CN1151253C - Mycose base hydrolase and its preparation and use - Google Patents

Abstract

The present invention discloses novel acidophilic heat resistant fucose base hydrolase, a preparation method thereof and an application thereof. The hydrolase can be obtained from microorganisms of escherichia, bacillus and saccharomyces, and can hydrolyze a bond between fucose and the rest of fucose bases in non-reducing polysaccharide with the fucose polymerization degree of equal to or more than 3, which has a fucose structure and is used as an end unit under the high-temperature acid condition with high efficiency. The molecular weight of the hydrolase is about 55000 to 65000 daltons, and the isoelectric point is about 5.5 to 6.6. The hydrolase is easily prepared on a large scale through microorganism fermentation, and is used for industrially preparing fucose. The fucose can be widely used in a composition of food, cosmetics and medicine.

Description

Mycose base hydrolase and preparation thereof and purposes

Technical field

The present invention relates to a kind of mycose base hydrolase and preparation thereof and purposes, particularly relate to a kind of acidophilic thermostable mycose-base lytic enzyme, it can be under acid hot environment the key between trehalose part and all the other glycosyl parts in the specificity hydrolysis irreducibility polysaccharide, it is that a terminal unit and glucose polymerization degree are 3 or higher that described irreducibility polysaccharide contains trehalose structure.The invention still further relates to and can produce the microorganism of described enzyme and the preparation of this enzyme, the trehalose that obtains with described enzyme, and contain the composition of described trehalose.

Background technology

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.Academic Press, 1993,18:201-225 and Applied and Environmental Microbiology.1990, as described in the 56:3213-3215, trehalose extensively is present in organic sphere, all have this sugar among comprising animal, plant and microorganism, but content is lower.Trehalose is a kind of non-reducible disaccharide, is white crystal under the normality, tasty and refreshing sweetness and do not have aftertaste, and its sugariness is 45% of a granulated sugar; Because this sugar has the unique biological function; multiple biomacromolecule and cytolemma there is significant provide protection; its purposes and latent effect are very extensive, and the protective material and the stablizer that can be used as biological products and active bacteria formulation use, and also can be used as natural additive in food and makeup.Therefore an urgent demand can large-scale industrialization prepare trehalose.

Traditional trehalose preparation method comprises as the open No.154 of Japanese Patent, utilize the open No.216 of the method for yeast extract and Japanese Patent in 485/75, report unites the method that transforms maltose generation trehalose with maltose phosphorylase and trehalose phosphorylase in 695/83.Yet, because the former is used as in the yeast body of parent material, the content of trehalose is usually less than 15W/W% (unless stated otherwise, the 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: (I) form through Cori ester owing to trehalose, can not adjust to satisfactory high level as the maltose concentration of substrate: (II) enzymatic reaction system of Phosphoric acid esterase is a transfer reaction, and the very difficult stable continuous enzymatic reaction system that obtains in the actual production, therefore the trehalose productivity ratio is lower, makes this method can not be as industrialized preparing process.

Consider these factors, everybody strives to find a kind of new way of utilizing the starch hydrolysis to prepare trehalose now.Result such as Japanese patent application No.362,131, disclosed in 92, it is found that isolating root nodule bacterium from soil (Rhizobium sp.) M-11 (CCTCC M94031) and Arthrobacter (Arthrobacter sp.) Q36 (CCTCC M94030) can produce a kind of mycose base hydrolase, at normal temperatures with can form terminal unit of tool trehalose and glucose polymerization degree be 3 or the mycose-base synthetase of higher non-reduced polysaccharide use hydrolyzed starch direct production trehalose.The hydrolysis at normal temperatures of this mycose base hydrolase the terminal unit of trehalose is arranged and have 3 or the irreducibility polysaccharide of higher glucose polymerization degree in, the key between trehalose part and all the other glycosyl parts discharges trehalose.Therefore with this trehalose preparation method, produce trehalose method than before from the irreducibility starch partial hydrolysate and have greatly improved.But owing to the microorganism culturing cycle in this method is long, it is low to produce enzyme level, particularly the poor heat stability of this enzyme, not acidproof, cause complex manufacturing, sugared transformation efficiency is low, numerous unfavorable factors such as production cost is higher, can not adapt to modern mashing produce in high temperature, efficient, high-quality demand.This is because in actual production, and when the starch hydrolysis temperature was lower than 55 ℃, as easy as rolling off a log microbiological contamination causes to produce polluted; PH value of solution can constantly descend and causes acid nonfast enzyme inactivation in the reaction process in addition, but under the neutrallty condition, starch is aging easily again, forms insoluble precipitation, and influence is produced; These all are significantly not enough in this method.For addressing this problem, press for a kind of novel alga glycosyl hydrolase of screening, it can have high hydrolysis vigor and ideal thermostability, non-inactivation in the environment more than 55 ℃, also require to possess ideal pH stability, under acidic conditions, also can possess stable enzyme activity especially.

Summary of the invention

In order to achieve the above object, the inventor has extensively screened the microorganism that can produce this new enzyme, described enzyme can be in the high-temperature acidic environment from have trehalose structure as a terminal unit and glucose polymerization degree be 3 or higher irreducibility polysaccharide hydrolysis discharge trehalose.As a result, we microorganism strains (E.coli) MTH-11 that found Escherichia can produce hydrolysis have trehalose end structure and glucose polymerization degree be 3 or higher non-reduced polysaccharide form the mycose base hydrolase of trehalose; We find, under the high-temperature acidic condition when using jointly with same heat-resisting acid proof mycose-base synthetase, this novel alga glycosyl hydrolase endonuclease capable forms trehalose with gratifying high yield catalytic hydrolysis reaction, and by allowing this novel alga glycosyl hydrolase and mycose-base synthetase to the effect of reductibility starch partial hydrolysate, and reclaim the high relatively reaction mixture of content of trehalose, be easy to prepare trehalose.We also find, the same with microorganism from Escherichia, bacillus, saccharomycetic microorganism also can produce similar hydrolysis have trehalose end structure and glucose polymerization degree be 3 or higher non-reduced polysaccharide form the mycose base hydrolase of trehalose, we have also carried out same research and have finished the present invention to this.We have also developed the composition that contains by the trehalose of above-mentioned preparation method's preparation simultaneously, as food, makeup and medicine, and have finished the present invention.

The inventor is with these microorganisms difference called afters " intestinal bacteria (Escherichia coli) MTH-11 " and " yeast (Saccharomyces) MTH-8 ", and be preserved in Chinese microorganism strain management committee common micro-organisms center (China GeneralMicrobiological Culture Collection Center on December 27th, 2000, be CGMCC) (China, Beijing, the Zhong Guan-cun).Preserving number be respectively CGMCC No 0526 (colon bacillus, MTH-11) and CGMCC No 0525 (yeast, MTH-8).

Except that mentioned microorganism, other bacterial strain of Escherichia, bacillus, yeast belong and its mutant; As long as can produce acidophilic thermostable mycose-base lytic enzyme of the present invention, just be applicable to the present invention, described mycose base hydrolase can be under the high-temperature acidic condition specificity hydrolysis have trehalose structure as terminal unit and glucose polymerization degree be 3 or higher non-reduced polysaccharide in key between trehalose part and all the other glycosyl parts.

