CN1946853A - Sugar with high content of lactose and its production method and use - Google Patents

Abstract

The present invention provides a method for producing a lactosucrose high-content sugar containing 70% or more of lactosucrose, 1-kestose and fructosyl lactosucrose in total of less than 3% as a sugar composition by allowing a sucrose non-utilizable yeast and β -fructofuranosidase derived from a microorganism belonging to the genus Bacillus to act in an aqueous solution containing sucrose and lactose, a method for producing a high-purity lactosucrose containing 90% or more of lactosucrose as a sugar composition by subjecting the lactosucrose high-content sugar to chromatography, a method for producing crystalline lactosucrose, and a lactosucrose high-content sugar and a high-purity lactosucrose obtained by the production methods.

Description

Sugar with high content of lactose and its production method and use

technical field

The present invention relates to sugar with high content of lactose and its production method and application. More specifically, it relates to a sugar composition containing greater than or equal to 70% of lactose, less than 3% of 1-kestose and kestose in total. Lactose-rich sugar of fructose-based lactose-sucrose and method for producing the same, further relating to extracting the lactose-rich sugar as a fraction containing 90% or more of lactose as a sugar composition by chromatography using a resin The invention relates to a method for producing high-purity lactose, and further relates to a method for producing crystalline lactose.

Background technique

In recent years, the functions of various sugars such as oligosaccharides have been gradually clarified. Among the functional oligosaccharides, lactosucrose [β-D-galactosyl-(1,4)-α-D-glucosyl-(1,2)-β-D-fructoside] is indigestible It is an oligosaccharide useful in a wide range of applications such as food, cosmetics, and medicine due to its excellent functions such as promoting the growth of bifidobacteria, preventing cariogenicity, and moisturizing properties. Lactose-sucrose is industrially produced by allowing β-fructofuranosidase derived from a microorganism belonging to the genus Arthrobacter to act on an aqueous solution containing sucrose and lactose, and through a sugar transfer reaction catalyzed by the β-fructofuranosidase (see Kaiping No. 3-27285 Bulletin). However, only by the transfer reaction of β-fructofuranosidase, the lactose content of the obtained reactant is as low as about 30% as a sugar composition, and the reactant contains a relatively large amount of unreacted lactose, sucrose , glucose and fructose as reaction residues, and 1-kestose and fructosyl lactosucrose as by-products, so it is difficult to say that the sugar composition can fully exert the original function of lactose. Therefore, in order to improve the purity of lactose, unreacted lactose is partially crystallized after the enzymatic reaction, and then either filtered and removed, or during the enzymatic reaction, invertase-deficient yeast is added to carry out monosaccharides such as glucose. Utilize the method, to manufacture lactose-containing sugar (registered trademark "milk fruit Oligo") (refer to Fujita Xiaohui, "Biosepare-ヨンヨンプロセス handbook" (biochemical separation process handbook), 196-201 pages, published by Kyoritsu Publishing, 1996 Year). In particular, in the transfer reaction of β-fructofuranosidase, the method of using invertase to delete yeast not only removes monosaccharides such as glucose from the reaction solution, but also removes monosaccharides, and the transfer reaction tends to produce lactosucrose, so there is lactosucrose. The amount of production of lactose can also improve such benefits, and the produced lactose can reach about 65% of the sugar composition as a reactant (see JP-A-4-293494). In addition, the reactant whose lactose content has been increased is subjected to chromatography using an ion exchange resin in the raw material to increase the purity of lactose to about 80%, and then, by using octadecyl silica resin (ODS) Just can obtain the refined lactosucrose of purity about 99% (refer to Koki Fujita, "Biosepare-ヨンヨンプロセス handbook" (biochemical separation process handbook), 196-201 pages, published by Kyoritsu Publishing, 1996).

However, β-fructofuranosidase derived from microorganisms belonging to the genus Arthrobacter catalyzes, in addition to the reaction of transferring the fructosyl group of sucrose to lactose to produce lactose, as a side reaction, the transfer of the fructosyl group to sucrose itself to produce lactose. 1-The reaction of kestose, or the reaction of catalyzing the transfer of fructosyl to the already formed lactosucrose to produce fructosyl lactosucrose. Production of 1-kestose and fructosyl lactosucrose as by-products is undesirable from the standpoint of production of lactose, and causes a decrease in the amount of lactose produced. In addition, the above-mentioned by-products are not utilized by invertase-deficient yeast, so they accumulate in the reaction product obtained by simultaneously acting β-fructofuranosidase and invertase-deficient yeast, and their content reaches 5 to 10%. Furthermore, when high-purity lactosucrose is produced by using the reactants of 1-kestose and fructosyl lactosucrose containing a large amount of by-products as raw materials, it is necessary to use the above-mentioned ion exchange resin and octadecyl Various purification methods such as silica resin (ODS) chromatography have the problem that the yield of high-purity lactosucrose obtained by purification is low, and it is considered unsuitable for industrial production of high-purity lactosucrose.

On the other hand, it is known that lactose-containing powder can be obtained by increasing its purity and reducing its hygroscopicity to obtain a highly stable powder (JP-A-4-281795). However, in subsequent studies, it has become clear that the powder is also highly hygroscopic and unstable in high-temperature and high-humidity environments in summer, such as at a temperature of 27°C and a relative humidity of 75%, and it is difficult to handle. It would be desirable to provide more stable lactose solids.

On the other hand, Gad Avigad disclosed in Journal Biological Chemistry, 1957, Vol. 229, pp. 121 to 129, the crystallization of lactosucrose, which is crystalline lactose containing pentahydrate obtained from 90% (V/V) aqueous ethanol. However, the crystals are highly hygroscopic, and crystallization is often difficult, so crystalline lactose sugar has not been produced on an industrial scale.

Contents of the invention

In view of such a situation, the object of the present invention is to provide a low content of 1-kestose and fructosyl lactosucrose as by-products, and a high lactose content sugar containing 70% or more of lactosucrose as a sugar composition, and in addition , to provide a method for producing high-content lactose sugar suitable for industrial production and a high-purity lactose sugar containing greater than or equal to 90% lactosucrose as a sugar composition, and to provide a method for producing crystalline lactose sugar and crystals containing low hygroscopicity A solid composition of lactose.

