CN1151253C - Trehalose hydrolase and its preparation and use - Google Patents

Abstract

The invention discloses a novel acidophilic heat-resistant trehalose hydrolase and its preparation and application. The enzyme can be obtained from microorganisms of the genus Escherichia, Bacillus and Saccharomyces, and can be highly efficient under high temperature and acidic conditions. Hydrolyzes the bond between the trehalose moiety and the remaining glycosyl moiety in a non-reducing polysaccharide having a trehalose structure as one terminal unit and a glucose polymerization degree of 3 or higher. The molecular weight of the enzyme is about 55,000 to 65,000 Daltons; the isoelectric point is about 5.5 to 6.6. The enzyme is easily produced in large quantities through microbial fermentation, and is used for industrial preparation of trehalose. The trehalose can be widely used in food, cosmetic and pharmaceutical compositions.

Description

Trehalose hydrolase and its preparation and use

technical field

The present invention relates to a trehalose hydrolase and its preparation and use, in particular to a novel acidophilic heat-resistant trehalose hydrolase, which can specifically hydrolyze the trehalose part of non-reducing polysaccharides in an acidic high-temperature environment and the remaining glycosyl moieties, the non-reducing polysaccharide contains a trehalose structure as a terminal unit and the degree of glucose polymerization is 3 or higher. The invention also relates to the microorganism capable of producing the enzyme, the preparation of the enzyme, the trehalose obtained by using the enzyme, and the composition containing the trehalose.

Background technique

Trehalose or α,α-trehalose is known as a non-reducing polysaccharide composed of glucose units. As described in Advances in Carbohydrate Chemistry. Academic Press, 1993, 18: 201-225 and Applied and Environmental Microbiology. 1990, 56: 3213-3215, trehalose widely exists in the biological kingdom, including among animals, plants and microorganisms This sugar is present in all foods, but the content is low. Trehalose is a non-reducing disaccharide, white crystal in normal state, refreshing and sweet without aftertaste, its sweetness is 45% of that of granulated sugar; because this sugar has unique biological functions, it is effective for a variety of biological macromolecules and cell membranes It has obvious protective effect, and its use and potential effect are very extensive. It can be used as a protective agent and stabilizer for biological products and live bacterial preparations, and can also be used as a natural additive in food and cosmetics. Therefore, it is urgently required to be able to industrially prepare trehalose on a large scale.

Traditional trehalose preparation methods include the method of using yeast extract as in Japanese Patent Publication No. 154,485/75 and the joint conversion of maltose with maltose phosphorylase and trehalose phosphorylase reported in Japanese Patent Publication No. 216,695/83 to produce trehalose method. However, since the former is used as a starting material in yeast, the content of trehalose is usually lower than 15W/W% (unless otherwise specified, the term "W/W%" in this specification is abbreviated as "%"), and the extracted And the purification steps are complicated, so it is not suitable for industrialized production. The latter has the following disadvantages: (I) because trehalose is formed through glucose-1-phosphate, it is impossible to adjust the concentration of maltose as a substrate to a high level that meets the requirements: (II) the enzyme reaction system of phosphatase is transfer reaction, but it is difficult to obtain a stable continuous enzyme reaction system in actual production, so the yield of trehalose is relatively low, so that this method cannot be used as an industrial production method.

Considering these factors, everyone is now trying to find a new way to prepare trehalose by hydrolysis of starch. As a result, as disclosed in Japanese Patent Application No. 362,131,92, it was found that Rhizobium sp. M-11 (CCTCC M94031) and Arthrobacter sp. Q36 (CCTCC M94030) isolated from soil could produce A fucosyl hydrolase is used together with a fucosyl synthetase capable of forming a non-reducing polysaccharide with a trehalose terminal unit and a glucose polymerization degree of 3 or higher at normal temperature to directly produce trehalose by hydrolyzing starch. The fucosyl hydrolase can hydrolyze the bond between the trehalose moiety and the remaining glycosyl moieties in non-reducing polysaccharides having trehalose terminal units and having a glucose polymerization degree of 3 or higher at normal temperature to release trehalose. Therefore, using this trehalose preparation method, the production of trehalose from non-reducing starch partial hydrolyzate has a great improvement compared with the previous method. However, due to the long microbial culture period and low enzyme production level in this method, especially the poor thermal stability of the enzyme and its intolerance to acid, the production process is complicated, the sugar conversion rate is low, and the production cost is high. Many unfavorable factors such as high production costs cannot adapt to modern starch saccharification. High-temperature, high-efficiency, and high-quality production needs. This is because in actual production, when the starch hydrolysis temperature is lower than 55°C, it is very easy to be contaminated with bacteria and cause production pollution; in addition, the pH of the solution will continue to drop during the reaction process, resulting in the inactivation of acid-resistant enzymes, but under neutral conditions, Starch is easy to age again, forms insoluble precipitate, affects production; These are all obvious deficiencies in this method. In order to solve this problem, it is urgent to screen a new type of trehalose hydrolase, which can not only have high hydrolytic activity and ideal thermal stability, but also require ideal pH stability properties, especially under acidic conditions, can also have stable enzyme activity.

Contents of the invention

In order to achieve the above object, the present inventors extensively screened microorganisms capable of producing this new enzyme that can be obtained from non-reducing enzymes having a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher in a high-temperature acidic environment. Hydrolysis releases trehalose from sexual polysaccharides. As a result, we found that a microbial strain of the genus Escherichia (E.coli) MTH-11 is capable of hydrolyzing a non-reducing polysaccharide having a trehalose terminal structure and a glucose polymerization degree of 3 or higher to form trehalose. Enzyme; We found that this novel trehalosyl hydrolase can catalyze the hydrolysis reaction to form trehalose in a satisfactorily high yield when used together with the same thermostable and acid-stable fucosyl synthetase under high-temperature acidic conditions, Furthermore, trehalose can be easily prepared by allowing the novel fucosyl hydrolase and trehalosyl synthetase to act on the reduced starch partial hydrolyzate and recovering a reaction mixture having a relatively high trehalose content. We also found that, like microorganisms from the genus Escherichia, microorganisms from the genus Bacillus, Saccharomyces can also produce similar hydrolysis of non-reducing polysaccharides with a trehalose terminal structure and a glucose polymerization degree of 3 or higher to form trehalose trehalosyl hydrolase, we also conducted the same research and completed the present invention. We have also developed compositions containing trehalose prepared by the above-mentioned preparation method, such as foods, cosmetics and medicines, and completed the present invention.

The inventor has named these microorganisms as "Escherichia coli (Escherichia coli) MTH-11" and "Yeast bacterium (Saccharomyces) MTH-8" respectively, and on December 27, 2000, they were preserved in the General Committee of the Chinese Microbiological Strains Management Committee. China General Microbiological Culture Collection Center (CGMCC) (China, Beijing, Zhongguancun). The preservation numbers are respectively CGMCC No 0526 (Escherichia coli, MTH-11) and CGMCC No 0525 (yeast, MTH-8).

In addition to the above-mentioned microorganisms, other bacterial strains and mutants thereof of the genus Escherichia, Bacillus, and Saccharomyces; as long as they can produce the novel acidophilic heat-resistant trehalose hydrolase of the present invention, they are suitable for the present invention. The fucosyl hydrolase can specifically hydrolyze the bond between the trehalose moiety and the rest of the glycosyl moiety in a non-reducing polysaccharide having a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher under high-temperature acidic conditions.

Any nutrient medium can be used in the present invention as long as the above-mentioned microorganisms can grow thereon and produce the fucosyl hydrolase of the present invention: for example, synthetic and natural nutrient media can be used freely. Any carbon-containing substance can be used in the present invention as a carbon source as long as it is utilized by the microorganism: examples of the carbon source are sugars such as glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, Starch and starch partial hydrolysates; organic acids such as citric acid and succinic acid. The concentration of these carbon sources in the nutrient medium can be appropriately selected: for example, when glucose is used, the preferred concentration is usually 10% or less, preferably 1% or more from the viewpoint of the growth and reproduction of the microorganisms. Low. Nitrogen sources that can be used in the present invention include inorganic nitrogen compounds such as ammonium salts and nitrates; organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract and beef extract. Inorganic salts useful in the present invention include calcium, magnesium, potassium, sodium, phosphate and other salts of zinc, iron, copper, molybdenum and cobalt. Amino acids and vitamins can be added if desired.

The microorganisms usable in the present invention are cultured under aerobic conditions, usually in the range of 4-50°C, preferably in the range of 25-45°C; and pH 4-10; preferably pH 5-9. The culture time applicable in the present invention is adjusted to be longer than the time required for the growth of the microorganism to start, preferably, 8-100 hours. The concentration of dissolved oxygen (DO) in the nutrient medium is not particularly limited, and it is generally satisfactory to use DO in the range of 0.5-20 ppm. By controlling the aeration rate, stirring the nutrient medium, aerating to supplement oxygen, and increasing the internal pressure of the fermentation vessel, the DO concentration can be maintained within this range. The culture method of the microorganism can be batch culture or continuous culture.

After the microorganism culture is completed, the enzyme of the present invention is recovered. Active enzymes of the invention are found in both cell and cell-free supernatants, which can be recovered separately and used as crude enzymes; the resulting culture can also be used as crude enzymes. In the present invention, conventional liquid-solid separation methods can be used to separate cells from culture. For example, a method of centrifuging the resulting culture directly, and a method of filtering it with a pre-coated filter or separating cells by membrane filtration using a plate and frame filter or hollow fiber, the cell-free filtrate thus obtained can be kept intact. It can be used as an enzyme solution or concentrated before use, and the obtained cells can also be directly crushed to extract the enzyme solution. Concentration methods usable in the present invention are, for example, heating method, salting out using ammonium sulfate, precipitation method using acetone and alcohol, concentration method using eg plate and frame filter and hollow fiber membrane.