Any nutritional medium needs only mentioned microorganism and can grow thereon, and can produce mycose base hydrolase of the present invention, just can be used for the present invention: for example can arbitrarily use synthetic and natural nutrition substratum.Any carbonaceous material is as long as it is utilized by described microorganism, and be used for the present invention with regard to can be used as carbon source: the example of described carbon source is a carbohydrate, as glucose, and fructose, lactose, sucrose, N.F,USP MANNITOL, sorbyl alcohol, molasses, starch and starch partial hydrolysate; Organic acid such as citric acid and succsinic acid.Can suitably select the concentration of these carbon sources in nutritional medium: for example, when using glucose, from the angle of described microorganism growth and breeding, preferred concentration normally 10% or lower, preferably 1% or lower.Can be used for nitrogenous source of the present invention has inorganic nitrogen compound, 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 salt of the present invention has calcium salt, magnesium salts, sylvite, sodium salt, phosphoric acid salt and zinc, iron, copper, other salt of molybdenum and cobalt.If desired, can add amino acid and VITAMIN.

Will be in the present invention the available microorganism under aerobic condition, usually 4-50 ℃ scope, preferred 25-45 ℃ scope; And pH4-10; Cultivate under the preferred pH5-9 condition.The incubation time that is suitable in the present invention is adjusted to begin the required time than described microorganism growth longer, preferably, 8-100 hour.Be not particularly limited the concentration of dissolved oxygen in the nutritional medium (DO), the DO that uses the 0.5-20ppm scope usually is gratifying.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.The training method of this microorganism can be a batch culture, also can be cultured continuously.

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, it can be reclaimed and be used as thick enzyme respectively; Also the culture of gained intactly can be used as thick enzyme.In the present invention, can use traditional liquid-solid separation method, so as from culture isolated cell.For example, can be with the direct centrifugation method of the culture of gained, and the precoating filter is with the method for its filtration or the membrane filtration isolated cell by using sheet frame filter or hollow fiber, therefore the cell-free filtrate that obtains intactly can be used as enzyme solution or before use that it is concentrated, also can be with the directly broken extraction enzyme of the cell liquid that obtains.The available concentration method uses saltouing of ammonium sulfate as, heating method among the present invention, uses the precipitator method of acetone and alcohol, uses the method for enrichment as sheet frame filter and hollow-fiber film.

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 bonding absorption method 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 liquid calcium chloride, 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 can be used as thick enzyme solution.By from cell, extracting enzyme of the present invention, comprise and use ultrasonic wave that use the Mechanical Crushing of granulated glass sphere and aluminum oxide, (Frenchpress) fragmentation etc. is pressed in France, make the extract that obtains through heat pre-treatment, 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 be purified with traditional method before use.For example can concentrate by crude enzyme liquid, dialysis, use chromatography on the anion-exchange column of " DEAE-Fast Flow " (a kind of anionite) then; Hydrophobic chromatography with " Phenyl-Sepharose " (a kind of hydrophobic resin); And the gel permeation chromatography of use " Sephaeryl S-200 " (a kind of gel-filtration resin), thereby prepare the pure enzyme preparation that is a band on the electrophoresis.

The mycose base hydrolase of the present invention that obtains thus has following physics-chem characteristic;

1. effect:

The specificity hydrolysis have trehalose structure as 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 about 55,000 to 65,000 dalton at sodium laurylsulfonate-polyacrylamide gel electrophoresis (SDSPAGE);

3. iso-electric point (PI)

With amphotericeledrolyte on the iso-electric point electrophoresis about 5.5 to 6.6;

4. optimum temps

When pH5.5 preserves 30 minutes, 60～85 ℃;

5. best pH

When preserving 30 minutes for 65 ℃, 5.0～6.0;

6. thermostability

When pH5.5 preserved 60 minutes, temperature that can be stable was about 30～80 ℃;

7.pH stability

Preserved 16 hours at 25 ℃, but stable p H is 4.0～11.0.

Follow these steps to detect the activity of marine alga base hydrolase of the present invention: the 0.1ml enzyme solution is added in the 50mm acetate buffer solution (pH5.5) of 0.4ml 1.25w/w% maltotriose glycosyl trehalose (another name α-maltotetrose base alpha-glucosaccharase), mixture was cultivated 30 minutes at 75 ℃.The reaction mixture of gained is added to carries out the Somogyi reaction in the Somogyi copper solutions, to stop enzyme reaction; Determine reducing power with the Somogyi-Nelson method then.In contrast, 100 ℃ with enzyme solution preheating 15 minutes so that enzyme deactivation, by and above-mentioned similar methods detect.Enzymic activity with 1 unit among the present invention is defined as: under this condition, per minute increases the required 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 the glucosyl group trehalose, the malt-base trehalose, the maltotriose glycosyl trehalose, maltotetrose base trehalose and maltopentaose base trehalose, these sugar are to act on trisaccharide maltose by mycose-base synthetase, maltotetrose, maltopentaose, MALTOHAXAOASE and Fructus Hordei Germinatus seven sugar and forming.Except that these substrates, by prepare as starch, amylopectin and amylose starch with amylase or acid moieties hydrolyzed starch class material, have terminal trehalose structure and glucose polymerization degree be 3 or higher low reductibility starch partial hydrolysate also be applicable to the present invention.

The described diastatic example of partially hydrolysed starch is (by Pergamon Press at Handbook of Amyloses andRelated Enzymes; Tokyo, Japan (1988) publishes) in disclosed α-amylase, maltopentaose forms amylase and MALTOHAXAOASE forms amylase.These amylase and debranching factor such as Starch debranching enzyme 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 soluble suspended substance that contains excessive 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 80 ℃, preferably at 55-80 ℃.Available reaction pH is 4.5-8.0 in enzyme reaction of the present invention, preferably at pH 5-7.Should 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 the g substrate 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 having the irreducibility starch partial hydrolysate that relatively low DE value has high relatively glucose polymerization degree again simultaneously, 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 mycose-base synthetase is used in combination with glucoamylase) gained in 131/92.Preparation method of the present invention, wherein, mycose-base synthetase is used in combination with mycose base hydrolase of the present invention, can about 70% or higher productive rate 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: at first the reductibility starch partial hydrolysate that will have a high relatively glucose polymerization degree with mycose-base synthetase is transformed into 1 mole of non-reduced polysaccharide with trehalose structure as terminal unit, then, with mycose base hydrolase of the present invention 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, be transformed into 1 mole trehalose and starch partial hydrolysate with mycose base hydrolase.Therefore, repeat the enzyme reaction of aforementioned mycose-base synthetase and mycose base hydrolase, can form many moles trehalose molecule and irreducibility starch partial hydrolysate from 1 mole of reductibility starch partial hydrolysate.(as Fig. 1)

In preparation method of the present invention, can use mycose-base synthetase and mycose base hydrolase of the present invention 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 the irreducibility starch partial hydrolysate continuously with this order.In order further to improve the trehalose productive rate, the reaction mixture that also 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, then with the solution decolouring of gac with gained, with the ion-exchanger desalination of H-and OH-form, further be condensed into the pulpous state product after, can optionally syrupy product be dried to powder-like product.