In order to solve the above-mentioned problems, the inventors of the present invention focused not only on the use of sucrose non-utilizing yeast but also on the origin and reaction conditions of β-fructofuranosidase in the production of lactose-sucrose using β-fructofuranosidase. The manufacturing method of lactose cane was studied. As a result, it became clear that although it was previously thought that even if β-fructofuranosidase and invertase-deficient yeast were used together, the content of lactose in the reactant produced by this reaction only reached a maximum of about 65% as a sugar composition, but it was completely unexpected. Yes, in an aqueous solution containing sucrose and lactose, β-fructofuranosidase derived from microorganisms of the genus Bacillus described in JP-A No. 9-224665 belonging to the same patent applicant and sucrose non-utilizing yeast are used for the reaction , by controlling the pH of the reaction solution at 4.0 to 5.5, the content of lactose in the obtained reactant becomes 70% or more as a sugar composition. In addition, examination of the obtained reaction products revealed that 1-kestose and fructosyllactosucrose as by-products were as extremely low as a total of less than 3% as a sugar composition. Furthermore, it is also clear that if the reactant is used as a raw material and purified by chromatography, lactosucrose of higher purity can be easily produced at a high yield. In addition, it was unexpectedly found that crystals of lactosucrose (containing pentahydrate crystals) were easily precipitated from a supersaturated aqueous solution of lactosucrose with a low content of the above-mentioned by-product 1-kestose and fructosyl lactosucrose, and it was found that the obtained crystalline lactosucrose, Even in a high-temperature and high-humidity environment in summer, for example, when the temperature is 27°C and the relative humidity is 75%, the hygroscopicity is low, and it is a solid product of lactose sugar that is easy to handle. In addition, it is clear that it can be advantageously used in In the manufacture of various solids containing crystalline lactose sucrose.

That is, the present invention provides lactose-containing high-content sugar containing greater than or equal to 70% lactose, 1-kestose, and fructosyl lactose in a total of less than 3% as a sugar composition; wherein β-fructofuranosidase derived from microorganisms belonging to the genus Bacillus and sucrose non-utilizing yeast act to form a sugar composition containing 70% or more of lactosucrose, 1-kestose, and fructosyllactosucrose A method for producing a lactose-rich sugar characterized by extracting a reactant with a total of less than 3%; and providing a process for obtaining a lactose-rich sugar as a sugar composition containing greater than or equal to A method for producing high-purity lactosucrose characterized by a fraction of 90% lactosucrose; and further providing a method for producing crystalline lactosucrose from the high-content lactose or high-purity lactosucrose obtained by the above-mentioned method and various solid-state products containing the lactosucrose composition, thereby solving the above-mentioned problems.

According to the present invention, starting from sucrose and lactose, even without using purification methods such as column chromatography, it is possible to produce a product containing 70 or more as a sugar composition only by the production reaction of simultaneously using β-fructofuranosidase and sucrose non-utilizing yeast. % lactosucrose, and the content of the by-product 1-kestose and fructosyl lactosucrose as the sugar composition is as low as a total of less than 3% lactose high content sugar. Furthermore, high-purity lactosucrose containing 90% or more of lactosucrose as a sugar composition can be easily produced by chromatography using a resin for this lactose-rich sugar. In addition, according to the present invention, it is possible to easily crystallize lactsucrose, which has been difficult to crystallize in the past, from a supersaturated aqueous solution of lactose, and the obtained crystallized lactose has no hygroscopicity even in a high-temperature and high-humidity environment in summer. It is also low and easy to handle, and by containing this crystalline lactosucrose, various stable solid compositions with low hygroscopicity can be provided.

Detailed ways

The sugar with high lactose content of the present invention contains 1-kestose and fructosyl lactosucrose greater than or equal to 70% lactose, 1-kestose and fructosyl lactosucrose totaling less than 3% as a sugar composition , which is characterized in that the content of 1-kestose and fructosyl lactosucrose produced by the side reaction of β-fructofuranosidase is extremely low compared with conventional lactose-containing sugars.

Sugars other than lactose, 1-kestose, and fructosyl lactose contained in the lactose-rich sugar of the present invention include unreacted sucrose, lactose, and lactulose, an alkaline isomer of lactose , Glucose and fructose, the decomposition products of sucrose produced by the action of β-fructofuranosidase, and glycerol, the fermentation product of glucose and lactose produced by the action of sucrose non-utilizing yeast, etc. As will also be shown in Examples described later, most of these sugars are lactose that is not decomposed by β-fructofuranosidase and is not used and decomposed by sucrose non-utilizing yeast.

The production method of lactose-sucrose-high-content sugar of the present invention (hereinafter, sometimes simply referred to as "the production method of the present invention" or "the production method") utilizes β-fructofuranosidase and sucrose derived from microorganisms of the genus Bacillus Characterized by non-utilizing yeast. The β-fructofuranosidase mentioned in the present invention means at least the β-fructofuranosidic bond hydrolysis of sucrose, raffinose and glucopyranosyl sucrose, and the reaction of freeing fructose and the reaction with these β-fructofuranosidases. An enzyme that catalyzes a reaction in which a sugar of a fructofuranosidic bond is used as a sugar donor to transfer a β-fructofuranosyl group to an acceptor selected from sugars, sugar alcohols, and alcohols other than the sugar donor. The β-fructofuranosidase that can be used in the present invention is defined as follows: when it is derived from a microorganism belonging to the genus Bacillus and acts in combination with the sucrose non-utilizing yeast used in the present invention described in detail below , as long as it can be produced as a sugar composition containing greater than or equal to 70% lactosucrose, and the content of by-product kestose and fructosyl lactosucrose as a sugar composition contains as low as a total of less than 3% lactosucrose high content sugar, the preparation method etc. are not particularly limited. For example, the natural type β-fructofuranosidase from the origin of Bacillus species V230 (FERM BP-5054) disclosed in Japanese Unexamined Patent Publication No. Hei 9-224665 of the same patent applicant as the present application, and the same as the present application The recombinant β-fructofuranosidase disclosed in the applicant's Japanese Patent Application Laid-Open No. 10-66586, using the DNA encoding the above-mentioned β-fructofuranosidase and prepared by gene recombination technology, can be advantageously used in this invention. invention. Needless to say, in the case of recombinant enzymes, β-fructofuranosidase obtained from host cells other than microorganisms belonging to the genus Bacillus can also be used in the present invention. Furthermore, mutated enzymes obtained by applying protein engineering techniques to the DNA encoding β-fructofuranosidase disclosed in the same publication can also be used in the present invention as long as the expected transfer function is not substantially lost.

