CN1946853A - Sugar with high content of lactosucrose, process for producing the same and use thereof - Google Patents

Abstract

A process comprising causing beta-fructofuranosidase derived from a microbe belonging to the genus Bacillus and sucrose unassimilating yeast to act on an aqueous solution containing sucrose and lactose to thereby produce a sugar with a high content of lactosucrose wherein with respect to sugar composition, lactosucrose is contained in a proportion of >= 70% while the sum of 1-kestose and fructosyllactosucrose is contained in a proportion of < 3%. Further, there is provided a process comprising subjecting this sugar with a high content of lactosucrose to chromatography to thereby produce a lactosucrose of high purity wherein with respect to sugar composition, lactosucrose is contained in a proportion of >= 90%. Still further, there is provided a process for producing crystalline lactosucrose. Moreover, there are provided, produced by the above processes, a sugar with a high content of lactosucrose and lactosucrose of high purity.

Description

Sugar with high content of lactosucrose and manufacture method thereof and purposes

Technical field

The invention relates to sugar with high content of lactosucrose and manufacture method thereof and purposes, in more detail, be about forming as sugar, contain lactosucrose, add up to less than 3% the 1-kestose and the sugar with high content of lactosucrose and the manufacture method thereof of fructosyl lactosucrose more than or equal to 70%, and then be about with this sugar with high content of lactosucrose by using the chromatography of resin, extraction contains the manufacture method that is divided into the high purity lactosucrose of feature more than or equal to the level of 90% lactosucrose as the sugar composition, further relates to the manufacture method of crystallization lactosucrose.

Background technology

In recent years, the function that has of various carbohydrates such as oligose becomes clear and definite just successively.In functional oligose, lactosucrose [β-D-galactosyl-(1,4)-α-D-glucosyl-(1,2)-and β-D-fructoside] have good functions such as indigestibility, bifidus bacterium breeding promotion property, difficult cariogenicity, moisture retention, be useful oligose in extensive uses such as food, makeup, medicine.Lactosucrose is in the aqueous solution that contains sucrose and lactose, make from the saccharase of the microorganism that belongs to genus arthrobacter and have an effect, (the opening flat 3-27285 communique) that industry is made by the catalytic sugared shift reaction of this saccharase with reference to the spy.But, only use the shift reaction of saccharase, the content of the lactosucrose of resulting reactant, form low about 30% degree that arrives as sugar, in addition, this reactant contains the unreacted lactose of more amount, sucrose, the glucose of reaction residues, fructose, the 1-kestose of by product, fructosyl lactosucrose etc., thereby the sugar composition that can give full play to the original function of lactosucrose hard to say.Therefore, in order to improve the purity of lactosucrose, after enzyme reaction, make partly crystallization of unreacted lactose, then or filter removal, or when enzyme reaction, add the method that saccharase disappearance yeast implements to make monose such as glucose to be utilized, make and contain lactosucrose carbohydrate (registered trademark " breast fruit Oligo ") (with reference to rattan Tian Xiaohui, " バ イ オ セ パ レ-シ ヨ Application プ ロ セ ス brief guide " (biochemical separating technology brief guide), the 196-201 page or leaf, upright altogether publication and distribution, 1996).Especially, in the shift reaction of saccharase and with saccharase, lack yeast method, not only remove monose such as glucose from reaction solution, and by removing monose, shift reaction is tended to the generation of lactosucrose, therefore have the growing amount of lactosucrose also to improve such benefit, the lactosucrose of generation reaches about 65% (opening flat 4-293494 communique with reference to the spy) of forming as the sugar of reactant.Have again, the reactant that in raw material this lactosucrose content has been improved makes the chromatography of spent ion exchange resin, lactosucrose purity is increased to about 80%, then, by the chromatography that uses octadecyl silica resin (ODS) just can access purity about 99% refining lactosucrose (with reference to rattan Tian Xiaohui, " バ イ オ セ パ レ-シ ヨ Application プ ロ セ ス brief guide " (biochemical separating technology brief guide), 196-201 page or leaf, upright altogether publication and distribution, 1996).