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

If desired, according to the present invention, the polysaccharide product that can contain trehalose with amylase, α-Dian Fenmei, glucoamylase, alpha-glucosidase and/or trehalase hydrolysis, or with cyclodextrine glucanotransferase and/or the catalytic polysaccharide-shift reaction of Transglucosylase to control its sugariness and reducing power and to reduce its viscosity.In addition, arbitrarily the hydrogenation polysaccharide product is converted into sugar alcohol, thereby reduces its reducing power.Can from the product of gained, remove glucose with the high component of preparation content of trehalose as ion-exchange chromatography with above-mentioned purification process.Can be easy to the component purifying that to obtain thus and be condensed into syrupy product, and can further syrupy product be condensed into supersaturation dope and crystallization as required to obtain moisture or the anhyrous crystalline trehalose.

Available ion-exchange chromatography technology comprises in the present invention, at the open No.23 of Japanese Patent, and the method for disclosed use strong-acid cation exchange resin in 799/83 and 72,598/83.Use these technology, can remove association sugar contained in thick trehalose product at an easy rate 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 on-crystallizer in, 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 water-containing crystal 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, with of the preparation of a small amount of cold water hydro-peening water-containing crystal trehalose with promotion high purity water-containing crystal trehalose.During spraying drying, has 60-85% concentration by penetrating from a high-pressure pump mouth, the massecuite of about 20-60% degree of crystallinity, with containing about 60-100 ℃ of hot-air dry that does not melt the gained crystal powder, gained powder limit was stirred logical the about 1-20 of warm air hour in 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 several hours or 3 days so that make whole inclusion crystallizations and be solidified into piece for the syrup of about 10-60%, 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 prepare the anhydrous crystal trehalose with dehydration by the aqueous crystalline trehalose of drying, but general method is to place crystallizer by moisture content being lower than 10% high content of trehalose solution, under agitation condition, in 50-160 ℃, preferred 80-140 ℃ scope, treatment soln is to obtain the massecuite of anhydrous crystal trehalose under the situation of interpolation crystal seed, under dry and high temperature with its crystallization, with traditional method such as piece comminuting method, fluid bed granulation method and spraying drying the anhydrous crystal trehalose of gained is pulverized, thus preparation anhydrous crystal trehalose.

The trehalose of the present invention that obtains thus is stable and do not have reducing power basically, and can mix with other material, particularly amino acid, polypeptide and protein, causes that coking is rotten and produces odour nuisance and needn't worry.Trehalose itself has gratifying quality and sugariness, and easily by intravital trehalase hydrolysis, can be assimilated by body when therefore oral, absorbs and utilization.In addition, trehalose is gone up the microbial fermentation that can not be brought out carious tooth substantially, so this can make it be used as anticariogenic sweeting agent.

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 to have been worried toxicity and side effect by live body metabolism and utilization.Therefore, help these products as the energy supplement agent that is used for body.

Trehalose is stable sweeting agent, and particularly 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 among the present invention and the polysaccharide composition that contains trehalose 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 among the present invention and the polysaccharide composition that contains trehalose intactly can be used as sweet taste condiment.Can be as required and one or more other sweeting agents of capacity, use together as atomizing syrup, glucose, fructose, maltose, sucrose, isomerose, honey, maple sugar, erythrose, sorbyl alcohol, N.F,USP MANNITOL, Saccharum lactis, stevioside glycosides, Ali's tower nurse (Alitame), glycyrrhizin, radix asparagi sweet extract (Asparmate), asccharin, glycine and L-Ala or weighting agent such as dextrin, starch and lactose.

Can intactly use the powder or the crystallization that contain trehalose of the present invention and contain the polysaccharide composition of trehalose to mix, and make particle, bat, flat, lozenge and tablet with other vehicle, weighting agent, thinner and tackiness agent.

Containing trehalose of the present invention can be compatible well with other material that tart flavour, saline taste, bitter taste, astringent taste and delicious food are arranged with the polysaccharide composition that contains trehalose, and high relatively acid resistance and heat resistance are arranged.Therefore 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 can be used for seasonings, as soy sauce, wine, mayonnaise, seasonings, vinegar, pickles, convenient flavouring, nucleic acid seasonings, mixed spices, sugar and the coffee candy of amino acid, peptide, soy sauce, powdered.

Trehalose of the present invention and the polysaccharide composition that contains trehalose arbitrarily can be used to increase the sweet taste of following food: cake, fruit peptone, flour paste, caramels; Sweet food such as dessert, biscuit, cheese, cooky, group, pudding, butter, eight treasure gruel, 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; Finished fruits and vegetables such as jam, preserved fruit, tomato-sauce; Salted vegetables and pickling food such as pickled radish, hot pickled mustard tube and salting cucumber salt down; Meat product such as ham and sausage; Fish flesh prod such as fish ham, fish intestines, dried fish; Dry vegetalbe such as laver, dry vegetable; Dairy products is as milk, soymilk; Canned and bottled product such as meat, the flesh of fish, fruits and vegetables can; Contain alcoholic beverage such as rice wine, yellow rice wine, grape wine and liquor; Soft drink such as coffee, tea, cocoa, fruit juice, soda pop, yogurt beverage and lactobacteria-containing feed; Ready-to-eat such as instant pudding mixture, instant hot pastry mixt and instant soup mixture; And Special food, as infant food, medical diets, space food, fully nutrient; Trehalose of the present invention and the polysaccharide composition that contains trehalose can improve the taste and the quality of above-mentioned food.

The feed and the pet food that also trehalose of the present invention and the polysaccharide composition that contains trehalose can be used for animal such as domestic animal, poultry, honeybee, silkworm and fish are to improve its taste hobby.Also trehalose and the polysaccharide composition that contains trehalose can be added into as tobacco, cigarette, toothpaste and toothpaste powder, lipstick, kermes, lipstick, face cream, medicine for oral administration, Oils,glyceridic,cod-liver, oral cooling agent, gargle, makeup and medicine as sweeting agent, flavor improvement agent, quality booster and stablizer.

Trehalose of the present invention and the polysaccharide composition that contains trehalose can be used for biologically active substance to its effective constituent and loss of activity sensitivity and the heath food and the pharmaceutical composition of matters of containing biological activities as quality booster and stablizer.The example of above-mentioned biologically active substance be lymphokine such as α-, β-, and gamma-interferon, α-Zhong Liuhuaisiyinzi (INF-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, toxinicide and human normal immunoglobulin, vaccinate for animals; 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, alpha-acetolactate decarboxylase, beta-amylase, isoamylase, poly-glucose enzyme and Sumylact L; Extract such as Radix Ginseng extract, testudinate extract, Algae Extract, Aloe extract, propolis extract; The microbial strains that can survive, as virus, milk-acid bacteria and yeast; Other biologically active substance such as royal jelly.The application of the invention trehalose and the polysaccharide composition that contains trehalose, can be easy to above-mentioned biologically active substance is processed into heath food and the pharmaceutical composition with gratifying stability and quality, and needn't worry its effective constituent and active forfeiture or lose.

By mentioned above, with trehalose of the present invention with contain trehalose and method that polysaccharide composition mixes above-mentioned substance comprises 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 higher amount, trehalose of the present invention and the polysaccharide composition that contains trehalose are mixed in the above-mentioned material and composition.

