5;

(6) thermostability

At pH7.0, when cultivating 30 minutes, can stable temperature reach about 30-45 ℃;

(7) pH is latent qualitative

Cultivated 16 hours for 25 ℃, stable in the pH of 5.0-10.0.

Follow these steps to detect the activity of trehalose of the present invention-release enzyme: the 1ml enzyme solution is added in the 50mM phosphoric acid buffer (pH7.0) of 4ml 1.25w/w% maltotriose glycosyl trehalose (another name α-maltotetrose base α-glucoside), mixture was cultivated 30 minutes at 40 ℃.The reaction mixture of gained is added to carries out the Somogyi reaction in the copper solutions,, then, determine reducing power with the Somogyi-Nelson method to stop enzyme reaction.In contrast, 100 ℃ with enzyme solution preheating 10 minutes so that enzyme deactivation, by and above-mentioned similar methods detect.The enzymic activity of the present invention of 1 unit is defined as: when detecting with aforesaid method, per minute increases the enzyme amount of 1 μ mol glucose reducing power.

Any material just can be used as the substrate of enzyme of the present invention as long as it is that a kind of trehalose structure that contains is 3 or higher non-reduced polysaccharide as a terminal unit and glucose polymerization degree.The example of described substrate is glycosyl trehalose such as glucosyl trehalose, the malt-base trehalose, the maltotriose glycosyl trehalose, maltotetrose base trehalose and maltopentaose base trehalose, these sugar are by non-reduced polysaccharide-form enzyme to act on trisaccharide maltose, maltotetrose, maltopentaose, MALTOHAXAOASE and Fructus Hordei Germinatus seven sugar and forming.Except that these substrates, also be applicable to the present invention by (having trehalose structure is 3 or higher as terminal unit and glucose polymerization degree) the relative low reductibility starch partial hydrolysate for preparing with amylase or acid moieties hydrolyzed starch material such as starch, a lotus starch and straight lotus starch.

The described diastatic example of partially hydrolysed starch is (by Pergamon Press at Handbook of Amyloses and Related Enzymes; Tokyo, Japan (1988) publishes) in disclosed α-Dian Fenmei, maltopentaose-formation amylase and MALTOHAXAOASE-formation amylase.These amylase and debranching factor such as amylopectin and isoamylase can be used in combination.

The concentration that is used as the reductibility starch partial hydrolysate of substrate is in the present invention had no particular limits.According to the present invention even can in the solution that contains 0.1% or 50% substrate, carry out enzyme reaction, form trehalose.Also can use the suspended substance that contains soluble substrate in the present invention.The available temperature of reaction can be the temperature of enzyme non-inactivation of the present invention in enzyme reaction of the present invention, promptly is up to about 55 ℃, preferably at 40-55 ℃.Available reaction pH is 5-10pH in enzyme reaction of the present invention, preferably 6-8pH.Only select the used time of enzyme reaction of the present invention according to the enzyme reaction condition, usually, when with 0.1-100 unit/when g substrate d.s.b. uses enzyme of the present invention, approximately need 0.1-100 hour.

About prepare the productive rate of trehalose from the material substrate, particularly, preparing under the situation of trehalose by the irreducibility starch partial hydrolysate with relatively low DE value (promptly have high relatively glucose polymerization degree those), trehalose preparation method of the present invention has the advantage that improves the trehalose productive rate, its productive rate is greater than using Japanese patent application No.362, the productive rate of disclosed preparation method (wherein non-reduced polysaccharide-formation enzyme is used in combination with glucoamylase) gained in 131/92.Preparation method of the present invention, wherein, non-reduced polysaccharide-formation enzyme is used in combination with trehalose-releasing enzyme of the present invention, with about 60% or higher high yield form trehalose, and the preparation method of Japanese patent application, the productive rate that forms trehalose only has an appointment 30%.

Enzyme mechanism of the present invention is as follows: the reductibility starch partial hydrolysate of at first using non-reduced polysaccharide-formation enzyme will have high relatively glucose polymerization degree is transformed into 1 mole of non-reduced polysaccharide with trehalose structure as terminal unit, then, with trehalose of the present invention-release enzyme the non-reduced polysaccharide hydrolysis of gained is become the reductibility starch partial hydrolysate of the glucose polymerization degree low 2 of 1 mole of trehalose and 1 mol ratio starting material reductibility starch partial hydrolysate.For the glucose polymerization degree of new formation is 3 or higher reductibility starch partial hydrolysate, it further can be converted into the non-reduced polysaccharide of a trehalose as terminal unit, then, become 1 mole marine alga sugar and starch partial hydrolysate with trehalose-release enzymic transformation.Therefore, repeat the enzyme reaction of aforementioned non-reduced polysaccharide-formation enzyme and trehalose-release enzyme, can form one or more trehalose molecules and quantity from 1 mole of reductibility starch partial hydrolysate and Duo 2 times than the trehalose molecule that forms, glucose polymerization degree is lower than the irreducibility starch partial hydrolysate of raw starch partial hydrolysate.

In preparation method of the present invention, can use non-reduced polysaccharide-formation enzyme and trehalose of the present invention-release enzyme simultaneously is 3 or higher irreducibility starch partial hydrolysate to act on glucose polymerization degree, or uses these two kinds of enzymes to act on irreducibility starch partial hydrolysate continuously with this order.In order further to improve the trehalose productive rate, the reaction mixture that can make gained is further through the effect of glucoamylase.

Filter and concentrate the reaction mixture that obtains thus to remove insoluble material with ordinary method, with the solution decolouring of gac with gained, the ion-exchanger desalination with H-and OH-form is being condensed into the pulpous state product then.Can optionally syrupy product be dried to powder-like product.

If desired, use one or more methods, as ion-exchange chromatography, carry out purifying with the column chromatography fractional separation of gac or silica gel; With organic solvent such as ethanol and acetone separation method; Alkaline purification method with degrading and removing remaining reduction polysaccharide is easy to powder-like product is processed into highly purified trehalose product.

If desired, according to the present invention, can contain the polysaccharide product of trehalose with amylase, α-Dian Fenmei, glucoamylase, alpha-glucosidase and/or trehalase hydrolysis, or make its polysaccharide-shift reaction to control its sugariness and reducing power and to reduce its viscosity through using cyclomaltodextrin glucanotransferase (CGTASE) and/or glycosyltransferase.In addition, can optionally make polysaccharide product hydrogenation being converted into sugar alcohol, thereby reduce its reducing power.Can from the product of gained, remove glucose with the high stream part of preparation content of trehalose as ion-exchange chromatography with above-mentioned purification process.Can be easy to the stream part purifying that will obtain thus and be condensed into syrupy product, and, if desired, can further syrupy product be condensed into supersaturation dope and crystallization to obtain aqueous crystalline trehalose or anhydrous crystalline trehalose.

Available ion-exchange chromatography technology comprises in the present invention, and is for example disclosed in 799/83 and 72,598/83 at the open No.23 of Japanese Patent, uses the method for strong-acid cation-exchange resin.Use these technology, can remove at an easy rate in thick trehalose product contained with companion's polysaccharide to obtain the product of high content of trehalose.In this case, can arbitrarily use fixed bed, moving-bed and half moving method.

In order to prepare aqueous crystalline trehalose, the aqueous trehalose of about 65-90% is placed in the crystallizer, in 95 ℃ or lower, in preferred 10-90 ℃ the scope, under the situation that has the 0.1-20% crystal seed, cool off gradually while stirring to obtain containing the massecuite of crystal shaped trehalose.Can use traditional method in the present invention, as partition method, piece porphyrization, fluidized-bed or granulation and spray-drying process so that prepare aqueous crystalline trehalose from massecuite or crystalline polysaccharide.

When separating, make massecuite through basket centrifugal so that from mother liquor, separate aqueous crystalline trehalose, if desired, contain the preparation of crystal shaped trehalose to promote high purity with the aqueous crystalline trehalose of a small amount of cold water hydro-peening.During spraying drying, by penetrate massecuite from a high-pressure pump nozzle with about 20-60% degree of crystallinity of 60-85% concentration (with dry weight basis) (with dry weight basis), with the about 60-100 ℃ of hot-air dry gains that do not melt the gained crystal powder, Bian Xiangqi blows about 30-60 ℃ warm air, the about 1-20 of powder hour of gained stirred on the limit, and being easy to preparation does not have or do not have substantially hygroscopic crystallization polysaccharide.When the piece porphyrization, to make moisture content be about 10-25% and degree of crystallinity puts youngster hour or 3 days so that make whole inclusion crystallizations and be solidified into piece for the syrup of about 10-60% (with dry weight basis), pulverize or cut the piece of gained, and with the gains drying, thereby preparation does not have or does not have substantially hygroscopic crystallization polysaccharide at an easy rate.Although can be by the aqueous crystalline trehalose of drying so that it is become anhydrous form, prepare anhydrous crystalline trehalose, but generally be by providing moisture content to be lower than 10% high content of trehalose solution, this solution is placed in the crystallizer, under agitation condition, in 50-160 ℃, preferred 80-140 ℃ scope, exist and keep solution under the situation of crystal seed to obtain containing the massecuite of anhydrous crystalline trehalose, with its crystallization, under dry and relative high temperature, usefulness traditional method such as piece comminuting method, fluid bed granulation method and spraying drying are pulverized the anhydrous crystalline trehalose of gained, thus preparation anhydrous crystalline trehalose.

The trehalose of the present invention that obtains thus is stable and does not have reducing power basically, and can with other material, particularly amino acid and contain amino acid whose material such as oligopeptides and protein mixes causes the rotten of tedious coking and smell and described material and needn't worry.Trehalose itself has gratifying high quality and sugariness.Because trehalose is easy to by the trehalase hydrolysis, therefore, when oral, can be assimilated by live body, absorb and utilization.In addition, trehalose is gone up the microbial fermentation that can not be brought out carious tooth substantially, and therefore, this can make it be used as sweeting agent and not bring out carious tooth basically.

Trehalose of the present invention can be made medicament, as, be used to transfuse blood and the nutrition medicament of gavage, can optionally it be applied to live body and be easy to and needn't be worried to cause toxicity and side effect by live body metabolism and utilization.Therefore, help these products as the energy supplement medicament that is used for live body.

Trehalose is stable sweeting agent, particularly, when with tackiness agent such as amylopectin, when hydroxyethylamyle or polyvinylpyrrolidone were used in combination, the crystal trehalose can arbitrarily be used as the sweet tablet agent of tablet.In addition, trehalose has numerous characteristics, as the ability of control osmotic pressure, give filler ability, give the glossy ability, keep the ability of humidity, the ability of giving viscosity, basic nonfermented, prevent the ability that gelation starch is degenerated and prevent other polysaccharide crystalline ability.

Therefore, can optionally trehalose of the present invention and the polysaccharide composition that contains trehalose of the present invention and polysaccharide be used for various compositions such as food, cigarette, tobacco, feed, makeup and medicine as sweeting agent, flavor improvement agent, quality booster, stablizer and weighting agent.