Cell-free filtrates and their concentrates can be used in conventional fixation methods. Examples of conventional methods are a binding method using an ion exchanger, a covalent bond adsorption method using a resin and a membrane, and an entrapment method using a high molecular weight substance. Cells isolated from the resulting culture broth can be used as crude enzyme without any further treatment or fixing before use. For example, cells are immobilized by mixing cells with alginate, dropping the resulting mixture in a calcium chloride solution, and gelling the beads into particles. The resulting particles can be immobilized with polyethyleneimine or glutaraldehyde. Enzyme preparations extracted from cells can be used as crude enzyme solutions. By extracting the enzyme of the present invention from cells, including ultrasonication, mechanical disruption using glass beads and aluminum oxide, Frenchpress (Frenchpress) disruption, etc., the resulting extract is subjected to heating pretreatment, centrifugation or membrane filtration, thereby obtaining the enzyme containing Crude Enzyme Clarification Solution of Enzyme of the Invention.

The crude enzyme solution thus obtained can be used as it is or purified by a conventional method before use. For example, it can be concentrated by crude enzyme solution, dialyzed, and then use "DEAE-Fast Flow" (an anion exchanger) anion exchange column chromatography; use "Phenyl-Sepharose" (a hydrophobic resin) hydrophobic column chromatography and gel filtration chromatography using "Sephaeryl S-200" (a gel filtration resin) to prepare a pure enzyme preparation as a single band on electrophoresis.

The thus obtained trehalosyl hydrolase of the present invention has the following physicochemical properties;

1. Function:

Specifically hydrolyzing the bond between the trehalose moiety and the rest of the glycosyl moiety in a non-reducing polysaccharide having a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher;

2. Molecular weight

About 55,000 to 65,000 Daltons on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDSPAGE);

3. Isoelectric point (PI)

About 5.5 to 6.6 on isoelectric point electrophoresis with ampholytes;

4. Optimum temperature

When stored at pH 5.5 for 30 minutes, 60-85°C;

5. Optimal pH

When stored at 65°C for 30 minutes, 5.0～6.0;

6. Thermal stability

When stored at pH 5.5 for 60 minutes, the stable temperature is about 30-80°C;

7. pH stability

Stored at 25°C for 16 hours, the stable pH is 4.0-11.0.

The activity of seaweed-based hydrolase of the present invention is detected by the following steps: add 0.1ml of enzyme solution to 0.4ml of 1.25w/w% maltotriosyl trehalose (alias α-maltotetraosyl α-glucoside) in 50mm acetate buffer solution (pH 5.5), the mixture was incubated at 75°C for 30 minutes. The resulting reaction mixture was added to Somogyi copper solution for Somogyi reaction to terminate the enzyme reaction; then the reducing ability was determined by the Somogyi-Nelson method. As a control, the enzyme solution was preheated at 100° C. for 15 minutes to inactivate the enzyme, and detected by a method similar to the above. In the present invention, 1 unit of enzyme activity is defined as: under this condition, the amount of enzyme required to increase 1 μmol of glucose reducing ability per minute.

Any substance can be used as the substrate of the enzyme of the present invention as long as it is a non-reducing polysaccharide containing a trehalose structure as a terminal unit and having a glucose polymerization degree of 3 or higher. Examples of such substrates are glucosyl trehalose, maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose and maltopentaosyl trehalose, which are produced by the action of trehalosyl synthetase on maltosyl trehalose. triose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. In addition to these substrates, low-reducing starches having a terminal trehalose structure and a glucose polymerization degree of 3 or more prepared by partially hydrolyzing starch-like substances such as starch, amylopectin, and amylose with amylase or acid Partial hydrolysates are also suitable for use in the present invention.

Examples of said amylases that partially hydrolyze starch are α-amylases, maltopentaose-forming amylases and maltohexaose-forming amylases disclosed in Handbook of Amyloses and Related Enzymes (published by Pergamon Press; Tokyo, Japan (1988)) enzyme. These amylases can be used in combination with debranching enzymes such as pullulanase and isoamylase.

There is no particular limitation on the concentration of the reducing starch partial hydrolyzate used as a substrate in the present invention. According to the invention it is even possible to carry out the enzymatic reaction to form trehalose in a solution containing 0.1% or 50% of the substrate. Insoluble suspensions containing excess substrate may also be used in the present invention. The reaction temperature usable in the enzyme reaction of the present invention may be a temperature at which the enzyme of the present invention is not inactivated, that is, up to about 80°C, preferably at 55-80°C. The usable reaction pH in the enzyme reaction of the present invention is 4.5-8.0, preferably at pH 5-7. The time for the enzyme reaction of the present invention should be selected according to the conditions of the enzyme reaction. Generally, when the enzyme of the present invention is used at 0.1-100 units/g substrate, it takes about 0.1-100 hours.

With regard to the yield of trehalose prepared from material substrates, especially in the case of trehalose prepared from non-reducing starch partial hydrolysates having a relatively low DE value while having a relatively high degree of glucose polymerization, the trehalose of the present invention The production method has the advantage of increasing the yield of trehalose higher than that obtained by the production method disclosed in Japanese Patent Application No. 362,131/92 in which fucosyl synthetase is used in combination with glucoamylase. The production method of the present invention, wherein the fucosyl synthetase and the fucosyl hydrolase of the present invention are used in combination can form trehalose in a yield of about 70% or higher, while the production method of the Japanese patent application forms trehalose The yield is only about 30%.

The enzyme mechanism of the present invention is as follows: first, the reducing starch partial hydrolyzate with a relatively high degree of glucose polymerization is converted into 1 mole of non-reducing polysaccharides with a trehalose structure as a terminal unit with trehalose synthetase, and then, the present invention is used to The fucosyl hydrolase hydrolyzes the obtained non-reducing polysaccharide into 1 mole of trehalose and 1 mole of reduced starch partial hydrolyzate whose degree of glucose polymerization is 2 lower than that of the raw material reduced starch partial hydrolyzate. For the newly formed reduced starch partial hydrolyzate with a degree of glucose polymerization of 3 or higher, it can be further converted into a non-reducing polysaccharide with a trehalose as the terminal unit, and then converted to 1 molar polysaccharide with a fucosyl hydrolase trehalose and starch partial hydrolyzate. Therefore, by repeating the enzymatic reaction of the aforementioned fucosyl synthetase and trehalosyl hydrolase, multiple moles of trehalose molecules and non-reducing partial starch hydrolyzate can be formed from 1 mole of reducing starch partial hydrolyzate. (Figure 1)

In the preparation method of the present invention, the fucosyl synthetase and the fucosyl hydrolase of the present invention can be used simultaneously to act on the non-reducing starch partial hydrolyzate with a glucose polymerization degree of 3 or higher, or used continuously in this order These two enzymes act on non-reducing starch partial hydrolysates. In order to further increase the yield of trehalose, the resulting reaction mixture can also be further subjected to the action of glucoamylase.

The reaction mixture thus obtained is filtered and concentrated by conventional methods to remove insoluble matter, and the resulting solution is then decolorized with activated carbon, desalted with H- and OH-form ion exchangers, and after further concentration into a syrupy product, which can be optionally The syrupy product is dried into a powdered product.

If necessary, purify by one or more methods, such as ion-exchange column chromatography, column chromatography fractionation with activated carbon or silica gel; separation method with organic solvents such as ethanol and acetone; alkali treatment method for degrading remaining reduced polysaccharides, etc. , it is easy to process powdered products into high-purity trehalose products.

If desired, according to the present invention, polysaccharide products containing trehalose may be hydrolyzed with amylase, α-amylase, glucoamylase, α-glucosidase and/or trehalase, or cyclodextrin dextran Polysaccharide-transfer reaction catalyzed by transferase and/or glucosyltransferase to control its sweetness and reducing power and reduce its viscosity. In addition, polysaccharide products can be optionally hydrogenated to convert them to sugar alcohols, thereby reducing their reducing power. Glucose can be removed from the resulting product by the above-mentioned purification method, such as ion exchange column chromatography, to prepare a fraction high in trehalose. The components thus obtained can be easily purified and concentrated into a syrup product, and the syrup product can be further concentrated into a supersaturated concentrate and crystallized to obtain hydrous or anhydrous crystalline trehalose as required.

Ion exchange column chromatography techniques usable in the present invention include methods using strong acid cation exchange resins disclosed in Japanese Patent Laid-Open Nos. 23,799/83 and 72,598/83. Using these techniques, associated sugars contained in crude trehalose products can be easily removed to obtain products with high trehalose content. In this case, fixed bed, moving bed and semi-mobile methods can be used at will.

In order to prepare aqueous crystalline trehalose, about 65-90% trehalose solution is placed in a crystallizer at 95°C or lower, preferably in the range of 10-90°C, in the presence of 0.1-20% seed crystals In this case, gradually cool while stirring to obtain a massecuite containing hydrous crystalline trehalose. Conventional methods such as separation, mass comminution, fluidized bed or granulation and spray drying can be used in the present invention to prepare aqueous crystalline trehalose from massecuite or crystalline polysaccharide.