Brief Description Of Drawings

Fig. 1 represents to generate the trehalose sketch by starch by mycose-base synthetase and mycose base hydrolase.Wherein, zero: glucosyl residue ●: the glucosyl residue reducing end-: α-1.4 glucoside bond～: α. α-1.1-glucoside bond d.p.n: the polymerization degree 〉=3.

Fig. 2 is intestinal bacteria MTH-11 mycose base hydrolase " DEAE-Fast Flow " gel chromatographic columns wash-out collection of illustrative plates.

Fig. 3 represents the optimal reactive temperature of the mycose base hydrolase of intestinal bacteria MTH-11.

Fig. 4 represents the optimum pH of the mycose base hydrolase of intestinal bacteria MTH-11.

Fig. 5 represents the temperature stability of the mycose base hydrolase of intestinal bacteria MTH-11.

Fig. 6 represents the pH value stabilization of the mycose base hydrolase of intestinal bacteria MTH-11.

Embodiment

The following example explanation is from microorganism Escherichia coli MTH-11 and yeast MTH-8, and produces in the hitherto known microorganism and purifying is of the present invention has a liking for sour thermostable mycose base hydrolase.

Embodiment 1

The preparation of mycose base hydrolase

Contain the 0.1w/v% peptone, the 0.05w/v% yeast extract, the liquid nutrient media of 0.1w/v% glucose and 0.1w/v% sodium-chlor is transferred pH to 7.0, and the liquid nutrient media of about 50ml equal portions is placed in the 250ml Erlenmeyer flask, at 15lbf/in ²(1.034 * 10 ⁵Pa) high pressure steam sterilization is 30 minutes, cooling, with the inoculation of intestinal bacteria MTH-11 inoculum, at rotating speed be on 220 rev/mins the shaking table 30 ℃ cultivated 12 hours, collect the culture of gained and as inoculum.

Go up in the fermentor tank that the liquid nutrient media of stating prepared fresh is placed on 5 liters about 3, sterilization is cooled to 28 ℃, the inoculum inoculation with 5%, and at 28-37 ℃, pH6.0-8.0, stir and the condition of ventilation under cultivation about 4-6 hour, as the OD of nutrient solution ₆₀₀During for 0.3-1.0, improve culture temperature and continue to cultivate 3-6 hour to 38-42 ℃.After cultivate finishing, with culture in 4 ℃, 6000 rev/mins centrifugal 10 minutes, be separated into somatic cells and supernatant liquor.

Embodiment 2

The purifying of enzyme

The thalline that is obtained by embodiment 1 method is handled, with 100ml acetate buffer solution (pH5.5) thalline that suspends again, ultrasonic disruption under condition of ice bath, up to thallus suspension liquid become limpid till; Gained suspension after 70 ℃ of insulation half an hour, 11, obtaining about 100mL supernatant liquor in centrifugal 20 minutes under the 000rpm is crude enzyme liquid.Use the post of 20mL " DEAE-Fast Flow " (a kind of anionite) filling to carry out column chromatography then; Target protein marine alga base hydrolase is adsorbed on the ion-exchanger, with the acetate buffer solution with different salt concn of prepared fresh it is eluted from post.Fig. 2 has represented the wash-out collection of illustrative plates of this column chromatography.When salt concn was 0.3M, mycose base hydrolase was eluted from post, merged to contain marine alga base hydrolase enzyme component alive and refining.

The component that contains mycose base hydrolase that merges is dialysed in the same acetate buffer solution that replenishes with 2M ammonium sulfate of prepared fresh.Dialyzate was removed insolubles in centrifugal 30 minutes under 10000rpm, the gained supernatant liquor carries out hydrophobic chromatography in order to the post of 10ml " Phenyl-Sepharose " (a kind of hydrophobic resin) filling.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 component with enzymic activity.Gained component post refining and that load with " Sephaeryl S-200 " (a kind of gel-filtration resin) is carried out gel permeation chromatography, reclaim component with enzymic activity.

Table 1 has shown total enzyme activity, specific activity and the yield of trehalose glycosyl hydrolase in each purification step.

Total enzyme activity is than yield alive

(U) (U/mg) (％)

Crude enzyme liquid 134,800 27

Thermal treatment 134,800 73 100

DEAE-Fast Flow ion column chromatography 109,180 153 81

Phenyl-Sepharose hydrophobic chromatography 68,748 220 51

Sephaeryl S-200 gel filtration chromatography 32,350 557 24

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

Embodiment 3

The character of mycose base hydrolase

Use contains the polyacrylamide gel of 10% sodium laurylsulfonate, will carry out electrophoresis by the pure enzyme preparation that embodiment 2 methods obtain, and by it and molecular weight of albumen mark are compared, record its molecular weight and be about 55,000-66,000 dalton.

Use contains 2w/v% amphotericeledrolyte " AMPHOLINE ", and the pure enzyme preparation that embodiment 2 methods are obtained carries 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 5.5-6.6.

According to the analysis of enzymic activity being studied temperature and pH influence to this enzymic activity.Fig. 3 (Temperature Influence) and Fig. 4 (influence of pH) have shown these results respectively.

When pH5.5 preserves 30 minutes, the optimal temperature of this enzyme is about 60-85 ℃, and when preserving 30 minutes for 65 ℃, the appropriate pH of this enzyme is about 5.0-6.0.

By will under differing temps, preserving 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 acetate buffer solution (pH5.5).By preserving 16 hours at 25 ℃ at the wide range buffered soln of different pH, adjust these damping fluids to pH5.5, analyze the pH stability that enzymic activity residual in every portion of damping fluid is measured this enzyme then.Fig. 5 and Fig. 6 have shown the heat and the pH stability result of this enzyme respectively.This enzyme is stable in temperature under up to about 80 ℃ and the about 4-11 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 as substrate, and it has terminal is that trehalose structure and glucose polymerization degree are 3 or higher.The mycose-base synthetase that adds 2 units/g substrate respectively in as the aqueous solution that contains 20% trisaccharide maltose, maltotetrose, maltopentaose, MALTOHAXAOASE or Fructus Hordei Germinatus seven sugar of substrate carried out enzyme reaction 48 hours with the gained mixture under 75 ℃ and pH5.5.With 100 ℃ of heating of this reaction mixture, make residual enzyme deactivation, filtration, decolouring, desalination also concentrate and obtain a kind of dense sugar soln, use " XT-1016 " (Na type, poly-and degree are a kind of ion-exchanger of 4%) that this solution is carried out column chromatography then.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 humidity remains on 55 ℃, and SV (volumetric velocity) be 0.13 time with 55 ℃ of hot water elutions, obtain highly purified non-reduced polysaccharide, it is that trehalose structure and glucose polymerization degree are 3 or higher that this sugar has terminal.In the goods that obtain, the purity that glucosyl trehalose goods contain the glucosyl trehalose is 97.6%, and maltose trehalose, maltotriose glycosyl trehalose, maltotetrose base trehalose and maltopentaose base marine alga and the purity of maltopentaose base trehalose in their high purity goods are respectively 98.6%, 99.6%, 98.3% and 98.1%.