The term "sucrose non-utilizing yeast" as used in the present invention means a yeast that can utilize monosaccharides but cannot utilize and/or decompose oligosaccharides of disaccharides or higher. The sucrose non-utilizing yeast used in the present invention is not particularly limited as long as it can utilize glucose and fructose, but cannot utilize and/or decompose sucrose, lactose and lactose. For example, yeast isolated from nature can be mutated with various mutagens to delete invertase, or yeast bred according to the purpose without the ability to utilize sucrose can also be used. In addition, as the invertase-deficient yeast, known known strains such as Saccharomyces cerevisiae ATCC56741 and Saccharomyces cerevisiae ATCC56742 can be advantageously used in the present invention.

The sucrose and lactose used as raw materials in the production method of the present invention are not particularly limited in their production methods or forms, and include, for example, commercially available products, preparations prepared by natural isolation, enzymatic or chemical methods, and synthetic preparations, Furthermore, as long as there is no hindrance in carrying out the enzymatic reaction in the production method of the present invention or in the utilization of monosaccharides by sucrose non-utilizing yeast, or in the utilization of the product produced by the production method, It may be a preparation containing such inclusions other than sugars, or a composition composed of a combination of the above preparations.

Simultaneous use of β-fructofuranosidase derived from microorganisms belonging to the genus Bacillus and sucrose non-utilizing yeast, if it contains sucrose and lactose (hereinafter, refers to either or both of sucrose and lactose, sometimes referred to as "substrate ”) in an aqueous solution, in addition to controlling the pH of the reaction solution at 4.0 to 5.5, under conditions appropriately selected according to the enzymatic properties of the β-fructofuranosidase used and the physiological properties of sucrose non-utilizing yeast When the effect occurs, a reactant can be obtained that contains greater than or equal to 70% lactosucrose as a sugar composition, and the content of the by-product 1-kestose and fructosyl lactosucrose is as low as less than 3% in total as a sugar composition. The lactosucrose content of the reactant obtained only by the lactosucrose production reaction is generally 70% or more, less than 80%, and less than 80% or more as a sugar composition. As conditions for the lactose-sucrose production reaction, for example, when using β-fructofuranosidase derived from microorganisms of the genus Bacillus disclosed in JP-A No. 9-224665 belonging to the same patent applicant and sucrose non-utilizing Saccharomyces cerevisiae ATCC56741, The enzyme is not completely inactivated, and the yeast can utilize the conditions of glucose and lactose, that is, the temperature is usually in the range of about 0 to 40°C, and it is desirable to be in the range of about 15 to 35°C, and the pH is usually controlled at 4.0 to 5.5 range is desirable. The concentration of the substrate in the reaction mixture is such that the desired reaction proceeds to obtain a reactant that contains greater than or equal to 70% lactose-sucrose as a sugar composition, and that by-products 1-kestose and fructosyl The total content of lactose and sucrose is less than 3%, so it is not particularly limited. For example, sucrose and lactose are usually in the range of 0.1 to 40% by mass, preferably in the range of 1 to 30% by mass. The molar ratio of the two is preferably 1: 0.65 to 1.3.

The amount of β-fructofuranosidase used is usually in the range of 0.1 to 50 units per 1 g of dry mass of sucrose in the reaction raw material (substrate), preferably in the range of 0.5 to 10 units. Furthermore, 1 unit of β-fructofuranosidase activity mentioned in this specification means that, according to the method described in Japanese Patent Application Laid-Open No. Hei 9-224665 of the same applicant, at pH 6.0, 40°C, sucrose When reacting as a substrate, the reduction ability is increased by the amount of enzyme corresponding to 2 μmol of D-glucose per minute.

The amount of sucrose non-utilizing yeast used is usually in the range of 0.01 to 1 g in terms of wet mass, preferably preferably in the range of 0.05 to 0.2 g, per 1 g of dry mass of sucrose in the reaction raw material (substrate). .

The method of using β-fructofuranosidase and sucrose non-utilizing yeast at the same time, as long as the obtained reactant contains lactose-sucrose greater than or equal to 70% as a sugar composition, and the by-products 1-kestose and fructosyl The total content of lactose and sucrose is less than 3%, and it is not particularly limited. For example, β-fructofuranosidase and yeast can be added to the aqueous solution containing sucrose and lactose simultaneously or at different times to make it work, or in the reaction Suitably, either or both of β-fructofuranosidase and yeast are added to make it act. Furthermore, in separate reaction vessels, the β-fructofuranosidase reaction and the treatment of sucrose non-utilizing yeast can be performed continuously by circulating the respective reaction liquids. In such a case, use Bioreactors of immobilized β-fructofuranosidase and immobilized sucrose non-utilizing yeast can be advantageously utilized.

The time for simultaneously using β-fructofuranosidase and non-utilizing yeast to carry out the reaction can be appropriately selected according to the degree of the method or reaction of simultaneously using β-fructofuranosidase and non-utilizing yeast, and is usually 2 to 200 hours The range of suitably is expected to be in the range of 10 to 80 hours.

The β-fructofuranosidase and/or sucrose non-utilizing yeast used in the reaction can be separated and recovered by methods such as centrifugation and membrane filtration as necessary, and can be advantageously used again in the reaction.