Trehalose of the present invention and the polysaccharide composition that contains trehalose of the present invention and polysaccharide intactly can be used as sweet taste condiment.If desired, can with one or more other sweeting agents of capacity, as atomizing syrup, glucose, fructose, maltose, sucrose, isomerose, honey, maple sugar, erythrose, sorbyl alcohol, N.F,USP MANNITOL, lactitol, dihydrocharcone, stevioside, alpha-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl L-phenylalanine methyl ester, asccharin, glycine and L-Ala; And/or weighting agent such as dextrin, starch and lactose use together.

Can intactly use the powder that contains trehalose of the present invention and polysaccharide or the trehalose of the present invention and the polysaccharide composition of crystalline form, if desired, before the use, it can be mixed with vehicle, weighting agent, thinner and tackiness agent, and make particle, ball, stub, flat tablet of (plate), lozenge and tablet.

The trehalose of the present invention and the polysaccharide composition that contain trehalose of the present invention and polysaccharide can be compatible well with other material that tart flavour, saline taste, bitter taste, astringent taste and delicious food are arranged, and high relatively sour tolerance and heat resistance are arranged.Therefore, general, can use it for food well, as sweeting agent, flavor improvement agent and quality booster.

Trehalose of the present invention and the polysaccharide composition that contains trehalose of the present invention and polysaccharide can be used for seasonings, as amino acid, peptide, soy sauce, the soy sauce of powdered, " miso ", " fummatsu-miso " (miso of powdered), " moromi " (a kind of refining Japanese sake), " hishio " (a kind of refining soy sauce), " fwrikake " (fish meal of a kind of seasoning), mayonnaise, seasonings, vinegar, " sanbai-zu " (sugar, the soy sauce of sauce oil and vinegar), " funmatsu-sushi-su " (vinegar that is used for the powdered of sashi), " chuka-no-moto " (the instant mixture that is used for Chinese dishes), " tentsuga " (being used for the soy sauce that Japanese fattiness is fried in shallow oil food), " mentsuyu " (soy sauce that is used for Japanese vermiui), soy sauce, catsup, " yakiniku-no-tare " (soy sauce that is used for Japanese barbecue), curry roux, instant stewed mixture, instant soup mixture, " dashi-no-moto " (instant soup stock mixture), the nucleic acid seasonings, mixed spices, " mirin " (sweet pure mellow wine), " shin-mirin " (synthetic mirin) sugar and coffee candy.

Can will contain the trehalose of the present invention of trehalose of the present invention and polysaccharide and the sweet taste that polysaccharide composition arbitrarily is used to increase following food: " wagashi " (Japanese cake) is as " sendei " (rice cracker), " arare-mochi " (rice cake sugar cube), " okosh " (millet-and-the rice cake), " mochi " (rice dough), " manju " (dessert that has beans sauce), " uiro " (sweet big rice cracker peptone), " an " (beans sauce), " yokan " (hesperidium peptone that bean or pea do), " mizu-yokan " (soft adzuki beans fruit peptone), " kingyoku " (a kind of yokan), the fruit peptone, paode Castella and " amedama " (Japanese caramels); Sweet food such as dessert, biscuit, cheese, cooky, group, pudding, butter, custard, butter puff, the sweet cake of milk egg grid, spongecake, a deep-fried dough cake circle, chocolate, chewing gum, caramel and candy; Frozen confection such as ice cream and sweet taste powdered juice, syrup is as " kajitsu-no-syrup-zuke " (fruit of preservation) and " korimitsu " (syrup that is used for water ice); Finished fruits and vegetables such as jam, marmalade, " syrup-zuke " (fruit salts down) and " toka " (preserved fruit); Salted vegetables and pickling food are as " fukujin-zuke " (Radix Dauci Sativae salts down), " bettare-zuke " (a kind of whole fresh radish that salts down), " senmai-zuke " (a kind of flaky bright radish that salts down) and " rakkyo-zuke " (that pickles is verdant); The premixture of salted vegetables and pickling food is as " takuan-zuke-no-moto " (premixture of pickled radish) and " hakusai-zuke-no-moto " (premixture of fresh white rape salted vegetables); By product such as ham and sausage; Fish flesh prod such as fish ham, the fish intestines, " kamaboko " (a kind of Steamed fish group), " chikuwa " (a kind of fishball) and " tenpura " (a kind of Japanese fattiness fried fish group), " chinmi " (appetizer) is as " uni-no-shiokara " (the salt intestines of sea urchin), " ika-no-shiokara " (salt intestines of cuttlefish), " su-konbu " (finished tangle), " sakic surmun " (dried cuttlefish bar) and " fugu-no-mirin-boshi " (filefish of the mirin seasoning of doing); " tsukudani " (food that boils in soy sauce) is as laver, edible wild plant, and dried cuttlefish, fish and Shellfish, daily dish is as " nimane " (fresh kidney beans of culinary art), tomato salad and " konbu-maki " (tangle roll); Dairy products; Canned and bottled product such as meat, the flesh of fish, fruits and vegetables; Contain alcoholic beverage such as synthetic pure mellow wine, grape wine and spirits; Soft drink such as coffee, tea, cocoa, fruit juice, soda pop, yogurt beverage and lactobacteria-containing beverage; Instant food such as instant pudding mixture, instant hot pastry mixt and " sokuseki-shirueo " (instant mixture of adzuki beans soup and rice cake) and instant soup mixture: and beverage, as infant food, treatment food, nutritious prod beverage; And improve the taste and the quality of above-mentioned food.

Also can the trehalose of the present invention of trehalose of the present invention and polysaccharide and feed and the pet food that polysaccharide composition is used for animal such as domestic animal, poultry, honeybee, silkworm and fish will be contained, to improve its taste hobby.Can in group and liquid form and other products, trehalose and polysaccharide composition be used as sweeting agent, flavor improvement agent, quality booster and stablizer.Oils,glyceridic,cod-liver, cachou, oral cooling agent, gargle, makeup and medicine as tobacco, cigarette, toothpaste and toothpaste powder, lipstick, kermes, lipstick, medicine for oral administration, tablet, lozenge, drops form.

Can be used for biologically active substance to its effective constituent and active forfeiture sensitivity and the heath food of matters of containing biological activities and pharmaceutical composition as quality booster and stablizer with containing the trehalose of the present invention of trehalose of the present invention and polysaccharide and polysaccharide composition.The example of above-mentioned biologically active substance be lymphokine such as α-, β-, and gamma-interferon, α-Zhong Liuhuaisiyinzi (TNF-α), β-tumour necrosis factor (TNF-β), macrophage migration inhibitory factor, G CFS, transfer factor and interleukin II (IL-2); Hormone such as Regular Insulin, tethelin, lactotropin, erythropoietin and follicular stimulating hormone; Biological products such as BCG vaccine, Japanese encephalitis vaccine, Measles Vaccine, the Poliomyelitis Vaccine of living, antismallpox vaccine, Toxoid,tetanus, Trimeresurus toxinicide and human normal immunoglobulin, microbiotic such as penicillin, erythromycin, paraxin, tsiklomitsin, Streptomycin sulphate and sulphuric acid kanamycin; VITAMIN such as thiamines, riboflavin, L-xitix, Oils,glyceridic,cod-liver, carotenoid, ergosterol and tocopherol; Enzyme such as lipase, elastoser, urea kinases, proteolytic enzyme, beta-amylase, isoamylase, poly-glucose enzyme and Sumylact L; Extract such as Radix Ginseng extract, dense tortoise extract, chlorella extract, Aloe extract light propolis extract; The microorganism that can survive, as virus, milk-acid bacteria and yeast; Other biologically active substance such as royal jelly.The trehalose of the present invention and the polysaccharide composition that contain trehalose of the present invention and polysaccharide by use, can be easy to above-mentioned biologically active substance is processed into heath food and the pharmaceutical composition with gratifying high stability and quality, and needn't worry forfeiture or its effective constituent of inactivation and activity.

By mentioned above, to contain the trehalose of the present invention of trehalose of the present invention and polysaccharide and method that polysaccharide composition is mixed people's above-mentioned substance and comprise traditional method, for example, mix, mediate, dissolve, melt, soak, permeate, spray, apply, coat, spray, injection, crystallization and curing.Usually with 0.1% or higher, preferred 1% or the amount of higher (with dry weight basis), the trehalose of the present invention and the polysaccharide composition that will contain trehalose of the present invention and polysaccharide are mixed in the people above-mentioned material and composition.

The following example illustrates from root nodule bacterium M-11 and Arthrobacter Q36 microorganism, and produces and purifying trehalose of the present invention-release enzyme in the hitherto known microorganism.

Embodiment 1

Produce trehalose-release sugar with root nodule bacterium M-11

To contain 2.0w/v% " PINE-DEX#4 " (Matstani ChemicalZnd.Co.Ltd.Kyoto, the starch product of Japan), 0.5w/v% peptone, the 0.1w/v% yeast extract, the 0.1w/v% Sodium phosphate dibasic, the liquid nutrient media of 0.1w/v% potassium hydrogen phosphate and water is transferred pH to 7.0.The liquid nutrient media of about 100ml equal portions is placed in the 500mlErlenmeyer flask, with sterilization, cooling was with the inoculation of root nodule bacterium M-11 (CCTCC M 94031) inoculum 120 ℃ of autoclavings 20 minutes, and under the agitation condition of 130rpm, cultivated 24 hours in 27 ℃.Collect the culture of gained and be used as inoculum.

The prepared fresh thing of about 20 liters of described liquid nutrient media used in above-mentioned cultivation is placed in 30 liters the fermentor tank, sterilization is cooled to 27 ℃, inoculum inoculation with 1w/v%, and at 27 ℃, pH6.0-8.0 cultivated about 72 hours under the condition of stirring and ventilation.

In culture, the non-reduced polysaccharide-formation enzyme of accumulation and the activity of trehalose of the present invention-release enzyme are respectively about 1.5 units/ml and about 2 units/ml.The part culture is centrifugal, be separated into cell and supernatant liquor, and cell suspension is obtained the equal volume part in 50mM phosphoric acid buffer (pH7.0), then detect the enzymic activity of cell suspending liquid and supernatant liquor.In cell suspending liquid, the activity of non-reduced polysaccharide-formation enzyme and trehalose of the present invention-release enzyme is respectively about 0.6 unit/ml and about 0.8 unit/ml, and supernatant liquor contains non-reduced polysaccharide-formation enzyme of 0.9 unit/ml and trehalose of the present invention-release enzyme of about 1.2 units/ml.

It is as follows that non-reduced polysaccharide forms the detection of enzyme: the 1ml enzyme solution is added in the 50mM phosphate buffer solution (pH7.0) of 4ml 1.25w/v% maltopentaose, this mixing solutions was cultivated 10 minutes in 40 ℃, and ended this enzyme reaction in 10 minutes 100 ℃ of heating.With a damping fluid this gained reaction mixture accurately is diluted to 10 times, and measures its reducing power with Somogyi-Nelson ' S method.In contrast, a kind of enzyme solution 100 ℃ of preheatings 10 minutes, makes this enzyme deactivation, detects with as above method again.The activity unit that this non-reduced polysaccharide forms enzyme is defined as when using aforesaid method to detect, and per minute is eliminated the enzyme amount of the reducing power of 1 micromolar maltopentaose.