Preparation contains the aqueous solution of 20% above-mentioned 5 kinds of non-reduced polysaccharide goods respectively, mycose base hydrolase with the purifying that obtains among this solution and the embodiment 2 mixes with the amount of 2 units/g substrate then, then gained solution is carried out enzyme reaction 48 hours in 75 ℃ and pH5.5.Analyze its composition the every kind of reaction mixture desalination that obtains and through high pressure liquid chromatography (HPLC) (using REZEX RNMCARBOHYDRATE post).In contrast, the same mycose base hydrolase of prepared fresh is acted on the maltose oligosaccharides, for example 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 2 shows this result.

Table 2

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
				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 mycose base hydrolase of the present invention hydrolysis specifically to have trehalose structure be 3 or greater than the key between part of the trehalose in 3 the non-reduced polysaccharide and the glycosyl part as an end and glucose polymerization degree, form trehalose and a kind of glucose polymerization degree and be 1 or greater than 1 non-reducing sugar;

2, Fructus Hordei Germinatus oligose is not by the hydrolysis of mycose base hydrolase of the present invention institute.

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

Be the trehalose in the various reaction mixtures of purifying, this reaction mixture is carried out column chromatography in order to the post of " XT-1016 (Na+ type) " (a kind of alkali metal type strong-acid cation-exchange resin) filling, reclaim afterwards and contain 97% or the component of higher trehalose.These components are merged and be 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 under these crystallizations and the vacuum.With glucosyl group trehalose, malt-base trehalose is that the yield that substrate prepares trehalose is respectively 9.5%, 14.9%, 16.0%, 18.5% and 17.7%.Fusing point, heat of fusion, specific rotation, infrared absorption spectrum, the X-diffracting spectrum of having studied the trehalose sample of buying on high purity trehalose goods and the market (making standard substance) reach the susceptibility to the hydrolytic action of trehalase (Sigma product).The result, the fusing point that various trehalose goods show is that 97.6 ± 0.5 ℃, heat of fusion are that 57.9 ± 1.2KJ/mol and specific rotation are+182 ± 1.1 °, very identical with the numerical value of standard marine alga sample, also very identical with standard marine alga sample of the infrared absorption spectrum of these trehalose goods and X-diffracting spectrum in addition.Similar to the trehalose sample of standard, these trehalose goods are easy to be become glucose monomer by the trehalase hydrolysis.

These results are unequivocally established, and are acted on by mycose base hydrolase of the present invention to have trehalose structure disconnected and glucose polymerization degree is 3 or is trehalose greater than the non-reduced polysaccharide that 3 non-reduced polysaccharide forms as an end.

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 ℃, mix with the amount of 4,000 units/g starch with a kind of isoamylase (isoamylase) sample, and make this enzyme reaction continue 20 hours.This reaction mixture is chilled to 60 ℃ in 120 ℃ of autoclavings 10 minutes, and the post in order to 750ml " Toropearl 50s gel " filling carries out gel permeation chromatography then, and obtaining glucose polymerization degree is the reductibility starch partial hydrolysate of 35-10.

With the above-mentioned reductibility starch partial hydrolysate that obtains or glucose polymerization degree is that 3 trisaccharide maltose is dissolved in 10mM acetate buffer solution (pH5.5) as substrate and makes 1% solution, then this solution is mixed with the amount of 4 units/g substrate with 4 units/mycose-base synthetase of g substrate and the mycose base hydrolase of purifying (with method preparation among the embodiment 2), and carried out enzyme reaction 24 hours in 75 ℃.After treating the reacting completely of enzyme, 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 the grape amylase sample mix of 50 units/g substrate.To top similar, the partial reaction mixture that obtains is carried out desalination and analyzes its composition with HPLC.Table 3 has shown this result.

Table 3

Reductibility starch partial hydrolysate glucose polymerization degree	Product	Form percentage (%) A B
				35	Trehalose	80.4	83.6
Glucose	0.3	16.4
			The reduction oligosaccharides		14.3	0
The glycosyl trehalose	5.0	0
			18		Trehalose	77.4	80.5
Glucose	0.3	19.5
				The reduction oligosaccharides	17.3	0
The glycosyl trehalose	5	0
				10	Trehalose	67.4	70.5
Glucose	0.2	29.5
			The reduction oligosaccharides		28.5	0
The glycosyl trehalose	4.9	0
			Trisaccharide maltose		Trehalose	4.3	23.5
	Glucose	2.2		76.5
					Trisaccharide maltose	64	0
	The glycosyl trehalose	28.3		0

A: mycose-base synthetase+this mycose base hydrolase

B: mycose-base synthetase+this mycose base hydrolase+grape amylase

As shown in table 3, using glucose polymerization degree is under 3 the situation of trisaccharide maltose as substrate, trehalose yield after mycose-base synthetase and this mycose base hydrolase generation enzyme reaction is lower, promptly 4.3%, but under the situation of starch partial hydrolysate of using glucose polymerization degree as 10-35 as substrate, the yield of this trehalose is than higher, i.e. 67.4-80.4%.And the glucose polymerization degree of finding the reductibility starch partial hydrolysate is high more, and the purity of gained trehalose is also high more.Also find by making the grape diastatic action in 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

1% glycine, 10% high purity trehalose (purity is 99.5%, by the method acquisition of embodiment 4) are dissolved in (pH7.0) in the 50mM phosphoric acid buffer,, cool off this solution then and measure its photoabsorption at 480nm wavelength place in 100 ℃ of preservations 90 minutes.With the glucose of same processing and maltose in contrast, the results are shown in Table 4.

Table 4

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

This result shows that trehalose goods of the present invention only show as more shallow color after Maillard reaction is induced, and its color intensity only is about 0.3-0.5% of glucose and maltose.Illustrating that this trehalose is gone up does not substantially participate in Maillard reaction, and 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

Mycose base hydrolase and character thereof by known microorganisms production

Yeast MTH-8 in the hitherto known microorganism strains and genus bacillus T-3 are confirmed that by the inventor they can produce mycose base hydrolase of the present invention, and be similar to embodiment 1, with these two kinds of bacterial strains respectively in fermentor tank 30 ℃ cultivated 12 hours.Make cytoclasis after the 2L nutrient solution of every kind of bacterial strain of gained collected thalline, and the centrifugal supernatant liquor that obtains, order is with chromatography on the anion-exchange column of " DEAE-Fast Flow " (a kind of anionite) then; Hydrophobic chromatography with " Phenyl-Sepharose " (a kind of hydrophobic resin); And the gel permeation chromatography that uses " SephaerylS-200 " (a kind of gel-filtration resin) obtains the enzyme preparation of purifying, studies its character then.The mycose base hydrolase of yeast MTH-8 and genus bacillus T-3 is basic similar to intestinal bacteria MTH-11.

Following embodiment A has been described the preparation of mycose base hydrolase of the present invention, and 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 intestinal bacteria MTH-11 is inoculated on the nutritional medium of 3L, and in the 5L fermentor tank, cultivated 7 hours.After having fermented, the gained fermented liquid is centrifugal, be separated into somatic cells and supernatant liquor.With 100ml acetate buffer solution (pH5.5) thalline that suspends again, ultrasonic disruption under condition of ice bath, up to thallus suspension liquid become limpid till; Gained suspension after 70 ℃ of insulation half an hour, 11, obtaining about 100mL supernatant liquor in centrifugal 20 minutes under the 000rpm is crude enzyme liquid, this solution contains the mycose base hydrolase of 1380 units/ml.