But, saccharase from the microorganism of genus arthrobacter, except that the fructosyl with sucrose is transferred on the lactose and generates the reaction of lactosucrose, also catalysis as side reaction fructosyl is transferred to sucrose from the reaction that generates the 1-kestose on one's body, or catalysis transfers to fructosyl on the lactosucrose that has generated and generates the reaction of fructosyl lactosucrose.As the generation of the 1-kestose of by product and fructosyl lactosucrose etc., from this viewpoint of manufacturing of lactosucrose, be worthless, become the reason that the lactosucrose growing amount is reduced.In addition, above-mentioned by product not by the utilization of saccharase disappearance yeast, therefore is accumulated in the reactant that saccharase and saccharase disappearance yeast is acted on simultaneously and obtain, and their content all reaches 5 to 10%.Have again, be raw material with what contain more amount as the 1-kestose of by product and the reactant of fructosyl lactosucrose, when making the high purity lactosucrose, owing to must use the above-mentioned ion exchange resin and the multiple refining means such as chromatography of octadecyl silica resin (ODS), there is the low such problem of the yield made from extra care the high purity lactosucrose that obtains, it is believed that the industry manufacturing that is not suitable for the high purity lactosucrose.

Known on the other hand, contain the lactosucrose powder, lower water absorbability by improving its purity, can obtain the higher powder (spy opens flat 4-281795 communique) of stability.But clear in research after this, this powder for example when 27 ℃ of temperature, relative humidity 75%, also very easily moisture absorption, is unsettled under the hot and humid environment in summer, and processing is difficult, thereby is desirable to provide more stable lactosucrose solids.

On the other hand, Gad Avigad is at Journal Biological Chemistry, and nineteen fifty-seven, the 229th volume discloses the crystallization of lactosucrose in 121 to 129 pages, is to contain 5 water crystallization lactosucroses from what 90% (V/V) aqueous ethanolic solution obtained.But this crystallization water absorbability is strong, and crystallization is difficulty often, thereby the crystallization lactosucrose does not reach plant-scale production as yet.

Summary of the invention

In view of such situation, problem of the present invention provides, 1-kestose and fructosyl lactosucrose content as by product are low, and form the sugar with high content of lactosucrose contain more than or equal to 70% lactosucrose as sugar, in addition, the sugar with high content of lactosucrose that is suitable for making is provided industrially and forms the manufacture method that contains more than or equal to the high purity lactosucrose of 90% lactosucrose, also be the solid-state composition that the manufacture method of crystallization lactosucrose is provided and contains the low crystallization lactosucrose of water absorbability as sugar.

In order to solve above-mentioned problem, the inventor etc. in the lactosucrose that uses saccharase is made, not only are conceived to and with the non-usability yeast of sucrose, and be conceived to saccharase origin and reaction conditions, studied the manufacture method of lactosucrose.Its result is clear, even although think in the past also with saccharase and saccharase disappearance yeast, by the lactosucrose content in the reactant of this reaction generation, also only reach the most about 65% as the sugar composition, but be fully unexpectedly, in the aqueous solution that contains sucrose and lactose, and react with deriving from saccharase and the non-usability yeast of sucrose that the spy who belongs to the same Patent applicant opens the microorganism of the bacillus of putting down in writing in the flat 9-224665 communique, be controlled at 4.0 to 5.5 by pH with reaction solution, lactosucrose content in the resulting reactant reaches more than 70% as the sugar composition.Have again, clear and definite when examining or check resulting reactant, as the 1-kestose and the fructosyl lactosucrose of by product, form the utmost point as sugar and be low to moderate total less than 3%.And clear, if with this reactant as raw material, make with extra care with chromatography, just can be easily with the more highly purified lactosucrose of high yield preparation.In addition, be surprised to find that, separate out lactosucrose (containing 5 water crystallizations) crystallization easily from the supersaturated aqueous solution of the low lactosucrose of the content of above-mentioned by product 1-kestose and fructosyl lactosucrose, find the crystallization lactosucrose of acquisition, even under the hot and humid environment in summer, for example when 27 ℃ of temperature, relative humidity 75%, water absorbability is also low, be to handle the solid-state product that is easy to lactosucrose, in addition, clear, can advantageously be used in the manufacturing of the various solids that contain the crystallization lactosucrose.