When separating, the massecuite is subjected to basket centrifugation to separate the hydrous crystalline trehalose from the mother liquor, and if necessary, the hydrous crystalline trehalose is spray-washed with a small amount of cold water to facilitate the preparation of high-purity hydrous crystalline trehalose. When spray drying, by injecting massecuite with 60-85% concentration and about 20-60% crystallinity from a high-pressure pump nozzle, drying with hot air containing about 60-100°C non-melting crystal powder obtained, while stirring the obtained powder While passing hot air for about 1-20 hours, it is easy to prepare crystalline polysaccharides with no or substantially no hygroscopicity. In block crushing, a syrup with a moisture content of about 10-25% and a degree of crystallinity of about 10-60% is allowed to stand for several hours or 3 days to allow the entire contents to crystallize and solidify into blocks, and the resulting blocks are crushed or cut , and drying the resultant, thereby easily preparing crystalline polysaccharides with no or substantially no hygroscopicity. Although anhydrous crystalline trehalose can be prepared by drying crystalline trehalose containing water to dehydrate, the general method is to place a high trehalose content solution with a moisture content of less than 10% in a crystallizer under stirring at 50 -160°C, preferably in the range of 80-140°C, treatment of the solution with addition of seed crystals to obtain a massecuite of anhydrous crystalline trehalose, which is crystallized in dryness and at high temperature, by conventional methods such as block crushing, fluidization Bed granulation and spray drying The obtained anhydrous crystalline trehalose was pulverized to prepare anhydrous crystalline trehalose.

The trehalose of the present invention thus obtained is stable and has substantially no reducing power, and can be mixed with other substances, especially amino acids, polypeptides and proteins, without worrying about causing scorched deterioration and producing unpleasant smells. Trehalose itself has satisfactory quality and sweetness, and is easily hydrolyzed by trehalase in the body, so it can be assimilated, absorbed and utilized by the body when taken orally. In addition, trehalose is substantially not fermented by caries-inducing microorganisms, thus allowing it to be used as an anti-caries sweetener.

The trehalose of the present invention can be made into medicaments, such as nutritional medicaments for blood transfusion and tube feeding, which can be freely administered to the living body and easily metabolized and utilized by the living body without worrying about toxicity and side effects. Therefore, it is advantageous to use these products as energy supplements for the body.

Trehalose is a stable sweetener, especially when used in combination with binders such as pullulan, hydroxyethyl starch or polyvinylpyrrolidone. Crystalline trehalose can optionally be used as a sugar coating for tablets. In addition, trehalose has many properties such as the ability to control osmotic pressure, the ability to impart fillers, the ability to impart luster, the ability to maintain humidity, the ability to impart viscosity, substantially non-fermentability, the ability to prevent degradation of gelatinized starch, and Ability to prevent crystallization of other polysaccharides.

Therefore, trehalose and the polysaccharide composition containing trehalose in the present invention can be freely used in various compositions such as food, cigarettes, tobacco, feed, cosmetics and medicines.

Trehalose and the polysaccharide composition containing trehalose in the present invention can be used as a sweet seasoning as it is. With sufficient amount of one or more other sweeteners as needed, such as powdered syrup, glucose, fructose, maltose, sucrose, isomerized sugar, honey, maple sugar, erythrose, sorbitol, mannitol, Lactitol, stevioside, Alitame, licorice, aspartame, saccharin, glycine, and alanine or fillers such as dextrin, starch, and lactose are used together.

Powder or crystals containing trehalose and trehalose-containing polysaccharide composition of the present invention can be used as they are mixed with other excipients, fillers, diluents and binders, and made into granules, ball sticks, flat tablets , Lozenges And Tablets.

The trehalose and trehalose-containing polysaccharide compositions of the present invention are well compatible with other substances that taste sour, salty, bitter, astringent and delicious, and have relatively high acid resistance and heat resistance. It can therefore be used very well in foodstuffs, as a sweetener, a taste improver and a quality improver.

The trehalose and trehalose-containing polysaccharide composition of the present invention can be used in flavoring agents, such as amino acids, peptides, soy sauce, powdered soy sauce, wine, mayonnaise, seasoning, vinegar, pickles, instant seasoning, nucleic acid seasoning , spice mix, sugar and coffee sugar.

The trehalose and trehalose-containing polysaccharide composition of the present invention can be optionally used to increase the sweetness of the following foods: cakes, pectin, pasta, toffee; sweets such as desserts, biscuits, cheese, cookies, pies, Puddings, butter, rice pudding, custard, custard, custard, sponge cake, donuts, chocolate, chewing gum, caramels and candies; frozen desserts such as ice cream and sweetened fruit juice powders, syrups; processed Fruits and vegetables such as jam, candied fruit, tomato paste; pickled vegetables and preserved foods such as pickled radish, pickled mustard and pickled cucumber; meat products such as ham and sausage; fish products such as fish ham, fish sausage, dried fish; dried vegetables such as seaweed , dehydrated vegetables; dairy products such as milk, soy milk; canned and bottled products such as meat, fish, canned fruits and vegetables; alcoholic beverages such as rice wine, rice wine, wine and liquor; soft drinks such as coffee, tea, cocoa, fruit juice, carbonated Beverage, yogurt drink and feed containing lactic acid bacteria; convenience food such as instant pudding mix, instant hot pastry mix and instant soup mix; and special food, such as baby food, medicinal food, space food, complete nutrition; trehalose and containing The polysaccharide composition of trehalose can improve the taste and quality of the above-mentioned foods.

The trehalose and trehalose-containing polysaccharide composition of the present invention can also be used in feed and pet food for animals such as livestock, poultry, bees, silkworms and fish to improve their taste preference. Trehalose and trehalose-containing polysaccharide compositions can also be used as sweeteners, taste improvers, quality improvers and stabilizers to be added into products such as tobacco, cigarettes, toothpaste and toothpaste powder, lipstick, rouge, lip balm, face cream, internal medicine, etc. Medications, cod liver oil, oral hypothermia, gargles, cosmetics, and medicines.

The trehalose and trehalose-containing polysaccharide composition of the present invention can be used as a quality improver and stabilizer for biologically active substances sensitive to the loss of their active ingredients and activity, as well as health food and pharmaceutical compositions containing biologically active substances. Examples of the aforementioned bioactive substances are lymphokines such as α-, β-, and γ-interferon, α-tumor necrosis factor (INF-α), β-tumor necrosis factor (TNF-β), macrophage migration Inhibitors, colony-stimulating factors, transfer factors, and interleukin-2 (IL-2); hormones such as insulin, growth hormone, prolactin, erythropoietin, and follicle-stimulating hormone; biologics such as BCG vaccine, Japanese encephalitis vaccine, measles Vaccines, live polio vaccine, smallpox vaccine, tetanus toxoid, antitoxin and human immunoglobulin, injectable veterinary vaccines; antibiotics such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin and kanamycin sulfate vitamins; vitamins such as thiamine, riboflavin, L-ascorbic acid, cod liver oil, carotenoids, ergosterol, and tocopherol; enzymes such as lipase, elastase, urea kinase, protease, alpha-acetolactate decarboxylase, beta-starch enzymes, isoamylase, polyglucose and lactase; extracts such as ginseng extract, turtle extract, algae extract, aloe extract, propolis extract; viable microbial strains such as viruses, lactic acid bacteria and Yeast; other biologically active substances such as royal jelly. By using the trehalose and trehalose-containing polysaccharide composition of the present invention, the above biologically active substances can be easily processed into healthy food and pharmaceutical compositions with satisfactory stability and quality without worrying about their active ingredients and activity loss or loss.

As described above, methods for incorporating the trehalose and the composition containing trehalose and polysaccharides of the present invention into the above-mentioned substances include conventional methods such as mixing, kneading, dissolving, melting, soaking, infiltrating, spraying, spreading, coating, Spray, inject, crystallize and solidify. The trehalose and trehalose-containing polysaccharide compositions of the present invention are generally incorporated into the above-mentioned substances and compositions in an amount of 0.1% or higher, preferably 1% or higher.

Brief description of the drawings

Fig. 1 shows a schematic diagram of the generation of trehalose from starch by trehalose synthetase and trehalose hydrolase. Among them, ○: Glucose residue ●: Glucose residue reducing end -: α-1.4 glucosidic bond ~: α.α-1.1-glucosidic bond d.p.n: Polymerization degree ≥ 3.

Figure 2 is the elution profile of the "DEAE-Fast Flow" gel chromatography column of Escherichia coli MTH-11 trehalose hydrolase.

Fig. 3 shows the optimum reaction temperature of trehalosyl hydrolase from Escherichia coli MTH-11.

Fig. 4 shows the optimum pH value of trehalose hydrolase of Escherichia coli MTH-11.

Fig. 5 shows the temperature stability of trehalosyl hydrolase from Escherichia coli MTH-11.

Figure 6 shows the pH stability of trehalosyl hydrolase from Escherichia coli MTH-11.

Detailed ways

The following examples illustrate the production and purification of the acidophilic thermostable trehalose hydrolase of the present invention from microorganisms Escherichia coli MTH-11 and yeast MTH-8, as well as hitherto known microorganisms.

Example 1

Preparation of trehalose hydrolase

Liquid nutrient medium containing 0.1w/v% peptone, 0.05w/v% yeast extract, 0.1w/v% glucose and 0.1w/v% sodium chloride, adjusted to pH 7.0, and about 50ml aliquots of liquid The nutrient medium was placed in a 250ml Erlenmeyer flask, autoclaved at 15lbf/in ² (1.034×10 ⁵ Pa) for 30 minutes, cooled, and inoculated with E. coli MTH-11 seed culture at 220 rpm After culturing at 30°C for 12 hours on a shaker, the resulting culture was harvested and used as a seed culture.