Add lime carbonate in 15% the potato starch suspension and make its final concentration reach 0.1%, gained solution is transferred to pH6.0, with 0.2% high-temperature sample 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 75 ℃, transfer pH to 5.5, with 1, the Pullulanase of 000 unit/g starch mixes with the mycose-base synthetase and the above-mentioned mycose base hydrolase solution of 4 units/g of 4 units/g starch, next carries out 48 hours enzyme reaction.The reaction mixture that so obtains kept 10 minutes in 100 ℃, 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%.

This syrup contains the maltotetrose and the senior oligosaccharides of 71.2% trehalose, 2.4% grape base trehalose, 3.3% malt-base trehalose, 0.7% glucose, 9.1% maltose, 12.1% trisaccharide maltose and 1.2%, it has tasty and refreshing moderate 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, 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 " XT-1016 " (Na ⁺Type, 4% the polymerization degree, a kind of alkali metal type strong-acid cation-exchange resin) separation of filling chromatography column.This process is as follows: use the stainless steel column loaded resin of 4 jacket layers of internal diameter 5.4cm, it is 20m that this post concatenated in order is made the total bed of a friendship with fixed attention degree of depth that obtains.This post is heated to 55 ℃ of 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, to remove the sugar of association, for example maltose and trisaccharide maltose are collected the cut of method for enriching trehalose afterwards.With this component merging of obtaining, purifying, concentrate, vacuum-drying and pulverize and obtain high density marine alga Icing Sugar, yield is about 58%.

The concentration of trehalose is about 97% in this product, and the tasty and refreshing moderate sweet taste of this product tool, 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 aqueous trehalose that obtains by embodiment A-2 method, use activated carbon decolorizing, 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, slowly cooling obtains a massecuite again, and its percent crystallization in massecuite is about 45%.This syrup obtains the trehalose crystalline powder through the dry spraying of high pressure, and is injected in the aging tower aging dry 10 hours and makes it complete crystallization, obtains Powdered crystalline trehalose, and yield is 90%.

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

Embodiment A-4

Similar to embodiment A-3, the high density aqueous trehalose with example A-2 method obtains places vaporizer, and vacuum-evaporation obtains humidity and is about 3.0% syrup.This syrup is placed in the crystallizing dish, adds about 1% hydrous crystalline trehalose,, product is placed aging tower, obtained block in aging 6 hours in 100 ℃ in 120 ℃ of following stirred crystallization 5 minutes.

The gained block is pulverized with pulverizer, and promptly obtained powdery hydrous crystalline trehalose (its humidity is about 0.4%) through fluidised bed drying, yield is about 88%.This product can be arbitrarily uses as the siccative of food, makeup, medicine, sweeting agent or weighting agent.

Embodiment A-5

Method by embodiment A-1, the inoculum of intestinal bacteria MTH-11 is inoculated on the 3L nutritional medium, cultivated 12 hours with fermentation container, after having cultivated, go cell to obtain about 3L filtrate with the SF-membrane filtration, this filtrate uses the SF-membrane concentration to the 100mL enzyme solution again, and this solution contains the mycose base hydrolase of 190 units that have an appointment/ml.

By heating 6% potato starch suspension gelatinization, transfer to pH4.5, be heated to 50 ℃, sneak into the isoamylase sample with the amount of 500 units/g starch, next carry out 20 hours enzyme reaction.This reaction mixture is transferred to pH6.5,, is chilled to 95 ℃, sneak into high-temperature, next carry out enzyme reaction in 15 minutes with the amount of 0.1%/g starch in 120 ℃ of autoclavings 10 minutes.This reaction mixture is chilled to 75 ℃ in 130 ℃ of autoclavings 30 minutes, transfers pH to 5.5, adds the mycose-base synthetase and the above-mentioned enzyme solution of 10 units/g starch, carries out enzyme reaction 64 hours afterwards.The gained reaction mixture is chilled to 50 ℃ in 100 ℃ of maintenances 20 minutes, transfers pH in 5.0, sneaks into grape amylase with the amount of 10 units/g starch, carried out enzyme reaction afterwards 40 hours, and heating makes residual enzyme deactivation.With the solution that so obtains with the ordinary method activated carbon decolorizing, with ion-exchanger desalination and about 60% solution of simmer down to (containing 80.5% trehalose).This solution concentration becomes 82% 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, with pulverizer block is pulverized and obtained powdery hydrous crystalline trehalose, yield 90% in room temperature.

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

Embodiment A-6

Press the method among the embodiment 1, the inoculum of inoculating yeast bacterium MTH-8 is inoculated in the nutritional medium, in fermentor tank, cultivated 72 hours.Gained 3L nutrient solution was removed cell in centrifugal 20 minutes in 10 under the 000rpm, and the supernatant liquor with the UF-membrane concentration obtains gets the 200mL enzyme solution, and it contains the mycose base hydrolase of 25 units/ml.

1 part of heavy potato starch is mixed stirring with the high-temperature of 6 parts of heavy water and 0.01 part of weight, 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 75 ℃, transfer pH to 5.5, with the isoamylase sample mix of 500 units/g starch, mix with the mycose-base synthetase and the above-mentioned enzyme solution of 10 units/g starch in addition, carried out enzyme reaction then 48 hours.After question response is complete, this reaction mixture heating 20min is made the residual enzyme inactivation, transfer to 50 ℃ and pH5.0, mix with glucoamylase, carried out enzyme reaction afterwards 40 hours, and heating makes remaining enzyme deactivation with 10 units/g amount of starch in 100 ℃.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.Identical with the method for implementing A-2, only be to use CG6000 (Na ⁺Type alkali metal type strong-acid cation-exchange resin) as ion-exchanger, spissated solution is carried out ion-exchange chromatography, obtain high density trehalose component, it contains 95% the trehalose of having an appointment.The gained component is concentrated to 75% concentration, places a flat plastic container crystallization, place and aging 3 days in room temperature, make caking, then this block is pulverized with pulverizer, obtain the powdery hydrous crystalline trehalose, yield is about 70%.

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 1, the seed thing of genus bacillus T-3 bacterial classification is inoculated on the nutritional medium, cultivated 36 hours in fermentation container, the 3L nutrient solution that obtains is handled with the cytoclasis instrument.The gained mixture is in 10, and centrifugal 30min removes residue under the 000rpm, with the supernatant liquor that the UR-membrane concentration obtains, obtains 100ml solution, and this solution contains the mycose base hydrolase of 12.5 units/ml.

33% tapioca (flour) suspension mixes with lime carbonate that to make its final concentration be 0.1%, and transfers its pH to 6.0, mixes with 0.3% α-Dian Fenmei, carries out enzyme reaction 20min at 95 ℃ then.The gained reaction mixture is at 2kg/cm ²Autoclaving 30min under the pressure is cooled to 75 ℃.With 200 units/g amount of starch and isoamylase sample mix, mix with the mycose-base synthetase and the above-mentioned enzyme solution of 10 units/g starch with the 0.2ml/g amount of starch, 75 ℃ were carried out enzyme reaction 48 hours afterwards again.The reaction mixture that so obtains is in 100 ℃ of maintenance 20min, 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%.