Embodiment 2

The purifying of enzyme

The about 18L nutrient solution that is obtained by embodiment 1 method is handled, with " MINI-RABO " (a kind of ultra-high voltage cell rupture instrument, by Tokyo Dainippcn Pharmaceutical Co., Ltd. sells) ruptured cell.Gained suspension is in 10, obtains about 16L supernatant liquor under the 000rpm in centrifugal 30 minutes.Be added to Tai-Ace S 150 in the supernatant liquor and make its dissolving obtain 0.2 saturation ratio, and the solution that obtains was placed 1 hour in 4 ℃,, obtained supernatant liquor under the 000rpm in centrifugal 30 minutes 10.

Tai-Ace S 150 is added in the supernatant liquor of gained and makes its dissolving obtain 0.6 saturation ratio, and with gained solution in 10, under the 000rpm centrifugal 30 minutes, obtain precipitation again and it be dissolved in the 10mM phosphoric acid buffer (pH7.0).The solution that so obtains was dialysed 24 hours in the identical phosphoric acid buffer of prepared fresh, in 10, removed insolubles under the 000rpm in centrifugal 30 minutes.The 360ml dialyzate of gained is divided into two parts, and every part of post that loads in order to 300ml " DEAE-Toyopearl " (by a kind of ion-exchanger of Tokyo Tosoh Corporation listing) respectively carries out column chromatography then.

The non-reduced polysaccharide of target forms enzyme and trehalose-releasing enzyme is adsorbed on the ion-exchanger, with they being eluted from post respectively with the additional phosphoric acid buffer of the salt of different salt concn of prepared fresh.Fig. 1 has represented the wash-out collection of illustrative plates of this post or column chromatography.When salt concn was about 0.2M, non-reduced polysaccharide formed enzyme and is eluted from post, and when salt concn was about 0.3M, trehalose-releasing enzyme was eluted from post.It is also refining to merge stream part of containing any target enzyme respectively.

The stream part of containing non-reduced polysaccharide formation enzyme that merges is dialysed in the same phosphoric acid buffer that replenishes with 2M ammonium sulfate of prepared fresh.Dialyzate was removed insolubles in centrifugal 30 minutes in 10 under the 000rpm, and the gained supernatant liquor is in order to 300ml " Butyl-Toyopearl ^650 " post of (a kind of hydrophobic gel is by Tokyo Tosoh Corporation listing) filling carries out the hydrophobicity column chromatography.The enzyme that will be adsorbed in gel with the linear gradient damping fluid from 2M to the 0M scope elutes from post, reclaims the stream part with enzymic activity.Gained is flowed part in order to 300ml " Toyopearl ^HW-55 " post of (a kind of gel chromatography resin is by Japan, Tokyo Tosoh Corporation listing) filling carries out gel permeation chromatography, reclaims the stream part with enzymic activity.

To " DEAE-Toyopearl ^" the active merging stream of the tool trehalose-releasing enzyme of post wash-out part; handle by adopting the purification step that forms in the enzyme preparation similar in appearance to non-reduced polysaccharide; this kind method is promptly dialysed in the damping fluid that contains 2M ammonium sulfate, carries out hydrophobicity column chromatography and gel permeation chromatography subsequently.

Table 1 has shown total enzyme activity, specific activity and the yield of non-reduced polysaccharide formation enzyme in each purification step, and table 2 has shown that trehalose sugar discharges the corresponding contents of enzyme.

Table 1

Purification step	Total enzyme *Active (unit)	Specific activity (unit/mg albumen)	Yield (%)
				The raw material nutrient solution	28,500	-	100
Supernatant liquor behind the cell rupture	22,900	0.12	80
				Dialysis solution after saltouing	21,100	0.43	74
The ion exchange column elutriant	15,200	6.2	53
				Hydrophobicity post elutriant	7,950	101	28
The gel-filtration column elutriant	5,980	197	21

Annotate: the non-reduced polysaccharide of " * " symbolic representation forms enzyme.

Table 2

Purification step	Total enzyme **Active (unit)	Specific activity (unit/mg albumen)	Yield (%)
				The raw material nutrient solution	37,400	-	100
Supernatant liquor behind the cell rupture	31,500	0.17	84
				Dialysis solution after saltouing	29,200	0.60	78
The ion exchange column elutriant	25,400	5.3	68
				Hydrophobicity post elutriant	18,700	98.5	50
The gel-filtration column elutriant	11,600	240	31

Annotate: " * * " symbolic representation trehalose-releasing enzyme.

The enzyme preparation that purifying is crossed (elutriant that is obtained by gel-filtration column in table 1 and 2) uses 7.5% polyacrylamide, measures its purity on electrophoresis.As a result, observe every kind of enzyme preparation and all be single albumen band, this shows that it is the electrophoresis single product with higher degree.

Embodiment 3

The character of trehalose-releasing enzyme

Use contains the polyacrylamide gel of 10% sodium laurylsulfonate, to carry out electrophoresis by the partially purified trehalose-releasing enzyme goods that method among the embodiment 2 obtains, and compare by sign albumen with it and Tokyo Japan Bio-Racl Laboratories listing, record its molecular weight and be about 57,000-68.000 dalton.

Use contains 2v/v% " AMPHOLINE " (a kind of amphotericeledrolyte of Sweden Uppsala Pharmacia LKB Biotechnology AB listing), and the purifying enzyme goods of other part are carried out iso-electric point electrophoresis (isoeletrohopresis).The gained gel is cut into slices, measure their pH value then, the pI that obtains this enzyme is about 3.3-4.3.

According to the analysis of enzymic activity being studied temperature and pH influence to this enzymic activity.Fig. 2 (Temperature Influence) and Fig. 3 (influence of pH) have shown these results respectively.

When pH7.0 cultivates 30 minutes, the optimal temperature of this enzyme is about 45 ℃, and when cultivating 30 minutes for 40 ℃, the appropriate pH of this enzyme is about 6.0-7.5.

By will under differing temps, cultivating 60 minutes, cool off the damping fluid in the test tube and measure enzymic activity residual in every portion of damping fluid with cold water then, thereby measure the thermostability of this enzyme at the enzyme in the 50mM phosphoric acid buffer (pH7.0).By cultivating 16 hours down in 25 ℃ at the enzyme in the 50mM of the different pH phosphoric acid buffer, adjust these damping fluids to pH7.0, analyze the pH stability that enzymic activity residual in every portion of damping fluid is measured this enzyme then.Figure 4 and 5 have shown the heat and the pH stability result of this enzyme respectively.This enzyme is stable in temperature under up to about 40 ℃ and the about 5-10 of pH.

Embodiment 4

From end be trehalose structure and glucose polymerization degree be 3 or higher non-reduced polysaccharide prepare trehalose

Press Japanese patent application No.362,131/92 described method prepares non-reduced polysaccharide, and this sugar is as substrate, and end and glucose polymerization degree that it has trehalose structure are 3 or higher.The enzyme preparation that adds the purifying that the people obtains by embodiment 2 methods in the aqueous solution that contains 20% trisaccharide maltose, maltotetrose, maltopentaose, MALTOHAXAOASE or Fructus Hordei Germinatus seven sugar as substrate, 2 units/g substrate (d.s.b.) carried out enzyme reaction 48 hours with the gained mixture under 40 ℃ and pH7.0.With this reaction mixture heating, make residual enzyme deactivation, filtration, decolouring, desalination also concentrate and obtain a kind of dense sugar soln, use then that " XT-1016 (Na type; poly-and degree is a kind of ion-exchanger of 4%; by Tokyo Tokyo Oranic Chemical Industries, the Ltd. listing) carries out column chromatography to this solution.In column chromatography, with internal diameter is 2.0cm, long is the stainless steel column filling ion-exchanger of 3 jacket layers of 1m, then with these post concatenated in order and be heated to 55 ℃ of post Nei Wenda, 5v/v% priming (to amount of resin) is gone up sample, when temperature remains on 55 ℃, and SV (volumetric velocity) be 0.13 time with 55 ℃ of hot water elutions (feed), obtain highly purified non-reduced polysaccharide, end and glucose polymerization degree that this sugar has trehalose structure are 3 or higher.In the goods that obtain, the purity that glucosyl trehalose goods contain the glucosyl trehalose is 97.6% (d.s.b.), and maltose trehalose, maltotriose glycosyl trehalose, maltotetrose base trehalose and the purity of maltopentaose base trehalose in their high purity goods are respectively 98.6%, 99.6%, 98.3% and 98.1% (d.s.b.).

Preparation contains the above-mentioned 5 kinds of non-reduced polysaccharide goods of 20% (d.s.b.), be a kind of aqueous solution in the glycosyl trehalose goods, trehalose-releasing enzyme with the purifying that obtains in this solution and the experimental example 2 mixes then, 2 units/g substrate (d.s.b.) carry out enzyme reaction 48 hour in 40 ℃ with pH7.0 with gained solution then.Analyze its composition the every kind of reaction mixture desalination that obtains and through high pressure liquid chromatography (HPLC) (using Tokyo Wako Pure Chemical Industries Ltd. " WAKOBEADSWB-T-330 post ").In contrast, the same trehalose-releasing enzyme of prepared fresh is acted on the maltose oligosaccharides, for example trisaccharide maltose, maltotetrose, maltopentaose, MALTOHAXAOASE and Fructus Hordei Germinatus seven sugar, and analyze the composition of the every kind of reaction mixture that obtains through HPLC.Table 3 shows 3 these results.

Table 3

Substrate	Product	The last elution time of HPLC (min)	Percentage (%)
				The glucosyl trehalose	Trehalose	27.4	17.5
Glucose	33.8	6.5
			The glucosyl trehalose		23.3	76.0
The malt-base trehalose	Trehalose	27.4					44.3
			Maltose	28.7	44.4
	The malt-base trehalose	21.6				11.3
			The maltotriose glycosyl trehalose	Trehalose	27.4		39.5
Trisaccharide maltose	25.9	60.0
				The maltotriose glycosyl trehalose	19.7	0.5
Maltotetrose base trehalose	Trehalose	27.4					34.2
			Maltotetrose	24.1	65.5
	Maltotetrose base trehalose	18.7				0.3

Table 3 (continuing)

Substrate	Product	The last elution time of HPLC (min)	Percentage (%)
				Maltopentaose base trehalose	Trehalose	27.4	29.1
Maltopentaose	22.6	70.6
			Maltopentaose base trehalose		17.8	0.3
Trisaccharide maltose	Trisaccharide maltose	25.9					100
			Maltotetrose	Maltotetrose	24.1	100
Maltopentaose	Maltopentaose	22.6					100
			MALTOHAXAOASE	MALTOHAXAOASE	21.8	100
Fructus Hordei Germinatus seven sugar	Fructus Hordei Germinatus seven sugar	21.0					100

Result in the table 3 shows:

1, according to trehalose-releasing enzyme of the present invention hydrolysis specifically to have trehalose structure end and glucose polymerization degree be 3 or be higher than trehalose part in 3 the non-reduced polysaccharide and the key between the glycosyl part, form trehalose and a kind of glucose polymerization degree and be 1 or greater than 1 non-reducing sugar; And

2, the maltose oligosaccharides is not by the hydrolysis of trehalose-releasing enzyme of the present invention institute.