Put about 3 liters of the above-mentioned freshly prepared liquid nutrient medium in a 5-liter fermenter, sterilize, cool to 28°C, inoculate with 5% seed culture, and inoculate at 28-37°C, pH 6.0-8.0 , cultivated for about 4-6 hours under stirring and aeration conditions, when the OD ₆₀₀ of the culture medium is 0.3-1.0, raise the culture temperature to 38-42°C and continue to culture for 3-6 hours. After the cultivation, the culture was centrifuged at 4°C and 6000 rpm for 10 minutes to separate bacterial cells and supernatant.

Example 2

Enzyme purification

The thalline obtained by the method of Example 1 is processed, resuspended thalline with 100ml acetic acid buffer (pH5.5), and ultrasonically disrupts under ice-bath conditions until the thalline suspension becomes clear; gained suspension is at 70 After incubation at ℃ for half an hour, centrifuge at 11,000 rpm for 20 minutes to obtain about 100 mL supernatant as crude enzyme solution. Then use 20mL "DEAE-Fast Flow" (an anion exchanger) to pack the column for column chromatography; the target protein algae-based hydrolase is adsorbed on the ion exchanger, and freshly prepared acetate buffer solution with different salt concentrations It was eluted from the column. Fig. 2 shows the elution profile of this column chromatography. When the salt concentration is 0.3M, the trehalose base hydrolase is eluted from the column, and the components containing the trehalose base hydrolase enzyme activity are combined and refined.

The pooled fractions containing trehalosyl hydrolase were dialyzed against freshly prepared same acetate buffer supplemented with 2M ammonium sulfate. The dialysate was centrifuged at 10,000 rpm for 30 minutes to remove insoluble matter, and the obtained supernatant was subjected to hydrophobic column chromatography with a column packed with 10 ml of "Phenyl-Sepharose" (a hydrophobic resin). The enzyme adsorbed on the gel was eluted from the column with a linear gradient buffer ranging from 2M to 0M, and the fraction with enzyme activity was recovered. The obtained fractions were purified and subjected to gel filtration chromatography using a column packed with "Sephaeryl S-200" (a gel filtration resin), to recover fractions having enzyme activity.

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

                                                        

(U) (U/mg) (%)

Crude enzyme solution 134800 27

Heat treatment 134800 73 100

DEAE-Fast Flow ion column chromatography 109180 153 81

Phenyl-Sepharose Hydrophobic Column Chromatography 68748 220 51

Sephaeryl S-200 gel column chromatography 32350 557 24

Purified enzyme preparations (eluates from gel filtration columns in Table 1) were electrophoretically determined for their purity using 7.5% polyacrylamide. As a result, the enzyme preparation was observed as a single protein band, which indicated that it was an electrophoretic single preparation with high purity.

Example 3

Properties of Trehalosyl Hydrolase

Using polyacrylamide gel containing 10% sodium dodecylsulfonate, the pure enzyme preparation obtained by the method of Example 2 was electrophoresed, and by comparing it with protein molecular weight markers, it was measured that its molecular weight was about 55,000- 66,000 Daltons.

Using "AMPHOLINE" containing 2w/v% ampholyte, the pure enzyme preparation obtained by the method of Example 2 was subjected to isoelectric point electrophoresis (isoeletrohopresis). The resulting gels were sliced and their pH values were then determined to give the enzyme a pi of about 5.5-6.6.

The effect of temperature and pH on the enzyme activity was studied based on the analysis of the enzyme activity. Figure 3 (effect of temperature) and Figure 4 (effect of pH) show these results, respectively.

When stored at pH 5.5 for 30 minutes, the enzyme has a suitable temperature of about 60-85°C, and when stored at 65°C for 30 minutes, the enzyme has a suitable pH of about 5.0-6.0.

The enzyme is assayed by storing the enzyme in 50 mm acetate buffer (pH 5.5) at various temperatures for 60 minutes, then cooling the buffer in test tubes with cold water and determining the residual enzyme activity in each buffer thermal stability. The pH stability of the enzyme was determined by storing broad range buffer solutions at different pH at 25°C for 16 hours, adjusting these buffers to pH 5.5, and then analyzing the residual enzyme activity in each buffer. Figure 5 and Figure 6 show the thermal and pH stability results of the enzyme, respectively. The enzyme is stable at temperatures up to about 80°C and at a pH of about 4-11.

Example 4

Preparation of trehalose from a non-reducing polysaccharide with a trehalose structure at the end and a glucose polymerization degree of 3 or higher

A non-reducing polysaccharide having a trehalose-terminated structure and a degree of glucose polymerization of 3 or higher was prepared as a substrate by the method described in Japanese Patent Application No. 362,131/92. Add 2 units/g of substrate fucosyl synthetase to aqueous solutions containing 20% maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose as substrates, and prepare the resulting mixture at 75 The enzyme reaction was carried out at pH 5.5 for 48 hours. The reaction mixture was heated at 100°C to inactivate residual enzymes, filtered, decolorized, desalted and concentrated to obtain a concentrated sugar solution, which was then treated with "XT-1016" (Na type, a 4% polymerization degree) ion exchanger) the solution was subjected to column chromatography. In column chromatography, the ion exchanger is packed with 3 jacketed stainless steel columns with an inner diameter of 2.0 cm and a length of 1 m, and then these columns are sequentially cascaded and heated until the inner temperature of the column reaches 55°C, and 5v/v% Concentrated sugar solution (to the amount of resin) is loaded, when the humidity is maintained at 55°C, and eluted with hot water at 55°C at a SV (volume velocity) of 0.13, high-purity non-reducing polysaccharides are obtained, and the sugar has seaweed at the end Sugar structure with a degree of glucose polymerization of 3 or higher. Among the products obtained, the glucosyl trehalose product contains glucosyl trehalose with a purity of 97.6%, maltose trehalose, maltotriosyl trehalose, maltotetraosyl trehalose and maltopentaose-based seaweed and malt five The purities of glycosyl trehalose in their high-purity preparations were 98.6%, 99.6%, 98.3% and 98.1%, respectively.

Prepare aqueous solutions containing 20% of the above five non-reducing polysaccharide products respectively, then mix the solution with the purified trehalosyl hydrolase obtained in Example 2 in an amount of 2 units/g substrate, and then place the resulting solution at 75 The enzyme reaction was carried out at ℃ and pH 5.5 for 48 hours. Each of the resulting reaction mixtures was desalted and analyzed for its composition by high pressure liquid chromatography (HPLC) using a REZEX RNMCARBOHYDRATE column. As a control, the freshly prepared same fucosyl hydrolase was acted on maltose oligosaccharides, such as maltose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose, and the obtained reaction mixture was analyzed by HPLC. composition. Table 2 shows the results.

Table 2 substrate product Elution time on HPLC (min) percentage(%) Glucosyl Trehalose Trehalose 27.4 17.5 glucose 33.8 6.5 Glucosyl Trehalose 23.3 76.0 Maltosyl trehalose Trehalose 27.4 44.3 maltose 28.7 44.4 Maltosyl trehalose 21.6 11.3 Maltotriosyl Trehalose Trehalose 27.4 39.5 Maltotriose 25.9 60.0 Maltotriosyl trehalose 19.7 0.5 Maltotetraosyl trehalose Trehalose 27.4 34.2 Maltotetraose 24.1 65.5 Maltotetraosyl Trehalose 18.7 0.3 Maltopentaosyl Trehalose Trehalose 27.4 29.1 maltopentose 22.6 70.6 Maltopentaosyl Trehalose 17.8 0.3 Maltotriose Maltotriose 25.9 100 Maltotetraose Maltotetraose 24.1 100 maltopentose maltopentose 22.6 100 Maltohexaose Maltohexaose 21.8 100 Maltoheptaose Maltoheptaose 21.0 100

The results in Table 3 show that:

1. The fucosyl hydrolase according to the present invention specifically hydrolyzes the bond between the trehalose moiety and the glycosyl moiety in a non-reducing polysaccharide having a trehalose structure as one end and a glucose polymerization degree of 3 or more to form seaweed Sugars and a non-reducing sugar with a glucose degree of polymerization of 1 or greater;

2. Maltooligosaccharides are not hydrolyzed by the fucosyl hydrolase of the present invention.

These results confirm that the fucosyl hydrolase of the present invention is a novel enzyme that has the ability to specifically hydrolyze non-reducing polysaccharides having a trehalose structure as one terminal and a glucose polymerization degree of 3 or more under high-temperature acidic conditions. The bond between the trehalose moiety and the glycosyl moiety, thereby releasing the trehalose from the non-reducing polysaccharide.

In order to purify trehalose in various reaction mixtures, the reaction mixture was subjected to column chromatography with a column packed with "XT-1016 (Na+ type)" (an alkali metal type strong acid cation exchange resin), and then 97% of trehalose was recovered. or higher trehalose components. These components were combined and concentrated to a concentration of about 65%, and the concentrate was left at 25°C for 2 days to crystallize hydrated crystalline trehalose, and then these crystals were separated and dried under vacuum to obtain a high purity of 99% or higher. Purity trehalose products. The yields of trehalose prepared from glucosyl trehalose and maltosyl trehalose were 9.5%, 14.9%, 16.0%, 18.5% and 17.7%, respectively. The melting point, melting heat, specific rotation, infrared absorption spectrum, X-ray diffraction pattern and the hydrolysis of trehalase (Sigma product) were studied for high-purity trehalose products and trehalose samples purchased on the market (as standard products) sensitivity. As a result, various trehalose products showed a melting point of 97.6±0.5°C, a heat of fusion of 57.9±1.2KJ/mol and a specific rotation of +182±1.1°, which were in good agreement with the values of the standard seaweed samples. The infrared absorption spectrum and X-ray diffraction pattern are also very consistent with those of the standard seaweed sample. Similar to standard trehalose samples, these trehalose preparations were easily hydrolyzed by trehalase into glucose monomers.