By adopting the method for producing high-lactose-sucrose-content sugar of the present invention as above, as a high-lactose-sucrose-content sugar, it is possible to obtain milk containing 70% or more as a sugar composition, and by-products 1-kestose and fructosyl milk The content of sucrose was as low as the reactants with a sugar composition of less than 3% in total. Such lactose-containing high-content sugar can be used in an intact state. In addition, the remaining lactose is partially isomerized into lactulose by alkali treatment, thereby reducing the lactose content and inhibiting the crystallization of the contained lactose. After the high sugar content of lactose can also be used freely. In addition, by conventional methods, such as decolorization by activated carbon treatment, desalination by ion exchange resin treatment, insolubilization of impurities by carbonic acid immersion, removal of lactose by crystallization, and use of auxiliary agents such as diatomaceous earth Filtration, concentration using an evaporator, etc., and if necessary, a purified product purified to a desired level by an appropriate process selected from spray drying and the like can be used for various purposes.

In addition, the sugar with high lactose content of the present invention can also be used as a raw material, and then purified by chromatography using an ion exchange resin, and the fraction containing greater than or equal to 90% lactose sugar as a sugar composition can be recovered and concentrated to form a syrupy high Pure lactose, or use a suitable drying process selected from spray drying, vacuum drying, freeze drying, etc. to form powdered powder or granular products for various purposes.

In addition, it is also possible to use the above-mentioned lactose-sucrose high-content sugar or high-purity lactosecane sugar to form a supersaturated aqueous solution of lactosecane sugar, and then crystallize the crystalline lactosecane sugar to extract the crystalline lactosecane sugar advantageously. As the crystallization conditions, any supersaturated aqueous solution of lactose can be used as long as the crystallized lactose can be precipitated. Generally, an aqueous solution having a lactose concentration of 75% or more is suitable. If it is specified, for example, lactose sugar with a purity of about greater than or equal to 75%, more preferably about 80 to 95%, forms a concentration of about 75 to 95% by mass or a concentration, and the temperature of its solution, as long as the solution does not freeze, and The crystallization temperature is lower than the melting point of crystallization, and it is not easy to cause browning and decomposition of lactose, for example, crystallization of a solution with a water content of about 5 to 25% by mass at 20 to 60°C. In addition, in order to adjust the degree of supersaturation, viscosity, etc. of the solution during crystallization, for example, methanol, ethanol, acetone, etc. are allowed to coexist.

In the crystallization method, usually a higher temperature solution containing a supersaturated lactosucrose solution is placed in a crystallization aid tank, and it is hoped that 0.1 to 20% by mass of seed crystals coexist, and slowly cool while stirring slowly to promote crystallization and become a massecuite (マスキツト) will do.

The method for producing crystalline lactose sugar from the crystallized massecuite is not limited as long as the crystalline lactose sugar can be obtained. For example, known methods such as the nectar separation method, the lump crushing method, the fluidized granulation method, and the spray drying method can be used.

For example, the honey separation method is usually a method of separating crystallized lactose and molasses by using a basket centrifugal separator. It is also easy to spray a small amount of cold water into the crystallization to clean it as needed, which is helpful for the production of higher purity Crystalline lactose is suitable. The other three methods do not separate molasses, so although the purity of the obtained crystal product cannot be improved, they have the characteristics of a large yield of product. Therefore, in the case of this product, crystalline products usually contain sugars derived from raw materials or manufacturing processes, such as sucrose, lactose, etc., in addition to lactose.

In the case of the spray drying method, massecuite having a crystallization rate of about 20 to 50% is usually sprayed from a nozzle with a high-pressure pump, and dried at a temperature at which the crystalline powder does not melt, for example, with warm air at about 40 to 65°C, and then, if Drying and aging with warm air can easily produce non-hygroscopic or non-hygroscopic powders containing crystals.

In addition, in the block pulverization method, the massecuite with a crystallization rate of about 10 to 60% is usually left for about 0.5 to 5 days to solidify the entire crystal into a block, then crush it by crushing or cutting, and dry it. Powders containing non-hygroscopic and poorly hygroscopic crystals can be easily produced.

In addition, according to a conventional method, the aqueous solution containing lactose sugar is heated and concentrated until the water content is less than 5% by mass to form a supersaturated aqueous solution of lactose sugar in a molten state, and kneading is carried out in the aqueous solution at a temperature lower than or equal to the melting point of the crystal. Seed crystals can be directly formed into various states, such as powders, granules, rods, plates, cubes, etc. It is also optional to produce non-hygroscopic or difficult-to-hygroscopic crystalline solids.

The high-content lactose sugar and high-purity lactose sugar obtained by the production method of the present invention, as well as crystallized lactose sugar, can be used in the same applications as conventionally known lactose sugar and lactose-containing sugars, such as sweeteners, indigestible Sexual sweeteners, low cariogenic sweeteners, humectants, starch aging inhibitors, intestinal regulators, mineral absorption promoters, etc., in the fields of food and beverages including health foods and beverages, cosmetics, medicine, and feed It can be advantageously used in various fields such as. In particular, crystalline lactose, in addition to the above-mentioned various functions, is substantially non-hygroscopic, is fluid, does not easily adhere or fix, is easy to handle, and has requirements for the management of its packaging, transportation, storage, etc. The feature that the expense of things and people can be greatly reduced.

In addition, the crystalline lactose sucrose of the present invention is a powder that is substantially non-hygroscopic or hardly hygroscopic, has high heat resistance, and good stability. Therefore, it can be used as various solid compositions that were extremely difficult to form in the past, such as powdered mixed sweet agent, chocolate, chewing gum, instant fruit juice, fast food soup, fixed inhibitors for granules, tablets, etc., excipients, bulking agents, powder bases, etc., it is desirable to make crystalline lactosucrose containing greater than or equal to 1% by mass, such as even if manufactured Various solid compositions such as food and beverages, cosmetics, medicines, molded products, etc., can also be used advantageously as reagents, chemical industry raw materials, and the like.

In addition, the lactose sugar of the present invention, and other crystalline sugars, such as maltose, lactose, maltitol, trehalose, palatinose, sucrose, etc., are desirably blended in an amount of 0.01 to 100 times the mass of the crystalline lactose sugar. Compared with combined use, it is also possible to advantageously implement production of various solid compositions with low hygroscopicity and easy handling.