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 tasty and refreshing moderate sweet taste, 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 basket centrifugation as crystal seed.Spraying into less water, to wash resultant purity be 99% high purity hydrous crystalline trehalose, and yield is about 50%.

Embodiment B-1

Sweeting agent

Add the stevioside glycosides of 0.01 part of heavy radix asparagi sweet extract and 0.01 part of weight in 1 part that obtains by the embodiment A-5 method heavy powdery hydrous crystalline trehalose, the 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 2/5 of a sucrose.

Because this product low in calories and have gratifying stability can not decomposed other sweeting agent of sneaking into, very suitable low calorie sweetener as the low calorific food that provides to obese person and diabetic subject.

When the bacteriological action of inducing carious tooth during in this product, it does not form acid and insoluble dextran basically, so can be used as sweeting agent and prevent the generation of carious tooth.

Embodiment B-2

Boiled sweet

100 parts heavy 55% sucrose solution is mixed with the trehalose syrup of 30 parts of weights (obtaining by embodiment A-7 method) heating, and vacuum concentration to humidity is lower than 2%.After in this strong solution, adding the citric acid perfume compound and tinting material mixing of the citric acid of 1 part of weight and capacity, make desired product with ordinary method.

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

3 parts of basic nurse glue are heated to the fusing deliquescing, mix 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 the high-content lactosucrose powder of 50 parts of weights (as the open No.281 of Japanese Patent, 795/92 in disclosed) are dissolved in the water of 1150 parts of weights.Gained solution is in 65 ℃ of sterilization 30min, is cooled to 40 ℃ and the milk-acid bacteria that inoculates 30 parts of weights as initiator, obtains desired product in 37 ℃ after cultivating 8 hours.

This product is the lactic drink of the gratifying fragrance of a kind of taste.Because contain oligosaccharides in this product, can keep the growth of the stable of milk-acid bacteria and promotion bifidus bacterium.

Embodiment B-6

Powder fruit juice

The amylopectin and the capacity powdery perfume compound of the high purity marine alga Icing Sugar (making by embodiment A-2 method) of the 33 parts heavy spray-dried powdery orange juices that obtain and 50 parts of weights, 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 of weight 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 obtains weighed and pack desired product.

The product that this kind contains 30% orange juice can keep it high-quality and can not produce peculiar smell in a long time.

Embodiment B-7

Caramel custard

After the salt of the marine alga syrup (obtaining by embodiment A-7 method) of 100 parts of heavy W-Gums, 100 parts of weights, 80 parts of heavy lactose, 20 portions of heavy sucrose and 1 part of weight mixed, the milk that boils that adds 280 parts of heavy eggs and 100 parts of weights more gradually, and continuation heated and stirred, when stopping heating in the mixture during the complete gelatinization of starch, obtain translucency, cool off this mixture and the past capacity vanilla that wherein adds then.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

Sweetened bean paste sauce

Soybean with 10 parts of weights is a raw material, mix with water and boil with ordinary method, after the astringent taste and smart rough sense and water-soluble impurity of removing beans, in gains, add 14 portions of heavy sucrose, the trehalose syrup (obtaining) of 5 parts of weights and the water of 4 parts of weights by embodiment A-1 method, and, mix and carefully mediate making beans not become pasty state with a small amount of salad oil with the gained mixture boiled.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 the raw material of sweetened bean paste sauce bread, sweetened bean paste sauce steamed stuffed bun, the rice dumpling, sweetened bean paste sauce wafer, rice cake and ice cream.

Embodiment B-9

Bread

The food-yeast powder mixing back of the powdery hydrous crystalline trehalose (obtaining by embodiment A-3 method) of 100 portions of heavy flours, 2 parts of heavy yeast, 5 portions of heavy sugar, 1 part of weight, 0.1 part of weight is added the water kneading by general method, in 26 ℃ of fermentations 2 hours, aging 30 minutes and baking.

This product is a kind of high-quality bread, has gratifying color, sugariness and soft property.

Embodiment B-10

Ham

Add the saltpetre of 15 portions of heavy salt and 3 parts of weights 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 cross liquid.Then, these ham sliced meat were soaked 7 days in salts solution prior to refrigeration, this salts solution contains the powder (by the preparation of embodiment A-6 method) and the capacity pepper of the non-reduced polysaccharide of 500 parts of heavy water, 100 portions of heavy salt, 3 parts of heavy saltpetre, 40 parts of weights, 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-11

Soymilk powder

1 portion of heavy soymilk is mixed with the crystalline trehalose powder (by the method preparation of embodiment A-6) of two parts of weights, and the gained mixture is put into plastic ware, obtains the powder soymilk powder in 50 ℃ of vacuum-dryings and pulverizing.

This product has gratifying taste and fragrance, can at random be used as candy, and for example the raw material of pre-composition, ice cream and ice cream is also fed shape worker's infant food and the raw material that nutritious prod is used in treatment as oral or feed.

Embodiment B-12

Powder yolk

Yolk with new fresh hen egg makes, mixes 1 part heavy gained liquid in 60～64 ℃ of sterilizations with panel heater with the powdered anhydrous crystalline trehalose of 4 parts of weights (obtaining by embodiment A-4).And change in the container, 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 pulverizer pulverizing.

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

Embodiment B-13

Cosmetic cream

The high purity marine alga Icing Sugar (obtaining) of the glyceryl monostearate (self emulsive) of 2 parts of heavy polyethylene glycol mono stearate of heating for dissolving, 5 parts of weights, 2 parts of weights, 1 part heavy alpha-glycosyl rutin, 1 part heavy liquid 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 the purified water of 2 parts heavy L-lactic acid, 5 parts of heavy 1,3 butylene glycols and 66 parts of weights, 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-14

The powder Radix Ginseng extract

The Radix Ginseng extract that moiety is heavy mixes with the powdered anhydrous crystalline trehalose of 1.5 parts of weights (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 pulverizer, portioning obtains the powder Radix Ginseng extract.

This product and capacity VITMAIN B1 and the B2 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-15

Solid medicine

With natural human's goods with the post that is fixed with anti-human's antibody on the ordinary method with the absorption alpha-interferon, and will remove excessive albumen then as a kind of damping fluid upper prop that contains bovine serum of stablizer.After this, with the physiological saline that contains 5% high-content marine alga Icing Sugar (obtaining by embodiment A-2 method) alpha-interferon is eluted from post, this moment, the pH of physiological saline changed.The gained elutriant is carried out membrane filtration, filtrate passing through adds the anhydrous crystal maltitol powder of 20 times of volumes, and pulverize product dehydration back, and the gained powder is passed through the pelleter film-making, obtain every tablet of tablet that contains about 150 unit natural human alpha-interferons, every heavily about 200mg.

This product can be used as Sublingual tablet and gives the patient oral, and dosage is everyone 1-10 sheet/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 marine alga in this and the maltose stablizer as the natural human alpha-interferon, its activity at room temperature also can the long period, keep preferably.

Embodiment B-16

Sugar coated tablet

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

This product had higher storage tolerance and can keep its high quality in considerable time.