These results confirm that trehalose-releasing enzyme of the present invention is a kind of new enzyme, it has hydrolysis specifically, and to have trehalose structure end and glucose polymerization degree be 3 or greater than the key between part of the trehalose in 3 the non-reduced polysaccharide and glycosyl part, thereby trehalose is discharged from non-reduced polysaccharide.

Be the trehalose in the various reaction mixtures of purifying, with this reaction mixture in order to " XT-1016 (Na ⁺Type) " post of (a kind of alkali metal type strong-acid cation-exchange resin, by Tokyo Tokyo Organic Chemical Industries, Ltd. listing) filling carries out column chromatography, reclaims afterwards to contain 97% or stream part of higher trehalose.These are flowed part merging and are concentrated into about 65% concentration, this enriched material is placed its crystallization of 2 angels in 25 ℃ go out hydrous crystalline trehalose, isolate then that the dry purity that obtains is 99% or higher high purity trehalose goods (d.s.b.) under these crystallizations and the vacuum.With glucosyl trehalose, malt-base trehalose, maltotriose glycosyl trehalose, maltotetrose base trehalose and maltopentaose base trehalose is that the yield that substrate prepares trehalose is respectively 9.5%, 14.9%, 16.0%, 18.5% and 17.7% (d.s.b.).The X-diffracting spectrum of fusing point, heat of fusion, specific rotation, infrared absorption spectrum, powder of the trehalose sample of buying on high purity trehalose goods and the market (making standard substance) and the easiness of trehalase sample (from U.S. St.Lauise, the Sigma Chemical Co. buys) hydrolysis that from the pig kidney, obtained have been studied.The result, the fusing point that various trehalose goods show is that 97.0 ± 0.5 ℃, heat of fusion are that 57.8 ± 1.2KJ/mol and specific rotation are+182 ± 1.1 °, the numerical value of these numerical value and standard trehalose sample is very identical, also has the infrared absorption spectrum and the powder X-ray-diffractogram of trehalose also very identical with standard trehalose sample.Similar to the trehalose sample of standard, these trehalose goods are decomposed into glucose monomer.

These results are unequivocally established, and are acted on that to have the disconnected and glucose polymerization degree in trehalose structure end be 3 or are trehaloses greater than the non-reduced polysaccharide that 3 non-reduced polysaccharide forms by trehalose-releasing enzyme of the present invention.

Embodiment 5

Prepare trehalose from the starch partial hydrolysate of irreducibility

Make the suspension gelatinization that contains 5% waxy corn starch through heating, transfer to pH4.5, be heated to 50 ℃, with a kind of isoamylase (isoamylase) sample (by Japanese Okayama, Hayashibara Biochemical Laboraloies Inc. listing) with 4, the amount of 000 unit/g starch (d.s.b.) is mixed, and makes this enzyme reaction continue 20 hours.This reaction mixture is chilled to 60 ℃, then in order to 750ml " Toyopearl in 120 ℃ of autoclavings 10 minutes ^The 50s gel " post of (by Tokyo Tosoh Corporation listing) filling carries out gel permeation chromatography, and obtaining glucose polymerization degree is the reductibility starch partial hydrolysate of 35-10.

To be that 3 trisaccharide maltose is dissolved in 10mM phosphoric acid buffer (pH7.0) and makes 1% solution as a kind of reductibility starch partial hydrolysate that so obtains of substrate or glucose polymerization degree, the trehalose-releasing enzyme (with method preparation among the embodiment 2) that then this solution and the non-reduced polysaccharide of purifying is formed enzyme and purifying mixes with the amount of 4 units/g substrate (d.s.b.), and carries out enzyme reaction 24 hours in 40 ℃.After treating enzyme reaction fully, the every kind of reaction mixture that obtains is carried out desalination and analyzes its composition through HPLC.

Remaining every kind of reaction mixture is heated to 50 ℃, transfers pH to 4.5,, carry out enzyme reaction in 24 hours then with the amount and glucoamylase sample (by Tokyo Seikagakukogyo Co., the Ltd. listing) mixing of 50 units/g substrate (d.s.b.).To top similar, the partial reaction mixture that obtains is carried out desalination and analyzes its composition with HPLC.Table 4 has shown this result.

Table 4

The glucose polymerization degree of reductibility starch partial hydrolysate	Reaction product	Form (%)
				A	B
		34.1	Trehalose glucose reduction oligosaccharides glycosyl trehalose			80.8 0.2 14.4 4.6	83.5 16.5 0.0 0.0
26.2	Trehalose glucose reduction oligosaccharides glycosyl trehalose			79.7 0.2 15.3 4.8	82.5 17.5 0.0 0.0
		18.1	Trehalose glucose reduction oligosaccharides glycosyl trehalose			77.7 0.2 17.0 5.1	80.7 19.3 0.0 0.0
15.2	Trehalose glucose reduction oligosaccharides glycosyl trehalose			75.0 0.3 18.6 6.1	78.5 21.5 0.0 0.0
		10.0	Trehalose glucose reduction oligosaccharides glycosyl trehalose			66.1 0.3 27.6 7.7	70.1 29.9 0.0 0.0
3 (trisaccharide maltoses)	Trehalose glucose trisaccharide maltose glycosyl trehalose			4.2 2.1 65.0 28.7	20.8 79.2 0.0 0.0

Annotate: the composition after the enzyme reaction of a kind of non-reduced polysaccharide formation enzyme of symbol " * " expression and trehalose-releasing enzyme of the present invention, the composition after the enzyme reaction of symbol " * * " expression glucoamylase." glycosyl trehalose " vocabulary shows that having trehalose structure end and glucose polymerization degree is 3 or greater than 3 non-reduced polysaccharide in the table.

As shown in table 4, using glucose polymerization degree is under 3 the situation of trisaccharide maltose as substrate, trehalose yield after non-reduced polysaccharide forms enzyme and this trehalose-releasing enzyme generation enzyme reaction is lower, promptly 4.2%, but under the situation of starch partial hydrolysate of using glucose polymerization degree as 10-34.1 as substrate, the yield of this trehalose is than higher, i.e. 66.1-80.0%.And the glucose polymerization degree of finding reductibility starch partial hydrolysate is high more, and the purity of gained trehalose is also high more.Also find by making glucoamylase act on the reaction mixture that makes by two kinds of enzymic hydrolysis reductibility starch partial hydrolysates, with association to have trehalose end and glucose polymerization degree be 3 or become trehalose and glucose molecule greater than 3 non-reduced polysaccharide hydrolysis, can improve the concentration of gained trehalose.

Embodiment 6

Maillard reaction

To contain 1% glycine, (purity is 99.5% to 10% high purity trehalose goods, d.s.b., method by embodiment 4 obtains) and the solution of 50mM phosphoric acid buffer (pH7.0) kept 90 minutes in 100 ℃, cool off this gained solution then and in the 1-cm groove, measure its absorption at 480nm wavelength place.In contrast, glucose and maltose are carried out and top similar processing, measure of the absorption of gained solution again in 480nm wavelength place.Table 5 has shown this result.

Table 5

Sugar prod	Color intensity (480nm)
		Trehalose (the present invention)	0.006
Glucose (contrast)	1.671
		Maltose (contrast)	0.926

The result of table 5 discloses trehalose goods of the present invention significantly and induces through Maillard reaction and only show more shallow color, and promptly its color intensity only is about 0.4-0.6% of glucose and maltose.

This result shows that trehalose of the present invention is gone up substantially and do not participated in Maillard reaction.Therefore, trehalose of the present invention is a kind of when them and amino-acid mixed fashionable, less to amino acid whose destruction menace a kind of sugar.

Embodiment 7

Utilize test in the body

According to Atsuji etc. at " Rinsho-Eiyo ", Vol.41, No.2, pp200-208 (1972) goes up reported method, (purity is 99.5% to the highly purified trehalose goods of 30g that method by embodiment 4 is obtained, d.s.b.) aqueous solution of making 20w/v% soluble in water gives this solution six healthy male volunteers (26,27,28,29,30 and 31 years old) oral then.The timed interval is gathered these volunteers' blood sample according to plan. analyze the blood sugar and the insulin level of every part of blood sample collection.With glucose in contrast.As a result, trehalose shows the kinetics identical with glucose, and promptly after they had oral back about 0.5-1 hour, the level of its blood sugar and Regular Insulin was the highest.Show also that these trehalose goods are easy to be digested, absorption, metabolism and by body as energy derive.

Embodiment 8

Acute toxicity test

(purity is 99.5%, d.s.b.) carries out its acute toxicity test to give the oral high purity trehalose goods that obtained by method among the embodiment 4 of mouse.The result shows that this trehalose is the lower material of a kind of toxicity, and does not have dead mouse when being applied to mouse with the highest possible dosage.Though not too accurate, the LD50 of these trehalose goods is 50g/kg or higher.

Embodiment 9

Produce trehalose-releasing enzyme by Arthrobacter Q36

Similar to embodiment 1, use a fermentation container, use the inoculum of Arthrobacter Q36 to replace the inoculum of root nodule bacterium M-11 (CCTCC M94031) to cultivate 72 hours.In the nutrient solution of gained, non-reduced polysaccharide forms enzyme and trehalose-releasing enzyme activity of the present invention is respectively about 1.3 units/ml and about 1.8 units/ml.Similar to embodiment 1, to analyzing by the cell suspending liquid and the nutrient solution supernatant liquor of resulting inoculum preparation.The activity that the former non-reduced polysaccharide formation enzymic activity is about 0.5 unit/ml and trehalose-releasing enzyme is about 0.5 unit/ml, and the latter's non-reduced polysaccharide formation enzymic activity is the active about 1.3 units/ml of being of about 0.8 unit/ml and trehalose-releasing enzyme.

Embodiment 10

The purifying of enzyme

With the 18L nutrient solution that obtains by method among the embodiment 9, the similar non-reduced polysaccharide that comes purifying to generate to embodiment 2 forms enzyme.Table 6 has been represented the result of each purification step.

Table 6

Purification step	Enzyme *Active	Specific activity (unit)	Yield (%) (unit/mg albumen)
				Nutrient solution	23,700	-	100
Supernatant liquor behind the cell rupture	22,400	0.15	95
				With the dialyzate behind the ammonium sulfate precipitation	20,200	0.51	85
The ion exchange column elutriant	15,100	6.5	64
				Mercapto water-based post elutriant	8,450	115	36
The gel-filtration column elutriant	6,120	217	26

Annotate: the non-reduced polysaccharide of symbol " * " expression forms enzyme.