Furthermore, the high-purity lactosucrose powder or crystalline product produced as above is prepared with a suitable water-soluble polymer, such as pullulan, water-soluble pullulan ether, water-soluble pullulan ester, gelatin, arabic Gum, carboxymethylcellulose, seaweed polysaccharides, etc. are used as binders and granulated by conventional methods to form granulated products with excellent fluidity and easy handling, which can also be advantageously implemented and utilized in various applications.

Hereinafter, the present invention will be described in more detail based on examples.

Example 1

Manufacture of sugar with high content of lactose

Example 1-1: Preparation of β-fructofuranosidase

According to the method described in Japanese Unexamined Patent Publication No. Hei 9-224665 of the same applicant, Bacillus sp. V230 (FERM BP-5054) was cultured at a culture scale of 40 L in a medium containing sucrose as a carbon source. Continue to follow the method described in the above bulletin, centrifuge the culture, extract about 38 L of its supernatant, and perform the activity measurement of β-fructofuranosidase, which confirms that the culture supernatant shows β-fructofuranosidase activity. The above-mentioned culture supernatant was concentrated by UF membrane to obtain 360 ml of crude enzyme solution having a β-fructofuranosidase activity of about 360 units per 1 ml. Continuing to follow the method described in the above publication, this crude enzyme solution was subjected to ion exchange chromatography using "DEAE-Toyopar-ru" gel (manufactured by Tosoh Corporation), and to ion-exchange chromatography using "Buchiru-toyopar-ru" gel (Tosoh Co., Ltd.). Cao Co., Ltd.) hydrophobic chromatography and ion exchange chromatography using "DEAE-ヨヨパ-ル" gel, in 7.5% (w/v) polyacrylamide gel electrophoresis, a single band was obtained. Refined standard of β-fructofuranosidase. The resulting refined standard had a specific activity of approximately 205 units per 1 mg of protein.

Example 1-2: Preparation of sucrose non-utilizing yeast

Put 200ml of liquid culture medium consisting of 2.0% (w/v) glucose, 2.0% (w/v) yeast extract and water in a 500ml capacity triangular flask, and sterilize in an autoclave at 121°C for 20 minutes. Bacteria, then cooled, inoculated with Saccharomyces cerevisiae ATCC56741, and cultured at 27°C with 230r/min for 24 hours with rotary shaking culture as a seed culture. Put the medium with the same composition as the seed culture in a 30L fermenter, heat-sterilize it, and cool it. After reaching a temperature of 27°C, inoculate 1% (capacity/capacity) seed culture solution while maintaining a pH of 4 at a temperature of 27°C. .0 to 7.0, while cultivating with aeration and agitation for 48 hours. After culturing, the culture solution was centrifuged (8000 rpm, 20 minutes), and about 680 g of the yeast cells were recovered by wet weight.

Embodiment 1-3: generation reaction of lactose

The aqueous solution containing 20% by mass of sucrose and lactose was adjusted to pH 6.0, and the β-furan prepared in Example 1-1 was added thereto at a ratio of 1 unit per 1 g of sucrose and 6.9% per solid mass Refined standard fructosidase and the yeast prepared in Example 1-2 were reacted at 30°C for 24 hours and 42 hours while controlling the reaction solution to pH 4.0 to 5.5 with 1N sodium hydroxide solution. After the reaction, the sugar composition of the reaction liquid was measured by high performance liquid chromatography (HPLC). HPLC was performed using a "TSK-GEL AMIDE-80" column (manufactured by Tosoh Corporation) at a column temperature of 35°C, using acetonitrile:water (71:29) as an eluent, and a flow rate of 1.0ml/min. Detection was performed with a differential refractometer "RI-8012" (manufactured by Tosoh Corporation). The results of the analysis of sugar composition are shown in Table 1. Furthermore, as a comparative experiment, the β-fructofuranosidase from Arthrobacter species K-1 was purified by the method described in Fujita Xiaohui et al., Agric Biol.Chem., volume 54, pages 913-919 (1990). , adding 10 units of refined β-fructofuranosidase per 1 g of sucrose, except that the reaction solution was controlled to pH 5.5 to 6.5, the same operation was carried out. The obtained results are shown in Table 1.

Table 1 source of yeast Response time (hours) Sugar composition (%) Gly DPl Suc Lac Kes LS F-LS UN V230 (this invention) twenty four 1.2 0.5 8.8 17.3 0.1 71.0 0.0 1.1 42 1.8 0.0 0.6 22.1 0.2 73.6 0.1 1.6 K-1 (comparison) twenty four 1.1 0.7 10.8 17.2 2.3 63.3 3.6 1.0 42 1.8 0.0 0.6 20.7 3.8 63.0 8.6 1.5

Gly, DPl, Suc, Lac, Kes, LS, F-LS and UN in the table mean glycerol, glucose and fructose collectively called monosaccharide, sucrose, lactose, 1-kestose, lactosucrose, fructosyl Lactose and other unknown sugars.

As shown in Table 1, it is clear that the β-fructofuranosidase of Bacillus species V230 used in the present invention produces 71.0% (reaction time 24 hours) to 73.6% (reaction time 42 hours) of β-fructofuranosidase as a sugar composition. Lactose, greater than or equal to 70%, and the by-product 1-kestose and fructosyl lactosucrose total only 0.1% (reaction time 24 hours) to 0.3% (reaction time 42 hours), less than 1%. On the other hand, it is the β-fructofuranosidase of Arthrobacter species K-1 of comparison, only produces 63.3% (reaction time 24 hours) to 63.0% (reaction time 42 hours) of lactosucrose as sugar composition, less than 65%, and it is clear that the by-products 1-kestose and fructosyl lactosucrose add up to 5.9% (reaction time 24 hours) to 12.4% (reaction time 42 hours), significantly generated.