These results clearly confirm that the non-reducing polysaccharide formed by the fucosyl hydrolase of the present invention acting on a non-reducing polysaccharide having a trehalose structure as one end and having a glucose polymerization degree of 3 or more is trehalose.

Example 5

Preparation of trehalose from non-reducing partial starch hydrolyzate

The suspension containing 5% waxy corn starch was gelatinized by heating, adjusted to pH 4.5, heated to 50°C, mixed with a sample of isoamylase (isoamylase) in an amount of 4,000 units/g starch, and the The enzymatic reaction lasted 20 hours. The reaction mixture was autoclaved at 120°C for 10 minutes, cooled to 60°C, and then subjected to gel filtration chromatography on a column packed with 750ml "Toropearl 50s gel" to obtain a reduced starch fraction with a glucose polymerization degree of 35-10 hydrolyzate.

The above-mentioned reduced starch partial hydrolyzate or maltotriose with a glucose polymerization degree of 3 was dissolved in 10 mM acetate buffer (pH 5.5) as a substrate to make a 1% solution, and then the solution was mixed with 4 units/g substrate The fucosyl synthetase and the purified fucosyl hydrolase (prepared by the method in Example 2) were mixed in an amount of 4 units/g substrate, and the enzyme reaction was carried out at 75° C. for 24 hours. After the enzyme reaction was complete, each reaction mixture obtained was desalted and its composition was analyzed by HPLC.

Each of the remaining reaction mixtures was heated to 50° C., adjusted to pH 4.5, mixed with glucoamylase samples in an amount of 50 units/g substrate, and then subjected to enzymatic reaction for 24 hours. Similar to above, a part of the reaction mixture obtained was desalted and its composition was analyzed by HPLC. Table 3 shows the results.

table 3 Glucose polymerization degree of reduced starch partial hydrolyzate product Composition percentage (%) A B 35 Trehalose 80.4 83.6 glucose 0.3 16.4 Reducing oligosaccharides 14.3 0 Glycosyl trehalose 5.0 0 18 Trehalose 77.4 80.5 glucose 0.3 19.5 Reducing oligosaccharides 17.3 0 Glycosyl trehalose 5 0 10 Trehalose 67.4 70.5 glucose 0.2 29.5 Reducing oligosaccharides 28.5 0 Glycosyl trehalose 4.9 0 Maltotriose Trehalose 4.3 23.5 glucose 2.2 76.5 Maltotriose 64 0 Glycosyl trehalose 28.3 0

A: Trehalose synthetase + this trehalose hydrolase

B: Trehalosyl synthetase + present trehalosyl hydrolase + glucoamylase

As shown in Table 3, when maltotriose with a glucose polymerization degree of 3 is used as a substrate, the yield of trehalose after the enzymatic reaction between the fucosyl synthetase and the present fucosyl hydrolase is relatively low, that is 4.3%, but in the case of using partial hydrolyzate of starch with a glucose polymerization degree of 10-35 as the substrate, the yield of trehalose is relatively high, namely 67.4-80.4%. It was also found that the higher the degree of glucose polymerization of the reduced starch partial hydrolyzate, the higher the purity of the obtained trehalose. It has also been found that the associated non-reducing polysaccharide having a trehalose terminal and a glucose polymerization degree of 3 or more is hydrolyzed to trehalose by allowing glucoamylase to act on a reaction mixture obtained by hydrolyzing a partial hydrolyzate of reducing starch with two enzymes And glucose molecules, can increase the concentration of trehalose obtained.

Example 6

Maillard reaction

1% glycine, 10% high-purity trehalose (99.5% purity, obtained by the method of Example 4) were dissolved in 50 mM phosphate buffer (pH7.0), stored at 100 ° C for 90 minutes, then cooled the solution and Its light absorption at a wavelength of 480 nm was measured. The same treated glucose and maltose were used as controls, and the results are shown in Table 4.

Table 4 sugar products Trehalose (the present invention) Glucose (control) Maltose (control) Color degree (480nm) 0.006 1.671 0.926

The results show that the trehalose product of the present invention only exhibits a lighter color after being induced by the Maillard reaction, and its color degree is only about 0.3-0.5% of that of glucose and maltose. It shows that the trehalose basically does not participate in the Maillard reaction, and the trehalose of the present invention is a kind of sugar that is less threatening to the destruction of amino acids when they are mixed with amino acids.

Example 7

Trehalosyl hydrolases produced by known microorganisms and their properties

Saccharomyces MTH-8 and Bacillus T-3 among the microbial strains known so far have been confirmed by the inventors that they can produce the trehalosyl hydrolase of the present invention, similar to Example 1, these two strains were respectively in Incubate at 30°C for 12 hours in a fermenter. After the 2L culture fluid of each bacterial strain of gained is collected thalline, cell is broken, and centrifugation obtains supernatant, then sequentially use " DEAE-Fast Flow " (a kind of anion exchanger) on the anion exchange column chromatography; Hydrophobic column chromatography of "Phenyl-Sepharose" (a hydrophobic resin); and gel filtration chromatography using "SephaerylS-200" (a gel filtration resin) were used to obtain a purified enzyme preparation, and then its properties were studied. The trehalose hydrolase of yeast MTH-8 and Bacillus T-3 is basically similar to that of Escherichia coli MTH-11.

The following Example A describes the preparation of the trehalose-based hydrolase of the present invention, trehalose and trehalose-containing sugar compositions prepared by using the enzyme, and Example B describes the preparation of trehalose and sugar compositions containing one or more such trehalose and sugars. Compositions:

Example A-1

According to the method of Example 1, the seed culture of Escherichia coli MTH-11 was inoculated on 3 L of nutrient medium, and cultivated in a 5 L fermenter for 7 hours. After the fermentation, the obtained fermentation broth is centrifuged to separate bacterial cells and supernatant. Use 100ml of acetic acid buffer (pH 5.5) to resuspend the bacteria, and ultrasonically break it in an ice bath until the suspension of the bacteria becomes clear; after the obtained suspension is incubated at 70°C for half an hour, it is centrifuged at 11,000rpm for 20 minutes to obtain About 100mL of the supernatant is the crude enzyme solution, which contains 1380 units/ml of trehalose hydrolase.

Calcium carbonate was added to the 15% potato starch suspension to make the final concentration 0.1%, the resulting solution was adjusted to pH 6.0, mixed with 0.2% high-temperature α-amylase sample, and then the enzyme reaction was carried out at 95°C for 15 Minutes, the reaction mixture was sterilized by autoclaving at 2kg/ ^cm2 pressure for 30 minutes, cooled to 75°C, and adjusted to pH 5.5, with 1,000 units/g starch of pullulanase and 4 units/g starch of trehalose Synthetic enzyme and 4 units/g of the above trehalosyl hydrolase solution were mixed, followed by enzymatic reaction for 48 hours. The reaction mixture thus obtained was maintained at 100° C. for 10 minutes, cooled and filtered, and the resulting filtrate was decolorized with activated carbon in the usual manner, desalted and purified with H- and OH-type ion exchangers, and the solution obtained was then concentrated to 60% Syrup, the yield is about 92%.

The syrup contains 71.2% trehalose, 2.4% glucosyl trehalose, 3.3% maltosyl trehalose, 0.7% glucose, 9.1% maltose, 12.1% maltotriose and 1.2% maltotetraose and higher oligosaccharides. Moderate sweetness, low reducibility, suitable viscosity and moisturizing properties. Due to these properties, it can be used arbitrarily in foods, cosmetics and pharmaceuticals as a sweetener, flavoring agent, thickening agent, stabilizer, diluent, filler and excipient.

Example A-2

The sugar solution obtained by the method of Example 1 is used as a raw material solution, and is separated by packing a chromatographic column with "XT-1016" (Na ⁺ type, 4% polymerization degree, an alkali metal type strong acid cation exchange resin). The process is as follows: 4 jacketed stainless steel columns with an inner diameter of 5.4 cm are used to pack resin, and the columns are sequentially cascaded so that the total coagulation bed depth obtained is 20 m. Heat the column to an internal temperature of 55°C, and at this temperature, put a 5V/V% sugar solution (to the resin) on the column, elute the sugar solution with 55°C hot water to remove the associated sugar , such as maltose and maltotriose, after which a trehalose-enriched fraction is collected. The obtained components were combined, purified, concentrated, vacuum-dried and pulverized to obtain high-concentration trehalose powder with a yield of about 58%.

The concentration of trehalose in the product is about 97%, and the product has a refreshing and moderate sweet taste, so it can be used in food, cosmetics and pharmaceuticals as a sweetener, flavoring agent, quality enhancer, stabilizer , excipients and fillers.

Example A-3

The high-concentration trehalose solution obtained by the method of Example A-2 is decolorized with activated carbon, desalted with an ion exchanger, and concentrated into a 70% solution, and then the solution is put into a crystallization dish and mixed with about 2% hydrated trehalose crystals as Seed crystals are cooled slowly to obtain a massecuite with a crystallization rate of about 45%. The syrup is dried and sprayed under high pressure to obtain trehalose crystalline powder, which is poured into an aging tower for aging and drying for 10 hours to completely crystallize to obtain powdery crystalline trehalose with a yield of 90%.