Table 7

Purification step	Enzyme **Active	Specific activity (unit)	Yield (%) (unit/mg albumen)
				Nutrient solution	32,500	-	100
Supernatant liquor behind the cell rupture	30,100	0.19	93
				With the dialyzate behind the ammonium sulfate precipitation	25,400	0.72	78
The ion exchange column elutriant	22,700	22.3	70
				Mercapto water-based post elutriant	15,200	215	47
The gel-filtration column elutriant	11,600	497	36

Annotate: symbol " * * " is represented trehalose-releasing enzyme of the present invention.

By adopting the electrophoresis method similar with embodiment 2, the non-reduced polysaccharide formation enzyme of the purifying that gel permeation chromatography post elutriant from table 6 and 7 is obtained and the activity of trehalose-releasing enzyme are studied.The result observes them respectively and is single albumen band, and this shows that they are the higher enzyme preparations of purity, demonstrates single bands of a spectrum on electrophoresis.

Embodiment 11

The character of enzyme

The molecular weight of the Purifing Trehalose release enzyme preparation that the method with embodiment 10 of measuring on SDS-PAGE obtains is about 57,000-67,000 dalton.PI value with this enzyme of iso-electric point electrophoresis technique determining in embodiment 3 is about 3.6-4.6.To embodiment 3 similar research temperature and pH influence and thermostability and pH stability to this enzymic activity.Fig. 6,7,8 and 9 have shown this temperature effect, pH influence, thermostability and pH stability respectively.

From these figure clearly the optimal temperature of this enzyme preparation be about 45 ℃; Appropriate pH is about 6.0-7.5; Thermostability is up to about 45 ℃; And pH stability is about 5.0-10.0.

Embodiment 12

From to have trehalose structure end and glucose polymerization degree be 3 or prepare trehalose greater than 3 non-reduced polysaccharide

Press the method for embodiment 4, come tentatively to have trehalose end and glucose polymerization degree be 3 or prepare trehalose greater than 3 non-reduced polysaccharide by using the purifying enzyme goods that obtain with the method among the embodiment 10.The result shows, this enzyme preparation from non-reduced polysaccharide (to obtain from root nodule bacterium M-11 sugared similar) discharged trehalose.

Embodiment 13

Trehalose-releasing enzyme and character thereof by known microorganisms production

Little yellow tyrothricin (ATCC11822) in the hitherto known microorganism strains and micrococcus roseus (ATCC186), confirmed that by the inventor they can produce trehalose-releasing enzyme of the present invention, similar to embodiment 1, these two kinds of bacterial strains were cultivated 72 hours in 27 ℃ in fermentation container respectively.The 18L nutrient solution of every kind of bacterial strain of gained is made cell rupture on the cell rupture instrument, and the centrifugal supernatant liquor that obtains, order obtains partially purified enzyme preparation through ion exchange column again with ammonium sulfate precipitation, dialysis then, studies its character then.Those results of root nodule bacterium M-11 and Arthrobacter Q36 list in the table 8 together.

Table 8

Microorganism	Change the enzymic activity (unit) of post elutriant from acid	Optimal temperature (℃)	Appropriate pH	Thermostability (℃)	PH stability
						Little yellow tyrothricin (ATCC 11822)	6,070	About 40	About 6.5～6.8	Up to about 40	About 5.5～9.5
Micrococcus roseus (ATCC 186)	3,010	About 35	About 6.8	Up to about 30	About 6.5～7.2
						Root nodule bacterium M-11 (CCTCC M 94031)	25,400	About 45	About 6.0～7.5	Up to about 40	About 5.0～10.0
Arthrobacter Q36 (CCTCC M 94030)	22,700	About 45	About 6.0～7.5	Up to about 45	About 5.0～10.0

Press the method for embodiment 12, tentatively to have trehalose structure end and glucose polymerization degree be 3 or prepare trehalose greater than 3 non-reduced polysaccharide.The result shows, to form enzyme similar to the trehalose that is obtained by root nodule bacterium M-11, and every kind of enzyme preparation of test has all generated trehalose from non-reduced polysaccharide.

Embodiment 14

Contain the N-terminal amino acids sequence

The partial purification enzyme preparation (obtaining) that will obtain by root nodule bacterium M-11 and by the partial purification enzyme preparation distilled water dialysis that Arthrobacter Q36 obtains by method among the embodiment 2, the about 80 μ g albumen that use every kind of goods that obtain as sample determination they contain the N-terminal amino acids sequence.With " PROTEIN SEQUENCER MODEL 473A " (a kind of U.S. Foster city Applied Biosystem, instrument of Inc.) analysis of amino acid sequence, 10 amino-acid residues of its N-end have been disclosed.What table 9 had been represented the part enzyme preparation contains the N-terminal amino acids sequence.

Table 9

Starter bacteria	The partial amino-acid series that contains the N-end
		Root nodule bacterium M-11 (CCTCC M 94031)	Ala-lys-Pro-Val-Gln-
Gly-Ala-Gly-Arg-Phe
	Arthrobacter Q36 (CCTCC M 94030)		Thr-Pro-Thr-Tyr-Pro-
Arg-Glu-Arg-Ala-lys

Table 9 shows that clearly the N-end of the enzyme preparation that obtains from root nodule bacterium M-11 is Ala., and the ensuing aminoacid sequence of Ala is Lys-Pro-Val-Gln-Gly-Ala-Gly-Arg-Phe.The enzyme preparation that obtains from Arthrobacter Q36, the N-end is Thr., and its ensuing aminoacid sequence is Pro-Thr-Tyr-Pro-Arg-Glu-Arg-Ala-Lys.

Embodiment 14 (2)

The internal amino acid sequence

Enzyme preparation that will obtain from root nodule bacterium M-11 (method by embodiment 2 obtains) and the enzyme preparation (method by embodiment 10 obtains) that obtains from Arthrobacter Q36 are dialysed 10mM Tris-Hcl damping fluid, and the dialyzate that obtains dilutes with freshly prepared identical damping fluid respectively and obtains about 1mg/ml concentration solution.In the solution of every part of 1ml equal portions of gained, add 10 μ g " LYSYL ENDOPEPIDASE " (a kind of Japan.Tokyo, Wako pure Chemcal Industries, the product of Ltd.), make it generate peptide in 22 hours in 30 ℃ of reactions, use reverse phase HPLC (reversed-phase HPLC) to isolate this peptide then.The equipment and the condition of the reversed-phase HPLC that the peptide of the enzyme preparation that separation obtains from root nodule bacterium M-11 is used are " CAPCELL PAK C18 posts ", and diameter is 4.6mm, long 250mm (Tokyo Shiseido Co., the product of Ltd.); Flow velocity, 0.6ml/min; Temperature, room temperature; Elution time, 60min; Gradient contains the linear gradient solution of 0.1V/V% trifluoroacetic acid and 16～48V/V% acetonitrile.The equipment of the reversed-phase HPLC that the peptide of the enzyme preparation that separation obtains from Arthrobacter Q36 is all and condition are " μ-BONDAPAK C18 post ", diameter 2.1mm, long 150mm (U.S. Milford, Chromatography Div., the product of MILLPORE Corp.); Flow velocity, 0.9ml/min; Temperature, room temperature; Elution time, 60min; Gradient contains the linear gradient solution of the acetonitrile of 0.1V/V% trifluoroacetic acid and 30～55V/V%.Detect the peptide that elutes from post by the absorption of measuring 210nm wavelength place.Three kinds of peptides that obtain from the enzyme preparation of root nodule bacterium M-11 are called the peptide (its retention time is respectively about 41,46 and 54) of RT41, RT46 and RT54 and are separated from the peptide of other association, three kinds of peptides (its retention time is respectively about 7,30 and 48) that are called AT7, AT30 and AT48 that obtain from the enzyme preparation of Arthrobacter Q36 are separated from the peptide of other association, this isolated peptide of vacuum-drying afterwards, and the peptide of gained is dissolved in the 200 μ l solution of 0.1～50V/V% different concns acetonitrile.On a protein sequence instrument, analyze the aminoacid sequence of 10 amino-acid residues of as many as of the peptide sample that so obtains.Table 10 has shown the internal amino acid sequence that analyzes.

Table 10

Starter bacteria	Peptide	The internal amino acid sequence
			Root nodule bacterium M-11 (CCTCC M 94031)	RT41	His-Gly-Glu-Gly-Asn-Thr-Try-Gly-Asp-Se
RT46	Asp-Glu-Arg-Ala-Val-His-Ile-Leu-Glu-Glu
		RT54		Leu-Asp-Try-Ala-Glu-Ala-Ser-Ala-Gly-Asp
Arthrobacter Q36 (CCTCC M 94030)	AT30				Gln-Gly-Glu-Gly-Asn-Thr-Try-Gly-Asp-Ser
		AT48	Asp-Glu-Arg-Ala-Val-His-Ile-Leu-Glu-Asp
	AT7			Leu-Asp-Try-Ala-Glu-Ala-Ala-Glu-Gly-Asp

As shown in table 10, the internal amino acid sequence of the peptide RT41 of the enzyme that obtains from root nodule bacterium M-11 in 10 amino-acid residues 9 internal amino acid sequences with the peptide AT30 of the enzyme that obtains from Arthrobacter Q36 in 10 amino-acid residues 9 are consistent, and consistent among 9 in 10 amino-acid residues and the peptide AT48 among the peptide RT46.In peptide RT54 and AT7,8 in their 10 amino-acid residues is consistent.Therefore, can conclude from root nodule bacterium M-11 bacterial classification enzyme that obtains and the enzyme that obtains from Arthrobacter Q36 bacterial classification within it portion's aminoacid sequence have than higher homology, this sequence can be expressed as Leu-Asn-Trp-Ala-Glu-Ala-X ₁-X ₂-Gly-Asp (" X ₁" representative " Ser " or " Ala ", " X ₂" representative " Ala " or " Glu "); Asp-Glu-Arg-Ala-Val-His-Ile-Leu-Glu-X ₃(X ₃Representative " Glu " or " Asp "); And X ₄-Gly-Glu-Gly-Asn-Thr-Try-Gly-Asn-Ser (" X wherein ₄" representative " His " or " Gln ").

Following embodiment A has been described the preparation of trehalose-releasing enzyme of the present invention, with the trehalose of this enzyme preparation and contain the sugar composition of trehalose; Embodiment B has been described the composition that contains one or more these class trehaloses and sugar:

Embodiment A-1

Press the method for embodiment 1, the inoculum of root nodule bacterium M-11 (CCTCC M94031) is inoculated on the nutritional medium, and in fermentation container, cultivated 80 hours.After having fermented, the nutrient solution of gained is removed cell with-SF-membrane filtration, obtain about 18L filtrate, use the SF-film with the concentrated enzyme solution into about 1L of this filtrate again, this solution contains the non-reduced polysaccharide formation enzyme of 17.2 units/ml and the trehalose-releasing enzyme of 20.8 units/ml.