Example 2

Manufacture of sugar powder with high lactose content

The aqueous solution containing 24% by mass sucrose and 16% by mass of lactose is adjusted to pH 6.0, and 1 unit per 1g of sucrose and 7.5% of the wet mass of the raw material solids are respectively added to it to prepare The β-fructofuranosidase crude enzyme solution and the sucrose non-utilizing yeast prepared in Example 1-2 were adjusted to pH 4.0 to 5.5 with 1N sodium hydroxide solution, and at 30°C React for 45 hours. After the reaction, the sugar composition of the reaction solution was measured by HPLC, and it contained 2.0% glycerin, 2.1% glucose and fructose in total, 5.4% sucrose, 14.7% lactose, 1.4% 1-kestose, 73.0% lactose, 0.1% fructosyl Lactose and 1.3% other unknown sugars. The obtained reaction solution was heated at 90° C. for 30 minutes to inactivate the enzyme, and then filtered with activated carbon according to a conventional method. After the obtained filtrate was adjusted to 60°C, 1.5% hydrated lime was added per solid mass to adjust the pH to 11, and after keeping for 15 minutes, carbon dioxide was blown in to obtain a pH of 8 for carbonation. It was filtered according to a conventional method, desalted with an ion exchange resin, concentrated, and spray-dried to obtain a sugar composition containing 1.9% glycerin, 2.2% glucose and fructose in total, 5.5% sucrose, 10.8% lactose, 3.3% lactulose, Lactose high content sugar powder with 1.3% 1-kestose, 73.5% lactose, 0.1% fructosyl lactose and 1.4% other unknown sugars.

This product can be advantageously used in the field of food and beverages including health foods and beverages as sweeteners, indigestible sweeteners, low cariogenic sweeteners, moisturizers, starch aging preventers, intestinal regulators, etc. Various fields such as cosmetics, medicine, and feed.

Example 3

Production of Syrup with High Content of Lactose Cane Sugar

The aqueous solution containing 18% by mass of sucrose and 22% by mass of lactose was adjusted to pH 6.0, and 1 unit per 1g of sucrose and 5.0% of the wet mass per solid were added thereto prepared in Example 1-1. The β-fructofuranosidase crude enzyme solution and the sucrose non-utilizing yeast prepared in Example 1-2 were reacted at 32°C while adjusting the reaction solution to pH 4.0 to 5.5 with 1N sodium hydroxide solution 42 hours. After the reaction, the sugar composition of the reaction solution was measured by HPLC, and it contained 1.9% glycerin, 0.1% glucose and fructose in total, 1.2% sucrose, 22.6% lactose, 0.3% 1-kestose, 72.4% lactose, 0.1% fructosyl Lactose and 1.4% other unknown sugars. According to conventional methods, filter the reaction solution obtained by SF membrane to remove yeast, and then according to conventional methods, filter with UF membrane to remove polymer components such as β-fructofuranosidase, and then filter with activated carbon and dry under reduced pressure to obtain A concentrate of about 80% by mass of solids. After adjusting the concentrated solution to a temperature of about 25° C., crystalline lactose powder was added at about 3% by mass of the added lactose to partially crystallize the lactose. The crystallized lactose was removed by filtration according to a conventional method, desalted with an ion exchange resin, and concentrated to obtain a sugar composition containing 2.2% glycerin, 0.1% glucose and fructose in total, 1.4% sucrose, 10.1% lactose, 0.4% 1- Lactose high content syrup of kestose, 84.1% lactose, 0.1% fructosyl lactose and 1.6% other unknown sugars.

This product can be advantageously used in the field of food and beverages including health foods and beverages as sweeteners, indigestible sweeteners, low cariogenic sweeteners, moisturizers, starch aging preventers, intestinal regulators, etc. Various fields such as cosmetics, medicine, and feed.

Example 4

Manufacture of high-purity lactose powder

The aqueous solution containing 20% by mass of sucrose and 20% by mass of lactose was adjusted to pH 6.0, and 1 unit per 1g of sucrose and 5.0% of the wet mass per solid were added thereto prepared in Example 1-1. The β-fructofuranosidase crude enzyme solution and the sucrose non-utilizing yeast prepared in Example 1-2 were reacted at 30°C while adjusting the reaction solution to pH 4.0 to 5.5 with 1N sodium hydroxide solution 30 hours. After the reaction, the sugar composition of the reaction solution was measured by HPLC, and it contained 2.0% glycerin, 0.2% glucose and fructose in total, 1.6% sucrose, 19.2% lactose, 0.6% 1-kestose, 74.7% lactose, 0.1% fructosyl Lactose and 1.7% other unknown sugars. According to conventional methods, filter the reaction solution obtained by SF membrane to remove yeast, and then according to conventional methods, filter with UF membrane to remove polymer components such as β-fructofuranosidase, and then filter with activated carbon and dry under reduced pressure to obtain A concentrate of about 60% by mass of solids. Using this syrup with a high lactose content as raw syrup, in order to increase the purity of lactose, a strong acidic cation exchange resin (trade name "Amber-Light CR-1310" (Na ⁺ type), manufactured by Olgano Co., Ltd.) was used to perform column layering. analysis. That is, ten jacketed stainless steel columns with an inner diameter of 12.5 cm were filled with the above-mentioned resin, and these columns were connected in series so that the total length of the resin layer became 16 m. While maintaining the internal temperature of the column at 40°C, add 1.5% (v/v) of the above syrup to the amount of resin, and flow warm water at 40°C at SV (space velocity) 0.2, and monitor the elution by HPLC Separate the high-content fraction of lactose sugar while separating the sugar composition of the liquid, filter the fraction according to the conventional method, use ion exchange resin, activated carbon to carry out desalination, decolorization and refinement, after concentration, carry out spray drying, and make it as the sugar composition containing 0.1% sucrose , 2.3% lactose, 1.1% 1-kestose, 96.0% lactose, 0.2% fructosyl lactose and 0.3% other unknown sugars high-purity lactose powder.

This product can be advantageously used in the field of food and beverages including health foods and beverages as sweeteners, indigestible sweeteners, low cariogenic sweeteners, moisturizers, starch aging preventers, intestinal regulators, etc. Various fields such as cosmetics, medicine, and feed.