The product is basically non-hygroscopic, easy to store, and can be used as a sweetener, flavoring agent, thickening agent, stabilizer, excipient and filler in food, cosmetics and medicines arbitrarily.

Example A-4

Similar to Example A-3, the high-concentration trehalose solution obtained by the method of Example A-2 was placed in an evaporator, and vacuum evaporated to obtain a syrup with a humidity of about 3.0%. Put the syrup in a crystallization dish, add about 1% hydrated crystalline trehalose, stir and crystallize at 120°C for 5 minutes, place the product in an aging tower, and age at 100°C for 6 hours to obtain a lump.

The resulting block was pulverized with a pulverizer, and dried in a fluidized bed to obtain powdery hydrated crystalline trehalose (with a humidity of about 0.4%), with a yield of about 88%. The product can be arbitrarily used as a desiccant, sweetener or filler for food, cosmetics and medicines.

Example A-5

According to the method of embodiment A-1, the seed culture of Escherichia coli MTH-11 was inoculated on 3L nutrient medium, cultivated with fermenter for 12 hours, after cultivating, remove cells with SF-membrane filter to obtain about 3L filtrate, The filtrate was then concentrated with an SF-membrane to 100 mL of an enzyme solution containing about 190 units/ml of trehalosyl hydrolase.

The 6% potato starch suspension was gelatinized by heating, adjusted to pH 4.5, heated to 50° C., and mixed with isoamylase samples in an amount of 500 units/g starch, followed by enzymatic reaction for 20 hours. The reaction mixture was adjusted to pH 6.5, autoclaved at 120° C. for 10 minutes, cooled to 95° C., mixed with high-temperature α-amylase at an amount of 0.1%/g starch, followed by enzymatic reaction for 15 minutes. The reaction mixture was autoclaved at 130° C. for 30 minutes, cooled to 75° C., adjusted to pH 5.5, added with 10 units/g starch of trehalosyl synthase and the above enzyme solution, and then carried out the enzyme reaction for 64 hours. The resulting reaction mixture was maintained at 100°C for 20 minutes, cooled to 50°C, adjusted to pH 5.0, mixed with glucoamylase at an amount of 10 units/g starch, and then subjected to an enzyme reaction for 40 hours, and heated to inactivate the residual enzyme. The solution thus obtained was decolorized with activated carbon, desalted with an ion exchanger and concentrated to about 60% solution (containing 80.5% trehalose) in a conventional manner. The solution is concentrated to an 82% solution, and then placed in a crystallization dish and mixed with hydrated crystalline trehalose accounting for about 2% of the dry weight of the solution as a seed crystal, and the trehalose is crystallized under stirring. The obtained crystals were put into a flat plastic container, left at room temperature for 3 days to agglomerate, and pulverized with a pulverizer to obtain powdery hydrated crystalline trehalose, with a yield of 90%.

The product is basically non-hygroscopic and easy to preserve, so it can be used arbitrarily in various compositions such as food, cosmetics and in pharmaceuticals and biological products.

Example A-6

According to the method in Example 1, the seed culture of strain yeast MTH-8 was inoculated in the nutrient medium, and cultivated in a fermenter for 72 hours. The resulting 3 L of culture solution was centrifuged at 10,000 rpm for 20 minutes to remove cells, and the resulting supernatant was concentrated with a UF-membrane to obtain 200 mL of an enzyme solution containing 25 units/ml of trehalosyl hydrolase.

1 part by weight of potato starch is mixed with 6 parts by weight of water and 0.01 part by weight of high-temperature α-amylase, adjusted to pH 6.2 and heated to 85-90°C to gelatinize and liquefy the starch. The resulting liquid starch was autoclaved at 120°C for 10 minutes to inactivate the remaining enzymes, cooled to 75°C, adjusted to pH 5.5, mixed with 500 units/g starch of isoamylase sample, and 10 units/g starch of trehalose Base synthetase and the above-mentioned enzyme solution were mixed, and then the enzyme reaction was carried out for 48 hours. After the reaction is complete, heat the reaction mixture at 100° C. for 20 minutes to inactivate the remaining enzymes, adjust to 50° C. and pH 5.0, mix 10 units/g of starch with glucoamylase, and then carry out the enzyme reaction for 40 hours, and Heat inactivates the remaining enzymes. The reaction mixture thus obtained was decolorized with activated charcoal in a conventional manner, desalted with an ion exchanger and concentrated to a ca. 60% solution containing 78.3% trehalose. It is the same as the method of implementing A-2, except that CG6000 (Na ⁺ type alkali metal type strong acid cation exchange resin) is used as an ion exchange agent, and the concentrated solution is subjected to ion exchange column chromatography to obtain a high concentration trehalose component, which contains About 95% trehalose. Concentrate the obtained components to a concentration of 75%, place them in a flat plastic container for crystallization, place them at room temperature and age them for 3 days to make them agglomerate, and then crush the agglomerates with a pulverizer to obtain powdery hydrated crystalline trehalose , the yield was about 70%.

The product is basically non-hygroscopic and easy to store, and can be freely used in various compositions such as sweeteners, flavoring agents, enhancers, excipients, fillers and diluents in food, cosmetics and pharmaceuticals .

Example A-7

According to the method of Example 1, the seed material of the Bacillus sp. T-3 strain was inoculated on the nutrient medium, cultivated in a fermenter for 36 hours, and the obtained 3 L culture solution was treated with a cell disruptor. The resulting mixture was centrifuged at 10,000 rpm for 30 min to remove residues, and the resulting supernatant was concentrated with a UR-membrane to obtain 100 ml of a solution containing 12.5 units/ml of trehalosyl hydrolase.

33% cassava starch suspension was mixed with calcium carbonate to make the final concentration 0.1%, and its pH was adjusted to 6.0, mixed with 0.3% α-amylase, and then the enzyme reaction was carried out at 95°C for 20 minutes. The resulting reaction mixture was autoclaved for 30 min at a pressure of 2 kg/cm ² and cooled to 75 °C. Then 200 units/g starch was mixed with isoamylase sample, 0.2 ml/g starch was mixed with 10 units/g starch trehalosyl synthase and the above enzyme solution, and then the enzyme reaction was carried out at 75°C for 48 hours. The reaction mixture thus obtained was maintained at 100° C. for 20 min, cooled and filtered to obtain a filtrate, which was decolorized with activated carbon in a conventional manner, desalted with H- and OH-type ion exchangers, and then concentrated into a 60% syrup with a yield of about 90%.

The product contains 60.1% trehalose, 1.4% glucosyl trehalose, 1.5% maltosyl trehalose, 1.0% glucose, 6.5% maltose, 8.3% maltotriose, 21.2% maltotetraose and higher oligosaccharides. Moderate sweetness, also has low reduction and moderate moisturizing properties. Due to these factors, it can be arbitrarily used in various compositions such as foods, cosmetics and pharmaceuticals as sweeteners, flavoring agents, enhancers, stabilizers, excipients, fillers and diluents.

Example A-8

The high-concentration solution containing about 95% trehalose obtained by the method of Example A-6 was decolorized and desalted by conventional methods. Concentrate the resulting solution to a 75% solution, then transfer the solution to a crystallization dish, mix in 2% hydrated crystalline trehalose as a seed crystal, heat to 50°C, gradually cool to 25°C while stirring, and use a basket centrifuge to separate crystallized. The obtained high-purity hydrated crystalline trehalose with a purity of 99% was sprayed with a small amount of water for washing, and the yield was about 50%.

Example B-1

sweetener

Add 0.01 part by weight of aspartame and 0.01 part by weight of stevioside to 1 part by weight of powdery hydrated crystalline trehalose obtained by the method of Example A-5, and send the resulting mixture into a granulator to obtain Granular sweetener. The product has a satisfactory sweet taste, and the sweetness is 2.5 times that of sucrose, and the caloric value is 2/5 of that of sucrose.

Because the product is low in calories and has satisfactory stability, it will not decompose other sweeteners mixed in, and is very suitable as a low-calorie sweetener for low-calorie foods provided to obese people and diabetic patients.

When caries-inducing bacteria act on this product, it does not substantially form acid and insoluble glucans, and thus can be used as a sweetener to prevent caries.

Example B-2

hard candy

100 parts by weight of 55% sucrose solution and 30 parts by weight of trehalose syrup (obtained by the method in Example A-7) were heated and mixed, and concentrated in vacuum until the humidity was lower than 2%. After adding 1 part of heavy citric acid and a sufficient amount of citric acid fragrance and coloring agent to the concentrated solution, the desired product is prepared by conventional methods.

The product is a high quality hard candy with a satisfying taste and chewing properties without concern for sucrose crystallization.

Example B-3

chewing gum

3 parts of gum gum are heated to melt and become soft, then mixed with 4 parts of heavy sucrose and 3 parts of heavy hydrated crystalline trehalose powder (obtained by the method of Example A-3), and then mixed with sufficient fragrance and coloring agent mix. The resulting mixture is kneaded by a conventional method and packaged to obtain the desired product.

The product is a chewing gum with a pleasing texture and taste.

Example B-4

sweetened condensed milk

Three parts by weight of trehalose syrup and 1 part by weight of sucrose obtained by the method of Example A-1 were dissolved in 100 parts of fresh milk, and the resulting solution was heated and sterilized by a flat heater and concentrated to a concentration of 70%, and then obtained The concentrate is aseptically filled into the desired product.