The middle lime carbonate that adds of potato starch suspension (d.s.b.) toward 15% makes its final concentration reach 0.1% (d.s.b.), gained solution is transferred to PH6.0, " TERMAMYL 60L " (a kind of α-Dian Fenmei sample with 0.2% (weight), from Denmark, Copenhagen NoVo Industri A/S listing) mix, next carried out enzyme reaction 15 minutes in 95 ℃.This reaction mixture is at 2kg/Cm ²Autoclaving sterilization is 30 minutes under the pressure, be cooled to 45 ℃, with 1, the amount of 000 unit/g starch (d.s.b) and " PULLULANASE (EC3,2,1,41) " (a kind of Hayashibara Biochemical Laboratories by Japanese Okay-ama, Inc. Shang Shi enzyme sample) mix, and mix with above-mentioned enzyme solution with the amount of 0.2ml/g starch (d.s.b), next carry out 48 hours enzyme reaction.The reaction mixture that so obtains kept 10 minutes in 95 ℃, cooling is also filtered, and gained filtrate is with the ordinary method activated carbon decolorizing, with H-and ion-exchanger desalination of OH-type and purifying, again the solution concentration that obtains must be 60% syrup afterwards, yield is about 92% (d.s.b.).

This syrup contains the maltotetrose and the senior oligosaccharides (d.s.b.) of 70.2% trehalose, 2.4% glucosyl trehalose, 3.3% malt-base trehalose, 0.7% glucose, 10.1% maltose, 12.9% trisaccharide maltose and 0.4%, it has medium and high texture sweet taste, lower reductibility and suitable viscosity and performance of keeping humidity.Because these character, it can at random be used for food, makeup and medicine, as sweeting agent, correctives, increase matter agent (quality-improving agent), stablizer, thinner, weighting agent and vehicle.

Embodiment A-2

The sugar soln that method by embodiment 1 obtains is a material solution, in order to " post that XT-1016 (Na+-type; 4% the polymerization degree) (a kind of alkali metal type strong-acid cation-exchange resin is by Tokyo Tokyo Organic Chemical IndustriesLtd. listing) loads separates.This process is as follows: be the stainless steel column loaded resin of 4 jacket layers of 5.4Cm with internal diameter, it is 20m that this post concatenated in order is made the total gel bed degree of depth that obtains.It is 55 ℃ that this post is heated to the interior temperature of post, and under this temperature keeps, with sugar soln (to the resin) upper prop of 5V/V%, separate this sugar soln with 55 ℃ of hot water elutions (feed), to remove the sugar of association, for example maltose and trisaccharide maltose, the stream part of collecting method for enriching trehalose afterwards.With part merging of this stream, the purifying that obtain, concentrate, vacuum-drying and pulverize and obtain high density marine alga Icing Sugar, yield is about 56% (d.s.b.).

The concentration of trehalose is about 97% (d.s.b.) in this product, and this product tool is medium and the sweet taste of high texture, thus it can at random be used for food, makeup and medicine as sweeting agent, correctives, increase matter agent, stablizer, vehicle and weighting agent.

Embodiment A-3

The high density marine alga solution that obtains by embodiment A-2 method, with the ordinary method activated carbon decolorizing, use the ion-exchanger desalination, and be condensed into 70% solution, then this solution is put into crystallizing dish, sneak into about 2% hydration trehalose crystallization as crystal seed, cooling obtains a massecuite gradually again, and its percent crystallization in massecuite is about 45%.This syrup is at 150kg/Cm ²High pressure under through being installed on the nozzle spray at drying tower top.In spray process, with 85 ℃ of warm airs sending into from the drying tower top syrup is ventilated simultaneously, collect the crystalline powder that obtains with the wire cloth that is positioned at the drying tower bottom, and when 45 ℃ airflow is flowed through wire cloth from bottom to top, slowly shift out crystalline powder.The crystalline powder that obtains injects aging tower made complete crystallization and dry in aging 10 hours, obtained the powdery hydrous crystalline trehalose more again, and yield is about 90% (for high-content trehalose stream part, d.s.b.).

This product is non-hygroscopic basically, is convenient to preserve, and therefore makes it can at random be used for food, makeup and medicinal, as sweeting agent, correctives, increase matter agent, stablizer, vehicle and weighting agent.

Embodiment A-4

Similar to embodiment A-3, purifying places vaporizer by high density trehalose stream part that embodiment A-2 method obtains with gained solution, and vacuum-evaporation obtains syrup, and its humidity is about 3.0%.The gained syrup is placed in the crystallizing dish, sneaks into the hydrous crystalline trehalose that accounts for syrup dry weight 1%, under agitation in 120 ℃ of crystallizations 5 minutes, the mixture that obtains is placed an aluminium matter plane container, and wearing out in 100 ℃ obtained block in 6 hours.

The gained block is pulverized with a cutter, and obtained powdered anhydrous crystalline trehalose (its temperature is about 0.3%) through fluidised bed drying, yield is about 85% (for high density trehalose stream part).This product can be used as siccative arbitrarily in food, makeup, medicine and their raw material and intermediate, also can be used for various compositions, for example makes white powdery sweeting agent in food, makeup and the medicine.

Embodiment A-5

Method by embodiment A-1, the inoculum of root nodule bacterium M-11 (CCTCC M 94031) is inoculated on the nutritional medium, cultivated 70 hours with fermentation container, after having cultivated, go cell to obtain about 100L filtrate with the SF-membrane filtration, this filtrate uses the SF-membrane concentration to the 5L enzyme solution again, and this solution contains the non-reduced polysaccharide formation enzyme of 410 units that have an appointment/ml and the trehalose-releasing enzyme of 490 units/ml.

By heating with 6% potato starch suspension gelatinization, transfer to PH4.5, be heated to 50 ℃, sneak into the isoamylase sample (by the Hayashibara Biochemical Laborat-ories of Japanese Okayama with the amount (d.s.b.) of 500 units/g starch, Inc. listing), next carry out 20 hours enzyme reaction.This reaction mixture is transferred to PH6.5, in 120 ℃ of autoclavings 10 minutes, be chilled to 95 ℃, sneak into " TERMANYL 60L " (a kind of α-Dian Fenmei sample is by NoVo Industri A/S listing of Copenhagen, Denmark) with the amount (d.s.b.) of 0.1%/g starch and next carry out enzyme reaction in 15 minutes.This reaction mixture is chilled to 45 ℃ in 130 ℃ of autoclavings 30 minutes, sneaks into above-mentioned enzyme solution with the amount of 0.01ml/g starch (d.s.b.), carries out enzyme reaction afterwards 64 hours.The gained reaction mixture kept 10 minutes in 95 ℃, be chilled to 50 ℃, transfer PH to 5.0, sneak into " GLUCOZYME " (a kind of glucoamylase sample with the amount (d.s.b.) of 10 units/g starch, by Tokyo Nagase Biochemicals, Ltd. listing), carry out enzyme reaction 40 hours afterwards, and heating makes residual enzyme deactivation.The solution that so obtains with the ordinary method activated carbon decolorizing, (is contained 80.5% trehalose, d.s.b.) with ion-exchanger desalination and about 60% solution of simmer down to.This solution concentration becomes 84% solution, place crystallizing dish afterwards and sneak into account for this solution dry weight 2% hydrous crystalline trehalose as crystal seed, crystallization under agitation goes out trehalose.The flat plastic container is put in the gained crystallization, placed 3 angels caking in room temperature, with cutter this block is pulverized and obtained the powdery hydrous crystalline trehalose, yield 90% is (for raw starch, d.s.b.).

This product is non-hygroscopic basically, and is easy to preserve, and therefore makes it can be used for various compositions arbitrarily, for example in food, makeup and the medicine, as sweeting agent, correctives, increase matter agent, stablizer, vehicle and weighting agent.

Embodiment A-6

Press the method among the embodiment 9, the inoculum of bacterial classification Arthrobacter Q36 (GCTCG M 94030) is inoculated in the nutritional medium, in fermentation container, cultivated 72 hours.The gained nutrient solution was removed cell in centrifugal 30 minutes in 10 under the 000rpm, and the supernatant liquor with the UF-membrane concentration obtains gets the 1L enzyme solution, and it contains the non-reduced polysaccharide formation enzyme of 16.3 units/ml and the trehalose-releasing enzyme of 25.1 units/ml.

" NEOSPITASE " (a kind of α-Dian Fenmei sample with 1 part of heavy potato starch and 6 parts of heavy water and 0.01 part of weight, Nagase Biochemicals Ltd. listing by Japan, Tokyo) mixes, stir, transfer to PH6.2 and be heated to 85～90 ℃ and make starch gelatinization and liquefaction.The gained liquid starch makes remaining enzyme deactivation in 120 ℃ of autoclaving 10min, be chilled to 45 ℃, with the amount of 500 units/g starch (d.s.b.) and isoamylase sample (by Japanese Okayama, Haya-shibara Biochemical Laboratories, Inc. listing) mix, mix with above-mentioned enzyme solution with amount, carried out enzyme reaction then 48 hours with 0.2ml/g starch.After question response is complete, in 95 ℃ this reaction mixture heating 10min is made the residual enzyme inactivation, transfer to 50 ℃ and PH5.0, with 10 units/g amount of starch and " GLOCOZYME " (a kind of glucoamylase sample, by the Tokyo, NagaseBiochemicals Ltd. listing) mix, carry out enzyme reaction 40 hours afterwards, and heating makes remaining enzyme deactivation.The reaction mixture that so obtains is with the ordinary method activated carbon decolorizing, and with ion-exchanger desalination and concentrated into about 60% solution, this solution contains 78.3% trehalose (d.s.b.).Identical with the method for embodiment A-2, only be to use " CG6000 (Na+ type) " (a kind of alkali metal type strong-acid cation-exchange resin, by the Tokyo, Japan Organ Co.Ltd. listing) as ion-exchanger, spissated solution is carried out ion-exchange chromatography, obtain high density trehalose stream part, it contains 95% the trehalose (d.s.b.) of having an appointment.Gained stream part is concentrated to 75% concentration, place crystallizing dish, stirring down, adding 2% hydrous crystalline trehalose is that crystal seed makes its crystallization, crystallization is transferred in the flat plastic container, placed and aging 3 days, make caking in room temperature, then this block is pulverized with cutter, obtain the powdery hydrous crystalline trehalose, yield is about 70% (for raw starch, d.s.b.).

This product does not have water absorbability and is easy to preserve basically, can at random be used as various compositions, for example sweeting agent, the correctives in food, makeup and the medicine, increase matter agent, vehicle, weighting agent and thinner.

Embodiment A-7

Press the method for embodiment 13, the inoculum of little yellow tyrothricin (ATCC 11822) bacterial classification is inoculated on the nutritional medium, cultivated 72 hours in fermentation container, the nutrient solution that obtains is handled with the cell rupture instrument.The gained mixture is in 10, and centrifugal 30min removes residue under the 000rpm, with the supernatant liquor that the UF-membrane concentration obtains, obtains 700ml solution, and this solution contains the non-reduced polysaccharide generation enzyme of 8 units/ml and the trehalose-releasing enzyme of 12 units/ml.