Example 5

Manufacture of high-purity lactose granules

For 100 parts by mass of the high-purity lactose powder obtained in Example 4, pullulan (trade name "Pullulan PF-20", sold by Hayashibara Trading Co., Ltd.) was dissolved in advance so that the final concentration became about 3% (weight/volume ratio). About 6 parts by mass of the pullulan aqueous solution obtained in deionized water was sprayed and granulated by a conventional method. The obtained granulated product was classified with a sieve with a sieve aperture of 1.7 mm to obtain a high-purity lactose granulated product. When the particle size distribution of the granulated product was measured and compared with the particle size distribution of the high-purity lactose powder as the raw material, the particles with a particle diameter of less than or equal to 106 μm in the high-purity lactose powder as the raw material accounted for greater than or equal to 85%, and the high-purity milk In the granulated sucrose products, the particles with a particle diameter greater than or equal to 150 μm account for greater than or equal to 70%.

Since this product is a granulated product, it has good fluidity, is easy to handle, and has the characteristics that it does not easily turn into powder when dissolved. This product can be advantageously used in the field of food and beverages including health foods and beverages as sweeteners, indigestible sweeteners, low cariogenic sweeteners, moisturizers, starch aging preventers, intestinal regulators, etc. Various fields such as cosmetics, medicine, and feed.

Example 6

Crystalline lactose

According to conventional methods, the lactose sugar high-content fraction obtained by the method of Example 4 was filtered, desalinated and decolorized with ion exchange resins and activated carbon, refined, concentrated under reduced pressure to about 25% by mass of water, and added 1 The mass % seed crystals were slowly stirred, and slowly cooled to about 30° C. over 24 hours. Put it on a centrifuge to obtain 5-hydrate crystalline lactose with a yield of about 40%. This product is crystalline lactose sucrose with a purity of about 98% or more. It does not substantially exhibit hygroscopicity even in a high-temperature and high-humidity environment in summer, and is easy to handle. It is used as a sweetener, indigestible sweetener, Intestinal regulators and the like can be advantageously used in various fields such as food and drink, cosmetics, and medicine, and can be advantageously used in solid compositions containing crystalline lactose sucrose in particular.

Example 7

sweetener

0.05 parts by mass of α-glucosyl stevioside (sold by Toyo Seicho Co., Ltd., trade name "αG-Sweet") was uniformly mixed with 1 part by mass of the crystallized lactose sucrose obtained by the method of Example 6, and the Get a granulated sweetener. The sweetness of this product is excellent, with about 2 times the sweetness of granulated sugar, and the calories when the sweetness is equivalent, as low as about 1/2 of granulated sugar. This sweetener is suitable as a low-calorie sweetener for obtaining sweetness in low-calorie food and beverages for obese people, diabetics, and the like who restrict their calorie intake. In addition, the present sweetener is suitable for obtaining sweet taste in foods and beverages that suppress dental caries, since the production of acid by caries-inducing bacteria is small and the production of insoluble glucan is small. This sweetener is also suitable as an intestinal regulator for adjusting the intestinal flora.

Example 8

shaped sweetener

1 part by mass of crystalline lactose sugar obtained by the method of Example 6 and 2 parts by mass of granulated sugar were uniformly mixed, and an appropriate amount of water was sprayed thereinto, and shaped according to a conventional method to obtain a shaped sweetener. This product can be effectively used in obtaining sweetness of coffee, black tea, etc. as a shaped sweetener having an intestine-regulating function of adjusting the flora in the intestine.

Example 9

chocolate

Mix 40 mass parts of cocoa nibs, 10 mass parts of cocoa butter, 20 mass parts of granulated sugar, 30 mass parts of crystallized lactose sucrose obtained by the method of embodiment 6, pass in a refiner (レフアイナ-), after the particle size is reduced, put It was put into conchie and refined at 50°C for 2 days and nights. Meanwhile, 0.5 parts by mass of lecithin was added to fully mix and disperse. Next, adjust the temperature to 31°C with a temperature regulator, inject the cocoa butter into the mold just before it solidifies, degas it with a vibrator, and pass it under a cooling air duct at 10°C for 20 minutes to solidify. This was released from the mold and packaged to obtain a product. This product has no hygroscopicity, good color and luster, good internal structure, melts smoothly in the mouth, and has top-grade sweetness and satisfying flavor. In addition, this product is also suitable as a chocolate with the effect of regulating the intestines.

Example 10

chewing gum

3 parts by mass of gum base was heated and melted to a soft degree, and 4 parts by mass of anhydrous crystalline maltitol (sold by Hayashibara Trading Co., Ltd., registered trademark "Mabit") and 3 parts by mass obtained by the method of Example 6 were added thereto. Crystallized lactose, mixed with appropriate amount of spices and coloring materials, kneaded with rollers according to conventional methods, shaped and packaged to obtain products. This product is a chewing gum with good structure and flavor. In addition, this product is also suitable as a chewing gum with the effect of regulating the intestines.

Example 11

drinks

Add 100 parts by mass of yogurt, 50 parts by mass of high-purity lactose powder obtained by the method of Example 4, 10 parts by mass of trehalose, 0.25 parts by mass of yogurt seasoning and 0.1 part by mass of lemon essence in water, so that the whole is combined into 1000 parts by mass. parts by mass, according to a conventional method, to make a yogurt drink. This product is rich in flavor and has the function of regulating intestinal flora.

Example 12

enteral nutrition

10 parts by mass of crystalline lactose sucrose obtained by the method of Example 6, 10 parts by mass of trehalose, 1.1 parts by mass of glycine, 1 part by mass of sodium glutamate, 0.4 parts by mass of calcium lactate, 0.1 parts by mass of magnesium carbonate, 0.01 parts by mass of A complex composed of parts by mass of thiamine and 0.01 parts by mass of riboflavin. 24 g of this compound was filled in a laminated aluminum pouch and heat-sealed to obtain a product. Dissolve 1 bag of this product in about 33 to 500ml of water, and use it as a nutritional supplement solution through nasal cavity, gastrointestinal administration, etc. through the tube method. This product can be advantageously used as a parenteral nutritional supplement solution not only for humans but also for livestock and the like. In addition, this product has the effect of regulating intestinal flora.