The product has a milky sweetness and a satisfying taste which makes it arbitrarily used as a flavoring in baby food, newborn food, fruit, coffee, cocoa and tea.

Example B-5

Lactic acid drink

170 parts by weight of skimmed milk, 130 parts by weight of trehalose syrup (obtained by the method of Example A-1) and 50 parts by weight of high-content lactose powder (as disclosed in Japanese Patent Publication No.281,795/92 open) dissolved in 1150 parts by weight of water. The obtained solution was sterilized at 65° C. for 30 minutes, cooled to 40° C. and inoculated with 30 parts by weight of lactic acid bacteria as a starter, and cultivated at 37° C. for 8 hours to obtain the desired product.

The product is an aromatic lactic drink with a satisfying taste. Because the product contains oligosaccharides, it can maintain the stability of lactic acid bacteria and promote the growth of bifidobacteria.

Example B-6

powdered juice

33 parts of heavy powdered orange juice obtained through spray drying and 50 parts of heavy high-purity trehalose powder (made by the method of Example A-2), 10 parts of heavy sucrose, 0.65 parts of heavy anhydrous citric acid, 0.1 parts by weight of malic acid, 0.1 parts by weight of L-ascorbic acid, 0.1 parts by weight of sodium citrate, 0.5 parts by weight of pullulan and a sufficient amount of powdered fragrance are mixed evenly. The resulting mixture was pulverized and sent to a granulator for granulation while spraying with trehalose syrup (obtained by the method of Example A-1) as a binder and ventilating with air at 40°C. The obtained granules are weighed and packaged to obtain the desired product.

The product containing 30% orange juice can maintain its high quality for a long time without producing off-flavor.

Example B-7

Custard Jelly

After mixing 100 parts by weight of cornstarch, 100 parts by weight of trehalose syrup (obtained by the method of Example A-7), 80 parts by weight of lactose, 20 parts by weight of sucrose and 1 part by weight of salt, gradually add 280 parts part eggs and 100 parts boiling milk, and continue to heat and stir, stop heating when the starch in the mixture is completely gelatinized and a translucent mass is obtained, then cool the mixture and add enough vanilla essence to it. The resulting mixture is weighed and filled to obtain the desired product.

The product has a smooth and shiny surface and has a milky and sweet taste.

Example B-8

bean paste

Use 10 parts by weight of soybeans as raw material, mix them with water in a conventional way and boil them. After removing the astringency and roughness of beans and water-soluble impurities, add 14 parts by weight of sucrose and 5 parts by weight of seaweed to the resultant. Sugar syrup (obtained by the method of Example A-1) and 4 parts by weight of water, and the resulting mixture was boiled, mixed with a small amount of salad oil and kneaded carefully until the beans were not mushy. In this way, about 35 kg of the desired product were produced.

The product, which is not discolored by boiling, has a satisfying taste and aroma, which makes it useful as a raw material for red bean paste bread, red bean paste buns, glutinous rice balls, red bean paste crackers, rice cakes, and ice cream.

Example B-9

bread

100 parts of heavy wheat flour, 2 parts of heavy yeast, 5 parts of heavy sugar, 1 part of heavy powdery hydrated crystalline trehalose (obtained by the method of Example A-3), 0.1 part of heavy edible yeast powder are mixed and pressed General method Add water and knead, ferment at 26°C for 2 hours, age for 30 minutes and bake.

The product is a high-quality bread with pleasing color, sweetness and softness.

Example B-10

Ham

Add 15 parts by weight of salt and 3 parts by weight of potassium nitrate to 1000 parts by weight of sliced ham and grind them evenly, pile up the ham slices and put them in the refrigerator for overnight. Then, the ham slices were soaked in salt solution for 7 days prior to refrigeration, and the salt solution contained 500 parts of heavy water, 100 parts of heavy salt, 3 parts of heavy potassium nitrate, 40 parts of heavy non-reducing polysaccharide powder ( Prepared by the method of Example A-6) and a sufficient amount of pepper, then wash with cold water in a conventional manner, hang up for smoking, cook, and cool and pack to obtain the desired product.

The product is a high quality ham with a pleasing colour, taste and aroma.

Example B-11

soy milk powder

1 part by weight of soybean milk was mixed with two parts by weight of crystalline trehalose powder (prepared by the method of Example A-6), and the resulting mixture was put into a plastic container, dried in vacuum at 50° C. and pulverized to obtain powdered soybean milk powder.

The product has a satisfactory taste and aroma, and can be freely used as a raw material for confectionery such as premixes, sorbet and ice cream, and also as a raw material for oral or feed-fed baby food and therapeutic nutritional products.

Example B-12

powdered egg yolk

Egg yolks prepared from fresh eggs were sterilized with a flat heater at 60-64° C., and 1 part by weight of the obtained liquid was mixed with 4 parts by weight of powdery anhydrous crystalline trehalose (obtained by Example A-4). And transfer it into a container, let stand overnight to agglomerate, at this time the anhydrous crystalline trehalose is transformed into hydrated crystalline trehalose. The lump thus obtained was pulverized with a pulverizer to obtain powdered egg yolk.

The product can be used arbitrarily as a raw material for confectionery, such as premixes, ice cream and dairy products, and also as a raw material for oral or feeding forms of baby food and therapeutic nutrition. This product can also be used as a skin conditioner and hair restorer.

Example B-13

cosmetic cream

Heating and dissolving 2 parts of heavy polyethylene glycol monostearate, 5 parts of heavy glyceryl monostearate (self-emulsifying), 2 parts of heavy high-purity trehalose powder (through embodiment A- 2 method), 1 part by weight of α-glycosyl rutin, 1 part by weight of liquid petrolatum, 10 parts by weight of tri-2-ethyl cyclohexanoic acid glyceride and a sufficient amount of antibacterial agent. Mix the resulting solution with 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butanediol and 66 parts by weight of purified water, and emulsify the resulting mixture with a homogenizer, and then mix in a sufficient amount of essence under stirring to obtain a cosmetic Frost.

The product has high stability and can be freely used as a high-quality sunscreen, skin care agent and skin whitening agent.

Example B-14

Powdered Ginseng Extract

Half the weight of ginseng extract was mixed with 1.5 parts by weight of powdered anhydrous crystalline trehalose (obtained by the method of Example A-4), the resulting mixture was transferred to a flat container, and allowed to stand for 2 days. Anhydrous crystalline seaweed The sugar transforms into hydrated crystalline trehalose, which agglomerates. The resulting block was pulverized with a pulverizer, and the powdered ginseng extract was obtained in portions.

The product and sufficient vitamins B1 and B2 are loaded into a granulator to obtain vitamin-containing powdered ginseng extract.

The product thus obtained can be arbitrarily used as a tonic, a fatigue recovery agent and a tonic.

This product can also be used as a hair restorer.

Example B-15

solid medicine

The natural human α-interferon product is immobilized with the column of anti-human α-interferon antibody by conventional methods to absorb α-interferon, and a buffer containing bovine serum as a stabilizer is applied to the column, and then removed excess protein. Thereafter, α-interferon was eluted from the column with physiological saline containing 5% high-content trehalose powder (obtained by the method in Example A-2), and the pH of the physiological saline changed at this time. The obtained eluate is subjected to membrane filtration, and the filtrate is added with 20 times the volume of anhydrous crystalline maltose powder, the product is dehydrated and crushed, and the obtained powder is tableted by a tablet machine to obtain about 150 units of natural human α-interferon per tablet. Tablets, each weighing about 200mg.

The product can be administered orally to patients as a sublingual tablet, with a dose of 1-10 tablets per person per day, and is optionally used for the treatment of viral diseases, allergies, arthritis, diabetes and tumors. More specifically, the product is suitable for use as a medicament for the treatment of AIDS and hepatitis, the number of patients suffering from such diseases is significantly increasing. The seaweed and maltose mixed in it are used as a stabilizer of natural human alpha-interferon, and its activity can be maintained for a long time and well at room temperature.

Example B-16

sugar coated tablets

Unprocessed tablets weighing 150 mg were coated with a solution until the total weight of the tablet reached 230 mg. The coating solution consisted of 40 parts by weight of powdery hydrated crystalline trehalose (obtained by the method in Example A-3), 2 Parts by weight of vanilla essence (average molecular weight 200,000), 30 parts by weight of water, 25 parts by weight of talc and 3 parts by weight of titanium dioxide; the resulting tablet is then coated with a solution consisting of 65 parts by weight of fresh The same preparation of hydrated crystalline trehalose, 1 part by weight of vanilla essence and 34 parts by weight of water, and glazed with liquid paraffin gave sugar-coated tablets with a satisfactory gloss and appearance.

The product has a high storage tolerance and maintains its high quality for a considerable period of time.

Example B-17

toothpaste

Toothpaste is prepared in a conventional manner by mixing the following ingredients:

Calcium hydrogen phosphate 45.0%

Vanilla essence 2.95%

Sodium Lauryl Sulfonate 1.5%

Glycerin 20.0%

Polyoxyethylene sorbitan 0.5%

Antimicrobial agent 0.05%

Powdered hydrated crystalline trehalose (prepared by the method of Example A-3) 12.0%

Maltol 5.0%

Water 13.0%

The product was satisfactorily used as a toothpaste for newborns due to sufficient sweetness.