33% tapioca (flour) suspension mixes with lime carbonate that to make its final concentration be 0.1%, and transfer its PH to 6.0, mix with 0.3% " TERMAMYL 60L " (a kind of α-Dian Fenmei is by the NoVo Industri A/S listing of Copenhagen, Denmark), carry out enzyme reaction 20min at 95 ℃ then.The gained reaction mixture is at 2Kg/Cm ²Autoclaving 30min under the pressure, be chilled to 40 ℃, again with 200 units/g amount of starch and isoamylase sample (by the Hayashibara BiochemicalLaboratories of Japanese Okayama, Inc. listing) mix, mix with above-mentioned enzyme solution with the 0.2ml/g amount of starch, carried out enzyme reaction afterwards 48 hours.The reaction mixture that so obtains is in 95 ℃ of maintenance 10min, and cooling is also filtered, and obtains filtrate, and this filtrate is with the ordinary method activated carbon decolorizing, and with H-and the desalination of OH-type ion-exchanger, reconcentration becomes 60% syrup, and yield is about 90% (d.s.b.).

This product contains 60.1% trehalose, 1.4% glucosyl trehalose, 1.5% malt-base trehalose, 1.0% glucose, 6.5% maltose, 8.3% trisaccharide maltose, 21.2% maltotetrose and senior oligosaccharides, it has the sweet taste of medium and high texture, also has low reductibility and suitable performance of keeping humidity.Because these factors, it can at random be used for various compositions, for example in food, makeup and the medicine as sweeting agent, correctives, increase matter agent, stablizer, vehicle, weighting agent and thinner.

Embodiment A-8

To contain about 95% high concentration of trehalose solution by what embodiment A-6 method obtained, with ordinary method decolouring and desalination.The gained solution concentration is become 75% solution, then this solution is transferred in the crystallizing dish, sneak into 2% hydrous crystalline trehalose, be heated to 50 ℃, stir down and is chilled to 25 ℃ gradually, go out crystallization with blue formula centrifugation as crystal seed.Spraying into less water washing gained crystal, to obtain purity be 99% high purity hydrous crystalline trehalose, and yield is about 50%.

Embodiment B-1

Sweeting agent

In the 1 part heavy powdery hydrous crystalline trehalose that routine A-5 method obtains by experiment, add 0.01 part of weight " α G SWEET " (a kind of alpha-glycosyl stevioside product; by Tokyo Toyo Sugar Refining Co.; Ltd. buy) and 0.01 part of weight " ASPARTAME " (a kind of L-Asp-L-Phe methyl ester product; by Tokyo Ajinomoto Co.; Ltd. put goods on the market); this mixture that obtains is sent into nodulizer, obtain granular sweeting agent.This product has gratifying sweet taste, and sugariness is 2.5 times of sucrose, and caloric value is low to moderate 2/5 of sucrose.

Because this product has gratifying stability, can not decompose other sweeting agent of sneaking into, its suitable low calorie sweetener as the low calorific food that provides for obese person and diabetic subject, this two classes people is limited to take in less heat.

When the bacteriological action of inducing carious tooth during in this product, it does not form acid and insoluble dextran basically, so can make it can be used for confectionery and avoid the generation of carious tooth.

Embodiment B-2

Boiled sweet

During heating, 100 parts heavy 55% sucrose solution is mixed with the trehalose syrup of 30 parts of weights (obtaining by embodiment A-7 method), gained solution is lower than 2% in vacuum concentration to humidity.This strong solution is mixed with the citric acid of 1 part of weight and the lemon aromatiser and the tinting material of capacity, with ordinary method the mixture that this obtains is made desired product.

This product is a kind of high-quality boiled sweet, and it has gratifying taste and chew characteristics, and does not worry producing crystallization of sucrose.

Embodiment B-3

Chewing gum

The matrix of 3 parts of weights is heated to the fusing deliquescing, mixes with the hydrous crystalline trehalose powder (obtaining) of 4 portions of heavy sucrose and 3 parts of weights afterwards, mix with capacity perfume compound and tinting material again by embodiment A-3 method.With ordinary method with the gained mixture with roller kneading moulding and pack desired product.

This product is a kind of chewing gum with gratifying structure and taste.

Embodiment B-4

Sweet enriching milk

To be dissolved in 100 parts of fresh milks by heavy marine alga syrup and 1 portion of heavy sucrose by three portions that embodiment A-1 method obtains, gained solution is through the panel heater heat sterilization and be condensed into 70% concentration, subsequently the enriched material aseptic canning that obtains is become desired product.

This product has milk sweet taste and gratifying taste, and this can at random be used in infant food, newborn infant's food, fruit, coffee, cocoa and the tea it and makes condiment.

Embodiment B-5

Lactic drink

The trehalose syrup (method by embodiment A-1 obtains) of 170 portions of heavy skimmed milks, 130 parts of weights and high-content lactosucrose (lactoglucose) powder of 50 parts of weights (are disclosed No.281 as Japanese Patent, disclosed in 795/92) be dissolved in the water of 1,150 part of weight.Gained solution is in 65 ℃ of sterilization 30min, is cooled to 40 ℃ and inoculate 30 parts heavy milk-acid bacterias as initiator, cultivates in 37 ℃ afterwards and obtains desired product in 8 hours.

This product is a kind of lactic drink with gratifying taste and fragrance.Because this product contains oligosaccharides, so it can keep the stable of milk-acid bacteria and promote two to split the growth of bacterium.

Embodiment B-6

Powder fruit juice

The heavy high-content marine alga Icing Sugar (making by embodiment A-2 method) of 33 portions of spray-dried powdery orange juices that obtain and 50 portions, 10 portions of heavy sucrose, 0.65 part of heavy Citric Acid, usp, Anhydrous Powder, 0.1 part of heavy oxysuccinic acid, 0.1 part heavy L-xitix, 0.1 part of heavy Trisodium Citrate, 0.5 part heavy amylopectin and capacity powdery perfume compound are mixed.Nodulizer is pulverized and sent into to the gained mixture granulate, simultaneously with as trehalose syrup (obtaining) spraying of tackiness agent and with 40 ° of air drafts by embodiment A-1 method.The saccharoid that so obtains weighed and pack desired product.

The product that this kind contains 30% orange juice (d.s.b.) can keep it high-quality and can not produce taste unsatisfactory or niff in a long time.

Embodiment 7

Caramel custard

100 parts of heavy W-Gums, 100 parts heavy trehalose syrup (obtaining by embodiment A-7 method), 80 parts of heavy lactose, 20 portions of heavy sucrose and 1 part of geavy salt are mixed.Sneak into 280 parts of heavy eggs in the gained mixture, add 100 portions of milk that heavily boil gradually again.The mixture that obtains like this continues heated and stirred, when stopping heating during the complete gelatinization of starch in the mixture, obtains translucency, cools off this mixture then and toward wherein adding the capacity vanilla.The gained mixture weighed and annotate adorn desired product.

Smooth and the tool gloss of this product surface, and milk flavor and sweet taste are arranged.

Embodiment B-8

Uiro-no-moto (starch paste pre-composition)

90 parts of heavy ground rice, 20 parts of heavy W-Gums, 40 portions of heavy sucrose, 80 parts heavy powdery hydrous crystalline trehaloses (obtaining by embodiment A-3 method) and 4 parts heavy vanillas are mixed, obtain Uiro-no-moto.This product is kneaded with the matcha (green tea) and the water of capacity, places a container to steam 60min in this mixture again, obtains matcha-uiro.

This product has gratifying gloss and chew characteristics, also has gratifying fragrance and taste, also because it has suppressed the degraded of starch well, thereby long preservation period is arranged.

Embodiment B-9

An (beans paste)

Adzuki beans with 10 parts of weights are raw material, mix with water and boil with ordinary method, remove astringent taste and the harsh feeling and the water-soluble impurity of beans afterwards, obtain " adzuki-tsubu-an " of about 21 parts of weights.Water that add 14 portions of heavy sucrose, 5 parts of heavy trehalose syrup (obtaining by embodiment A-1 method) and 4 parts of weights in the gains, and with the gained mixture boiled mix also carefully to mediate making beans not become pasty state with a small amount of salad oil.Like this, just made about 35Kg desired product.

Do not have gratifying taste and fragrance because of boiling this product that fades, this makes it can be used as beans sauce bread, fills out beans sauce bag, dumplings, fill out the raw material of beans sauce wafer, ice cream and ice cream.

Embodiment B-10

Bread

100 portions of heavy flours, 2 parts of heavy yeast, 5 portions of heavy sugar, 1 part heavy powdery hydrous crystalline trehalose (obtaining by embodiment A-3 method), 0.1 part heavy inorganic edible yeast are pressed general method and water kneading, in 26 ℃ of fermentations 2 hours, aging 30 minutes and baking.

This product is a kind of high-quality bread, has gratifying color and rises by big and gratifying elasticity and medium sugariness.

Embodiment B-11

Ham

Add 15 parts of geavy salt and 3 parts heavy saltpetre in the 1000 parts heavy tableted meat of ham and evenly grind, refrigerating chamber is piled up and be placed on to these ham sliced meat spend the night.Then, these ham sliced meat were soaked in salts solution 7 days prior to refrigerating chamber, this salts solution contains the powder (by the preparation of embodiment A-6 method) and the capacity pepper of 500 parts of heavy water, 100 parts of geavy salt, 3 parts of heavy saltpetre, 40 parts of non-reduced polysaccharide of weight, again with the ordinary method cold wash, hang-up smokes system, boil, cooling packing gets desired product.

This product is a kind of high-quality ham, has gratifying color, taste and fragrance.

Embodiment B-12

Powdered peptide

With 1 part of weight " HINUES " (a kind of peptide solution that contains 40% edible soybean, Fuji oil Co. pushes market by the Tokyo) heavily contain powdery crystalline trehalose powder (the method preparation by embodiment A-6) and mix with two parts, the gained mixture is put into plastic ware, obtains the powder peptide in 50 ℃ of vacuum-dryings and pulverizing.

This product has gratifying taste and fragrance, can at random be used as candy, for example the raw material of pre-composition, ice cream and ice cream, the also raw material of using nutritious prod as the infant food and the treatment of oral or the form of feeding.

Embodiment B-13

Powder miso

In 1 part of heavy akamiso (a kind of miso), add 3 parts of powdered anhydrous crystalline trehaloses of weight (method by embodiment A-4 obtains).The gained mixture is put into the salver that the surface has the semisphere well, and kept at room temperature overnight, obtaining the miso solid, every part of heavily about 4g puts into a pulverizer with this miso then, obtains desired product.This product can at random be used as instant food and soup, also has the condiment of miso candy.

Embodiment B-14

Powder yolk

The yolk that makes with new fresh hen egg in 60～64 ℃ of sterilizations, mixes 1 part heavy gained liquid (for heavy this liquid of portion) with panel heater with 4 parts of powdered anhydrous crystalline trehaloses of weight (obtaining by embodiment A-4 method).The gained mixture is changed in the container, and placing spends the night makes caking, and this moment, the anhydrous crystal trehalose changed into hydrous crystalline trehalose.The block that obtains is like this obtained powder yolk with the cutter pulverizing.