Example 13

sugar-coated tablet

With the plain tablet of quality 150mg as core agent, on core agent, use 45 mass parts crystalline lactose sucrose, 2 mass parts pullulan (average molecular weight 200,000), 30 mass parts water, 25 mass parts obtained by the method of embodiment 6 The bottom layer liquid that mass parts talcum powder and 3 mass parts titanium oxides are formed, sugar-coating makes tablet quality be about 230mg, then, use the same crystalline lactosucrose that is made of 65 mass parts, 1 mass part pullulan and 34 mass parts water The supernatant liquid forms a sugar coating, and then polishes with a wax solution to obtain an excellent sugar-coated tablet with a glossy appearance. This product not only has good operability when coating sugar, but also has excellent impact resistance and maintains high quality for a long time. In addition, this product has the function of regulating intestinal flora.

Industrial Applicability

As described above, according to the method for producing lactose-sucrose-rich sugar of the present invention, even without using purification means such as column chromatography, only the reaction using β-fructofuranosidase and sucrose non-utilizing yeast at the same time can be obtained from Sucrose and lactose are manufactured as lactose high sugars whose sugar composition contains greater than or equal to 70% lactose. In addition, the lactosucrose obtained by the production method of the present invention has a high sugar content, and the content of the by-product 1-kestose and fructosyl lactosucrose is as low as less than 3% in total as a sugar composition, so if it is used as a raw material, the layer By analyzing and refining, lactose of higher purity can be easily produced at a high yield.

In addition, the high-purity lactose sugar obtained according to the present invention contains lactose sugar effective for exerting functions by 70% or more or 90% or more as a sugar composition. Compared with low-lactose sucrose-containing sugars, the excellent functions such as indigestibility, bifidobacteria growth promotion, cariogenic resistance, and moisturizing properties inherent in lactose can be exhibited with a relatively small amount. Because of this, when using the high-content lactose sugar or high-purity lactose sugar obtained in the present invention to manufacture various compositions such as food and drink, cosmetics, medicine, and feed, a wider range can be selected when blending with other raw materials. Match composition.

In addition, the crystallized lactose containing pentahydrate produced by crystallization from a supersaturated aqueous solution of lactose containing high-content sugar or high-purity lactose, and then extracting it is substantially non-hygroscopic, has good fluidity, and is not easy to stick , fixation, and easy handling, so it can be advantageously used in various solid compositions.

The present invention is an invention that achieves such remarkable effects, and is an invention that makes a very significant contribution in this field.

Claims (16)

1. sugar with high content of lactosucrose contains more than or equal to 70% lactosucrose, 1-kestose and fructosyl lactosucrose total less than 3% as the sugar composition.

2. sugar with high content of lactosucrose according to claim 1 also contains as other carbohydrate and to be selected from more than a kind or 2 kinds of glycerine, glucose, fructose, sucrose, lactose and lactulose.

3. the manufacture method of claim 1 or 2 described sugar with high content of lactosucrose, it is characterized in that, in the aqueous solution that contains sucrose and lactose, make from saccharase and the non-usability yeast of sucrose of the microorganism that belongs to bacillus and have an effect, formation is formed to contain more than or equal to 70% lactosucrose, 1-kestose and fructosyl lactosucrose as sugar and is added up to reactant less than 3%, as required, make again and mix the lactose isomerization that exists, after forming the mixture of lactose and lactulose, with its extraction.

4. the manufacture method of sugar with high content of lactosucrose according to claim 3 wherein, is a recombinase from the saccharase of the microorganism that belongs to bacillus.

5. according to the manufacture method of claim 3 or 4 described sugar with high content of lactosucrose, wherein, the microorganism that belongs to bacillus is bacillus kind V230 (FERM BP-5054).

6. according to the manufacture method of each the described sugar with high content of lactosucrose in the claim 3～5, wherein, the non-usability yeast of sucrose is yeast saccharomyces cerevisiae ATCC56741 or yeast saccharomyces cerevisiae ATCC56742.

7. according to the manufacture method of each the described sugar with high content of lactosucrose in the claim 3～6, wherein, as the ratio of the sucrose of the reaction raw materials in the lactosucrose formation reaction and lactose be 1: 0.65 to 1.3.

8. according to the manufacture method of the described sugar with high content of lactosucrose of arbitrary claim in the claim 3～7, wherein, the reaction solution pH in the lactosucrose formation reaction is adjusted to 4.0 to 5.5.

9. the manufacture method of high purity lactosucrose is characterized in that, claim 1 or 2 described sugar with high content of lactosucrose by using the chromatography of resin, are obtained as sugar and form the fraction that contains more than or equal to 90% lactosucrose.

10. high purity lactosucrose manufacturing method of power, it is characterized in that, claim 1 or 2 described sugar with high content of lactosucrose by using the chromatography of resin, are obtained as sugar and form the fraction that contains more than or equal to 90% lactosucrose, this fraction is concentrated, carry out spraying drying.

11., wherein, also comprise granulation process according to claim 9 or 10 described high purity lactosucrose manufacturing method of power.

12. the manufacture method of crystallization lactosucrose, it is characterized in that, the high purity lactosucrose that use is obtained by the manufacture method of claim 1 or 2 described sugar with high content of lactosucrose or the described high purity lactosucrose of claim 9, make the crystallization from the supersaturated solution of lactosucrose of crystallization lactosucrose, extract this crystallization then.

13. the manufacture method of crystallization lactosucrose according to claim 12, wherein, the crystallization lactosucrose is to contain 5 water crystallization lactosucroses.

14. solid-state composition contains the crystallization lactosucrose that the manufacture method by claim 12 or 13 described crystallization lactosucroses obtains.

15. solid-state composition according to claim 14 is characterized in that, when containing the crystallization lactosucrose, contains with respect to the quality of crystallization lactosucrose other the crystallised sugar with the scope of 0.01 to 100 times of amount.

16. according to claim 14 or 15 described solid-state compositions, this solid-state composition is any form of ingesta, makeup or medicine.