Example B-18

Complete Nutrients

A composition consisting of the following components was prepared: 500 parts by weight of powdery hydrated trehalose crystals (prepared by the method of Example A-6), 270 parts by weight of powdered egg yolk, 209 parts by weight of skimmed milk, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate, 0.01 parts by weight of thiamine, 0.1 parts by weight of sodium ascorbate, 0.6 parts by weight of vitamin E and 0.04 parts by weight of nicotinamide. The composition was filled into moisture-proof sachets in 25 g aliquots and heat sealed to produce the desired product.

Dissolve 1 bag of this product in about 150-300ml of water to make liquid food, and administer it to the nasal cavity, stomach or intestinal tract through oral or parenteral feeding to supplement the body's energy.

Example B-19

super nutrient solution

The high-purity hydrated crystalline trehalose prepared by the method of Example A-8 was dissolved in water to make a 10/w/v% trehalose aqueous solution, and then the aqueous solution was subjected to membrane filtration to remove pyrogens in a conventional manner, and loaded under sterile conditions Put it into a plastic bottle and seal it to get the desired product.

The product is a satisfactorily stable supernutrient solution that is essentially unchanged on storage and is suitable for intravenous and intraperitoneal administration. A 10w/v% solution of this product is isotonic with blood and has been shown to provide energy to the body at concentrations twice as high as glucose.

Example B-20

super nutrient solution

The high-purity hydrated crystalline trehalose prepared by the method of Example A-8 and the amino acid composition composed of the following components were dissolved in water under stirring to obtain solutions with concentrations of 5w/v% and 30w/v%, respectively, and conventionally Methods The aqueous solution was membrane-filtered to remove pyrogens, put into plastic bottles under aseptic conditions, and sealed to obtain the desired product.

Composition of Amino Acid Composition

Component mg/100ml

L-Isoleucine 180

L-Leucine 410

L-Lysine monohydrochloride 620

L-methionine 240

L-Phenylalanine 290

L-threonine 180

L-Tryptophan 60

L-Valine 200

L-Arginine Hydrochloride 270

L-Histidine monohydrochloride 130

Glycine 340

Although the product is a complex super nutrient solution containing trehalose and amino acids, it has satisfactory stability and basically does not change during storage. The product can be used to supplement energy and amino acids to the body at will.

Example B-21

ointment for wounds

200 parts of heavy trehalose powder (produced by the method of embodiment A-2) and 300 parts of heavy maltose are mixed with 50 parts of heavy methanol solution containing 3 parts of heavy sulfur, and this gained solution is mixed with 200 parts of Part by weight of 10 w/v/% aqueous solution of vanilla essence was mixed to obtain the desired product with satisfactory spreadability and stickiness.

The sulfur contained in this product exhibits bactericidal activity, the trehalose in this product acts as an energy supplement for living cells, thanks to these properties, this product shortens the healing period of the wound surface and allows a satisfactory recovery.

It can be seen from the above that when the fucosyl hydrolase of the present invention acts on the partial hydrolyzate of reducing starch together with the fucosyl synthetase, it can release trehalose from non-reducing polysaccharides with a higher yield. The non-reducing polysaccharide has a trehalose structure end and a glucose polymerization degree of 3 or higher. The trehalose obtained in this way can be easily separated and purified, and the obtained trehalose and the sugar composition containing it have satisfactory stability, high quality and moderate sweetness. The trehalose can be conveniently digested, absorbed and utilized by the body when the unprocessed product is taken orally or the infusion solution is administered through the gastrointestinal tract. Trehalose itself and sugar compositions containing it can be arbitrarily used as various compositions such as sweeteners, bulking agents, stabilizing agents, excipients and fillers in foods, cosmetics and pharmaceuticals.

Therefore, the present invention provides a new technology for preparing trehalose and sugar compositions containing it on an industrial scale, which uses the starch partial hydrolyzate prepared from cheap and abundant starch as raw material, and has a relatively low cost. Therefore, the present invention brings unpredictable great influence to many fields, these fields include such as starch, enzyme and biochemical science etc., other industrial fields have food, cosmetics, pharmaceutical industry, and forestry, fishery, agriculture, livestock and chemical industry. Therefore, the present invention has a profound and huge impact on these fields.

While the description herein is of preferred embodiments of the invention, it will be apparent that various modifications and additions may be made therein. The present invention therefore embraces the appended claims falling within the true spirit and scope of the present invention, as well as all such amendments and additions arising therefrom.

Claims (20)

1. An acidophilic thermostable trehalose hydrolase, said enzyme can specifically and efficiently hydrolyze non-alcohol having a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher at pH 5.5 and 75°C. Reducing the bond between the trehalose moiety and the rest of the sugar moiety in the polysaccharide, and has the following physical and chemical properties: (1) Function: Specifically hydrolyzing the glycosidic bond between the trehalose moiety and the rest of the glycosyl moiety in a non-reducing polysaccharide having a trehalose structure as a terminal structure and a glucose polymerization degree of 3 or higher; (2) Molecular weight About 55,000 to 65,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (3) Isoelectric point About 5.5 to 6.6 on isoelectric point electrophoresis with ampholytes; (4) Optimal temperature When stored at pH 5.5 for 30 minutes, 60-85°C; (5) optimal pH When stored at 65°C for 30 minutes, 5.0～6.0; (6) thermal stability When stored at pH 5.5 for 60 minutes, the stable temperature is about 30-80°C; (7) pH stability Stored at 25°C for 16 hours, the stable pH is 4.0-11.0; Wherein, the enzyme is obtained from Escherichia coli (Escherichia Coli) MTH-11 whose preservation number is CGMCC No.0526, or from Saccharomyces sp MTH-11 whose preservation number is CGMCC No.0525. 8 extracted. 2. A method for preparing the enzyme of claim 1, said method comprising: cultivating a microorganism capable of producing said enzyme in a nutrient medium to form said enzyme, and reclaiming the enzyme of gained; said microorganism is Escherichia coli (Escherichia Coli) MTH-11 with the preservation number CGMCC No.0526, or Saccharomyces sp MTH-8 with the preservation number CGMCC No.0525. 3. A method for preparing trehalose, comprising: (a) making the enzyme of claim 1 act on a non-reducing polysaccharide solution containing a trehalose structure as a terminal unit and having a glucose polymerization degree of 3 or higher, the enzyme being capable of specifically hydrolyzing the trehalose moiety in the non-reducing polysaccharide and the bond between the remaining glycosyl moieties; (b) Purification of the obtained trehalose. 4. The method according to claim 3, wherein the enzyme in step (a) is used together with a fucosyl synthetase capable of forming one or more trehalose structures as Non-reducing polysaccharides with one terminal unit and a glucose degree of polymerization of 3 or higher. 5. The method according to claim 3, wherein the step (a) further comprises the step of making the glucoamylase act on the solution obtained in the step (a). 6. The method according to claim 3, wherein the step (b) comprises the step of purifying the trehalose by subjecting the trehalose-containing solution obtained in the step (a) to column chromatography equipped with a strong acid cation exchange resin. 7. The method according to claim 3, wherein step (b) further comprises the step of making trehalose in the solution obtained in step (b) into hydrous or anhydrous crystalline trehalose. 8. The method of claim 3, wherein the glycosyl moiety consists of one or more glucose residues. 9. A method for preparing a trehalose-containing polysaccharide composition, comprising (a) making the enzyme according to claim 1 act on a non-reducing polysaccharide having trehalose as a terminal unit and a glucose polymerization degree of 3 or higher to form trehalose, said enzyme capable of specifically hydrolyzing the non-reducing polysaccharide the bond between the trehalose moiety and the rest of the glycosyl moiety in the sugar; (b) recovering the resulting polysaccharide composition containing trehalose and other polysaccharides. 10. The method according to claim 9, wherein the enzyme in step (a) is combined with an enzyme capable of forming one or more non-reducing polysaccharides having a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher. Use with fucosyl synthase. 11. The method according to claim 9, wherein step (a) further comprises the step of making glucoamylase act on the solution obtained in step (a). 12. The method according to claim 9, wherein step (b) further comprises crystallizing the trehalose in the solution obtained in step (a) into hydrous or anhydrous crystal trehalose. 13. The method of claim 9, wherein the glycosyl moiety consists of one or more glucose residues. 14. A method of preparing a trehalose-containing composition comprising: (a) making the enzyme according to claim 1 act together with a fucosyl synthetase on a reducing starch partial hydrolyzate containing one or more glucose polymerization degrees of 3 or higher to form trehalose, said trehalose The base synthetase can form a non-reducing polysaccharide with a trehalose structure as a terminal unit and a glucose polymerization degree of 3 or higher; (b) recovering the resulting trehalose with or without other polysaccharides; (c) Incorporating trehalose with or without other polysaccharides into the material composition. 15. The method of claim 14, wherein said composition is a food product. 16. The method of claim 14, wherein said composition is a cosmetic composition. 17. The method of claim 14, wherein said composition is a pharmaceutical composition. 18. A method for reducing the degree of glucose polymerization of reducing starch partial hydrolyzate without increasing its reducing ability, comprising the steps of: making the enzyme according to claim 1 and the trehalose-based synthetase act together on one or more A solution of a reduced starch partial hydrolyzate having a glucose polymerization degree of 3 or higher, said fucosyl synthetase being capable of forming one or more types of starch having a trehalose structure as a terminal unit and having a glucose polymerization degree of 3 or higher of non-reducing polysaccharides. 19. An Escherichia coli (Escherichia Coli) MTH-11 capable of producing acidophilic thermostable trehalose hydrolase, the preservation number is CGMCC No.0526. 20. A yeast (Saccharomycessp) MTH-8 capable of producing acidophilic thermostable trehalose hydrolase, the preservation number is CGMCC No.0525.

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