This product can at random be used as candy, and for example the raw material of pre-composition, ice cream, ice cream and milk-product also has the infant food of oral or the form of feeding and the raw material that nutritious prod is used in treatment.This product also can be used as skin-care agent (skin refiner) and hair growth promoter.

Embodiment B-15

Cosmetic cream

The heavy glyceryl monostearate (self emulsive) of 2 parts of reunion ethylene glycol monostearates of heating for dissolving, 5 parts, 2 parts heavy high-content marine alga Icing Sugar (obtaining), 1 part heavy alpha-glycosyl rutin, 1 part of heavy-fluid attitude Vaseline, 10 parts heavy three-2-ethyl cyclohexylenedinitrilotetraacetic acid glyceryl ester and capacity antiseptic-germicide according to a conventional method by embodiment A-2 method.Gained solution is mixed with 2 parts heavy L-lactic acid, 5 parts of heavy 1,3 butylene glycols and 66 parts heavy purified water, and, under agitation sneak into capacity essence again, obtain cosmetic cream with homogenizer emulsification gained mixture.

This product has higher stability, can at random be used as high quality sunscreen, skin-care agent and skin whitener.

Embodiment B-16

The powder Radix Ginseng extract

The heavy Radix Ginseng extract of moiety is mixed with 1.5 parts of powdered anhydrous crystalline trehaloses of weight (obtaining by embodiment A-4 method), and the gained mixture changes in the container of plane, makes it to place the anhydrous crystal trehalose to be converted into hydrous crystalline trehalose in 2 days caking.Pulverize the gained block with cutter, portioning obtains the powder Radix Ginseng extract.

This product and capacity vitamins B ₁And B ₂The nodulizer of packing into makes the powder Radix Ginseng extract that contains VITAMIN.

This product that obtains like this can at random be used as nourishing agent, fatigue recovery agent and corroborant.This product also can be used as hair growth promoter.

Embodiment B-17

Solid medicine

With natural human interferon-alpha goods (Cosmo Bio. pushes market by the Tokyo) with the post that is fixed with anti-human interferon-alpha antibody on the ordinary method with adsorptive hindrance element-α, and will remove excessive albumen then as a kind of damping fluid upper prop that contains bovine serum albumin of stablizer.After this, with the physiological saline that contains 5% high-content marine alga Icing Sugar (obtaining by embodiment A-2 method) interferon-' alpha ' is eluted from post, this moment, the PH of physiological saline changed.The gained elutriant is carried out membrane filtration, and filtrate is by adding " the FINETOSE of 20 times of volumes ^" (a kind of anhydrous crystal maltitol powder, by Japanese Okayama, Hayashibara Shoji, Inc. pushes market).Pulverize the dewatered product that obtains then, and the gained powder is passed through the pelleter film-making, obtain every tablet of tablet that contains about 150 unit natural human interferon-' alpha 's, every heavily about 200mg.

This product can be used as Sublingual tablet and gives the patient oral, and dosage is 1-10 sheet/or people/sky, and at random is used for the treatment of virus disease, transformation reactions, sacroiliitis, diabetes and tumour.More specifically, this product is suitable for use as the medicament of treatment AIDS and hepatitis, and patient's digital display work of suffering from this class disease increases.Be mixed in trehalose in this product and maltose stablizer, so it is active even at room temperature also can keep preferably the long time as the natural human interferon-' alpha '.

Embodiment B-18

Sugar coated tablet

Heavily unprocessed of 150mg, carry out always the weigh 230mg of dressing with solution until sheet, this dressing solution composition is 40 parts heavy powdery hydrous crystalline trehaloses (obtaining by embodiment A-3 method), 2 parts heavy vanillas (molecular-weight average is 200,000), 30 parts of heavy water, 25 parts of heavy talcum powder and 3 parts heavy titanium dioxide; The gained tablet is used a kind of solution dressing again, and this solution composition is the identical hydrous crystalline trehalose of 65 parts of prepared fresh, 1 part of heavy vanilla and 34 parts of heavy water, and uses the liquid paraffin glazing, obtains having the sugar coated tablet of satisfactory gloss and outward appearance.

This product had higher storage tolerance (shock tolerance) and can keep its high quality in quite long period.

Embodiment B-19

Toothpaste

By following component is mixed, prepare toothpaste with ordinary method:

One hydrogen calcium phosphate 45.0%

Vanilla 2.95%

Sodium laurylsulfonate 1.5%

Glycerine 20.0%

The polyethenoxy sorbitan moon 0.5%

The cinnamic acid ester

Antiseptic-germicide 0.05%

Powdery hydrous crystalline trehalose (logical 12.0%

Cross the preparation of embodiment A-3 method)

Maltitol 5.0％

Water 13.0%

This product can be used toothpaste as the newborn infant satisfactorily because enough sweet tastes are arranged.

Embodiment B-20

Feed and use solid articles

The composition that preparation is made up of following component: 500 parts heavy powdery hydration trehaloses crystallization (by the method preparation of embodiment A-6), 270 parts heavy powder yolk, 209 portions of heavy skimmed milks, 4.4 parts of heavy sodium-chlor, 1.8 parts of heavy Repone K, 4 parts of heavy sal epsom, 0.01 part of heavy VitB1,0.1 part of heavy sodium ascorbate, 0.6 part of heavy vitamin e acetate and 0.04 part heavy nicotinamide.The layer sample pouch and the heat-sealing of packing moistureproof with the every equal portions of 25g said composition make desired product.

The water that 1 bag of this product is dissolved in about 150～300ml is made liquid food, be administered to nasal cavity, stomach or enteron aisle, to replenish the body energy by feed per os or parenteral route.

Embodiment B-21

Super nutritive liquid

To with ordinary method this aqueous solution be carried out membrane filtration then and remove pyrogen by the high purity hydrous crystalline trehalose 10w/v% aqueous trehalose solution of making soluble in water of embodiment A-8 method preparation, in the Plastic Bottle of packing under the aseptic condition, seal desired product.

This product, it is a kind of gratifying stable super nutritive liquid, can not change basically when depositing, and is suitable for using in vein and the abdomen.Infiltrations such as the 10w/v% solution of this product and blood are, and show that the concentration that it can be higher than 2 times of glucose provides energy to body.

Embodiment B-22

Super nutritive liquid

Will be by the high purity hydrous crystalline trehalose of embodiment A-8 method preparation with by soluble in water under the amino-acids composition stirring that component is formed down, obtain concentration and be respectively 5w/v% and 30w/v%, and with Embodiment B-10 this solution that obtains of purifying similarly, obtain not contain the solution of pyrogen, with in its injected plastic bottle, seal and make desired product again.

The composition of amino-acids composition

Component mg/100ml

L-Isoleucine 180

L-leucine 410

L-Methionin mono-hydrochloric salts 620

L-methionine(Met) 240

L-phenylalanine 290

L-Threonine 180

L-tryptophane 60

L-Xie Ansuan 200

L-arginine monohydrochloride 270

L-Histidine mono-hydrochloric salts 130

Glycine 340

Though this product is a kind of trehalose and amino acid whose compound super nutritive medium of containing, it has gratifying stability, can not change basically when depositing, so can be suitable for being applied to body through vein and intraperitoneal.This product can at random be used for replenishing the energy and amino acid to body.

Embodiment B-23

Treatment damage ointment

200 portions of high-load marine alga Icing Sugar of weight (making by embodiment A-2 method) and 300 portions of heavy maltose are mixed with 50 parts that contain 3 parts of heavy iodine heavy methanol solutions, this gained solution again with the vanilla aqueous solution of the 10w/v% of 200 parts of weights, obtain having gratifying extensibility and adhesive desired product.

The iodine that contains in this product shows bacterial activity extremely, and the trehalose in this product is as the energy supplement agent of viable cell, because these character, this product has shortened the healing stage of wound surface, and makes its recovery satisfactory.

Therefore, when trehalose-releasing enzyme of the present invention generates enzyme with non-reduced polysaccharide, when acting on reductibility starch partial hydrolysate, it can discharge from non-reduced polysaccharide with higher yields and generate trehalose, and described non-reduced polysaccharide has the trehalose structure end and glucose polymerization degree is 3 or is higher than 3.The trehalose that obtains like this can be separated and purifying easily, and the trehalose of gained has gratifying stability and higher quality and moderate sweet taste with the sugar composition that contains it.When oral unprocessed product or when gi tract are used infusion solution, this trehalose can be easily by body digestion, absorption and utilization.Marine alga itself and the sugar composition that contains it can at random be used as various compositions, for example sweeting agent, the correctives in food, makeup and the medicine, increase matter agent, stability, vehicle and weighting agent.

Therefore, the invention provides the new technology that a kind of technical scale prepares trehalose and contains its sugar composition, this technology is a raw material with the starch partial hydrolysate of inexpensive and resourceful starch preparation, has lower cost.So the present invention has brought uncertain tremendous influence for many fields, these fields are starch, enzyme and biochemical science and other industrial circle for example, and system is not food, makeup and pharmaceutical industries, also has forestry, fishery and agricultural, family holds and chemical industry.Therefore, the present invention is abysmal to the influence in these fields, is huge.

Consider that the description at this is the preferred embodiments of the invention, but must understand wherein and can carry out various modifications, and the present invention is intended to cover all these type of modifications that fall into the appended claims in connotation of the present invention and the scope.

Claims (6)

1. a method for preparing trehalose comprises

(a) the partially hydrolysed starch material is to obtain the starch hydrolyzates of partial reduction, and it comprises amylose starch and/or Fructus Hordei Germinatus oligose;

(b) make the described enzyme that can form non-reducing saccharide as follows act on the starch hydrolyzates of partial reduction described in the step (a), form non-reducing saccharide:

Gn-T

Wherein, symbol " G " expression glucosyl residue: " n " represents one or more integers; " T " expression trehalose residue;

(c) make trehalose-releasing enzyme act on described non-reducing saccharide to form trehalose; With

(d) reclaim the gained trehalose.

2. the process of claim 1 wherein that trehalose-releasing enzyme in the step (c) and one or more are selected from following enzyme and unite use: α-Dian Fenmei, beta-amylase, glucoamylase and alpha-glycosidase.

3. the process of claim 1 wherein that step (c) comprises that also remaining recuding sugars is the step of sugar alcohol in the hydrogenation gained mixture.

4. the process of claim 1 wherein that step (c) also comprises the gained mixture through having the column chromatography of ion-exchanger to remove the step of impurity.

5. the process of claim 1 wherein that step (c) also comprises the described trehalose crystalline step in the gained mixture.

6. claim 1 or 3 method, wherein said trehalose contains one or more following members that are selected from: non-reducing saccharide, sugar alcohol, glucose, maltose, trisaccharide maltose, maltotetrose and other reduction oligosaccharides.

Images