Embodiment B-17

Toothpaste

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

Secondary calcium phosphate 45.0%

Vanilla 2.95%

Sodium laurylsulfonate 1.5%

Glycerine 20.0%

Polyethenoxy sorbitan 0.5%

Antiseptic-germicide 0.05%

Powdery hydrous crystalline trehalose (by the preparation of embodiment A-3 method) 12.0%

Maltol 5.0%

Water 13.0%

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

Embodiment B-18

Fully nutrient

The composition that preparation is made up of following component: 500 parts of weights the nicotinamide of powdery hydration trehalose crystallization (the method preparation by 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 and 0.04 part of weight.Said composition is made desired product with pack into moistureproof pouch and the heat-sealing of the every equal portions of 25g.

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-19

Super nutritive liquid

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

This product 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-20

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 the solution that concentration is respectively 5w/v% and 30w/v%, with ordinary method this aqueous solution is carried out membrane filtration and remove pyrogen, pack under the aseptic condition in the Plastic Bottle, seal desired product.

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 substantially when depositing, and this product can at random be used for replenishing the energy and amino acid to body.

Embodiment B-21

Treatment damage ointment

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

The sulphur 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 mycose base hydrolase of the present invention with mycose-base synthetase, 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.Trehalose itself and the sugar composition that contains it can at random be used as various compositions, for example the sweeting agent 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 of limit 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 brings uncertain tremendous influence for many fields, these fields comprise that other industrial circle has food, makeup, pharmaceutical industries as starch, enzyme and biochemical science etc., and forestry, fishery, agricultural, domestic animal and chemical industry.Therefore, the present invention has far-reaching and tremendous influence to these fields.

Consider that the description at this is the preferred embodiments of the invention, but very clearly wherein can carry out various modifications and replenish.Therefore the present invention had both comprised the appended claims that meet in connotation of the present invention and the scope, had also comprised all relevant therewith these type of modifications and had replenished the claim that produces.

Claims (20)

1. have a liking for sour thermostable mycose base hydrolase for one kind, described enzyme can be at pH5.5, under 75 ℃ of conditions the specific efficient hydrolysis have trehalose structure as a terminal unit and glucose polymerization degree be 3 or higher non-reduced polysaccharide in, key between trehalose part and all the other glycosyl parts, and have following physics-chem characteristic:

(1) effect:

The specificity hydrolysis have trehalose structure as an end structure and glucose polymerization degree be 3 or higher non-reduced polysaccharide in, the glycosidic link between trehalose part and all the other glycosyl parts;

(2) molecular weight

About 55,000 to 65,000 dalton on sodium laurylsulfonate-polyacrylamide gel electrophoresis;

(3) iso-electric point

With amphotericeledrolyte on the iso-electric point electrophoresis about 5.5 to 6.6;

(4) optimum temps

When pH5.5 preserves 30 minutes, 60～85 ℃;

(5) best pH

When preserving 30 minutes for 65 ℃, 5.0～6.0;

(6) thermostability

When pH5.5 preserved 60 minutes, temperature that can be stable was about 30～80 ℃;

(7) pH stability

Preserved 16 hours at 25 ℃, but stable p H is 4.0～11.0;

Wherein, described enzyme is to be Escherichia intestinal bacteria (Escherichia Coli) MTH-11 of CGMCC No.0526 from preserving number, or extracts from preserving number is yeast belong (Saccharomyces sp) MTH-8 of CGMCC No.0525.

2. method for preparing the described enzyme of claim 1, described method comprises: in nutritional medium, cultivate the microorganism that can produce described enzyme forming described enzyme, and the enzyme of recovery gained; Described microorganism is that preserving number is Escherichia intestinal bacteria (Escherichia Coli) MTH-11 of CGMCC No.0526, and perhaps preserving number is yeast belong (Saccharomyces sp) MTH-8 of CGMCC No.0525.

3. method for preparing trehalose comprises:

(a) enzyme of claim 1 being acted on contain trehalose structure is 3 or higher non-reduced polysaccharide soln as a terminal unit and glucose polymerization degree, described enzyme can the described non-reduced polysaccharide of specificity hydrolysis in key between trehalose part and all the other glycosyl parts;

(b) trehalose of purifying gained.

4. method according to claim 3, wherein the described enzyme in the step (a) is used with mycose-base synthetase, described mycose-base synthetase can form that one or more to have trehalose structure be 3 or higher non-reduced polysaccharide as a terminal unit and glucose polymerization degree.

5. method according to claim 3 wherein also comprises the step that makes glucoamylase act on gained solution in the step (a) in the step (a).

6. method according to claim 3, wherein step (b) comprises the column chromatography that contains the post of aqueous trehalose through strong-acid cation-exchange resin is housed that makes gained in the step (a) step with Purifing Trehalose.

7. method according to claim 3, wherein step (b) also comprises the trehalose in the gained solution in the step (b) is made step moisture or the anhyrous crystalline trehalose.

8. method according to claim 3, wherein said glycosyl part is made up of one or more glucosyl residues.

9. a method for preparing the polysaccharide composition that contains trehalose comprises

(a) the described enzyme of claim 1 is acted on have trehalose as a terminal unit and glucose polymerization degree be 3 or higher non-reduced polysaccharide forming trehalose, described enzyme can the described non-reducing sugar of specificity hydrolysis in key between trehalose part and all the other glycosyl parts;

(b) polysaccharide composition that contains trehalose and other polysaccharide of recovery gained.

10. method according to claim 9, wherein with the described enzyme in the step (a) with can form one or more have trehalose structure as terminal unit and glucose polymerization degree be 3 or the mycose-base synthetase of higher non-reduced polysaccharide use.

11. method according to claim 9, wherein step (a) also contains the step that makes glucoamylase act on gained solution in the step (a).

12. method according to claim 9, wherein step (b) also comprises and crystallizes into moisture trehalose in step (a) the gained solution or the anhydrous crystalline trehalose.

13. method according to claim 9, wherein said glycosyl part is made up of one or more glucosyl residues.

14. one kind prepares the method for compositions that contains trehalose, comprising:

(a) described enzyme of claim 1 and mycose-base synthetase one are reacted on contain one or more glucose polymerization degrees be 3 or higher reductibility starch partial hydrolysate forming trehalose, described mycose-base synthetase can form that to have terminal unit of trehalose structure work and glucose polymerization degree be 3 or higher non-reduced polysaccharide;

(b) reclaim the trehalose that has or do not have the gained of other polysaccharide;

(c) trehalose that will have or not have other polysaccharide mixes material formation composition.

15. method according to claim 14, wherein said composition is a food.

16. method according to claim 14, wherein said composition is a make-up composition.

17. method according to claim 14, wherein, described composition is a pharmaceutical composition.

18. one kind is reduced the glucose polymerization degree of reductibility starch partial hydrolysate and is not increased the method for its reducing power, comprise step: described enzyme of claim 1 and mycose-base synthetase one being reacted on contain one or more glucose polymerization degrees is 3 or the solution of higher reductibility starch partial hydrolysate, and described mycose-base synthetase can form one or more, and to have trehalose structure be 3 or higher non-reduced polysaccharide as a terminal unit and glucose polymerization degree.

19. one kind can produce Escherichia intestinal bacteria (Escherichia Coli) MTH-11 that has a liking for sour thermostable mycose base hydrolase, preserving number is CGMCC No.0526.

20. one kind can produce yeast (Saccharomycessp) MTH-8 that has a liking for sour thermostable mycose base hydrolase, preserving number is CGMCC No.0525.

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