That is, the invention provides to form to contain and add up to sugar with high content of lactosucrose less than 3% more than or equal to 70% lactosucrose, 1-kestose and fructosyl lactosucrose as sugar; Also provide so that the saccharase and the non-usability yeast of sucrose that derive from the microorganism that belongs to bacillus are had 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%, extracts the manufacture method that this reactant is the sugar with high content of lactosucrose of feature; And provide with the chromatography of this sugar with high content of lactosucrose by the use resin, obtain containing the manufacture method that is divided into the high purity lactosucrose of feature more than or equal to the level of 90% lactosucrose as the sugar composition; And then provide the sugar with high content of lactosucrose that obtains with aforesaid method or high purity lactosucrose to make the method for crystallization lactosucrose and contain the various solid-state compositions of this lactosucrose, solve above-mentioned problem thus.

According to the present invention, even do not use process for purification such as column chromatography, from sucrose and lactose, only with using the formation reaction of the non-usability zymic of saccharase and sucrose simultaneously, can make to form to contain to form as sugar and be low to moderate total less than 3% sugar with high content of lactosucrose more than or equal to the content of 70% lactosucrose and by product 1-kestose and fructosyl lactosucrose as sugar.Have again, this sugar with high content of lactosucrose by using the chromatography of resin, can easily be made as sugar and formed the high purity lactosucrose that contains more than or equal to 90% lactosucrose.In addition, according to the present invention, the lactosucrose that makes crystallization difficulty in the past from the supersaturated aqueous solution of lactosucrose easily crystallization become possibility, and resulting crystallization lactosucrose is even under the hot and humid environment in summer, water absorbability is also low, processing is easy, by containing this crystallization lactosucrose, just can provide water absorbability low, stable various solid-state compositions.

Embodiment

Sugar with high content of lactosucrose of the present invention, form as sugar, contain more than or equal to 70% lactosucrose, 1-kestose and fructosyl lactosucrose and add up to 1-kestose and fructosyl lactosucrose less than 3%, it is characterized in that the 1-kestose that generates by the side reaction of saccharase and the content of fructosyl lactosucrose, compare extremely low with the lactosucrose carbohydrate that contains in the past.

Carbohydrate in the sugar with high content of lactosucrose of the present invention beyond contained lactosucrose, 1-kestose and the fructosyl lactosucrose, the resolvent glucose of the sucrose that can enumerate the alkali isomerization thing lactulose of unreacted sucrose and lactose, lactose, produces because of the effect of saccharase and fructose and glucose that produces by the effect of the non-usability yeast of sucrose and lactose send out product glycerine etc. pure.As shown in the embodiment described later, the major part of these carbohydrates is not decomposed by saccharase yet, not the lactose that is decomposed by the utilization of the non-usability yeast of sucrose.

The manufacture method of sugar with high content of lactosucrose of the present invention (below, only be called " manufacture method of the present invention " or " this manufacture method " sometimes) is a feature with saccharase and the non-usability yeast of sucrose that is used to from the microorganism of gemma Pseudomonas.Said saccharase means among the present invention, at least make the β-fructofuranose glycosidic bond hydrolysis of sucrose, raffinose and erlose, and make fructose free reaction and with these carbohydrates with β-fructofuranose glycosidic bond as saccharide donor, catalytic enzyme is carried out in β-fructofuranose group-transfer to the reaction on the acceptor that is selected from other carbohydrates, sugar alcohol and alcohols beyond the saccharide donor.The saccharase that can utilize in the present invention defines in this wise: be derived from the microorganism that belongs to bacillus, and and the non-usability yeast of sucrose that in the present invention of as detailed below, uses and with and when having an effect, contain more than or equal to 70% lactosucrose so long as can make as the sugar composition, and the content of by product kestose and fructosyl lactosucrose forms to contain as sugar and is low to moderate total less than 3% sugar with high content of lactosucrose, and preparation method etc.s just do not limit especially.For example, open disclosed natural type saccharase in the flat 9-224665 communique the patent applicant's identical spy, and the applicant's identical with the application spy opens disclosed in the flat 10-66586 communique, as to use the above-mentioned saccharase of encoding DNA, the saccharase of the recombinant type that is prepared into gene recombination technology can both advantageously be used in the present invention from bacillus kind V230 (FERM BP-5054) origin with the application.Much less, in the occasion of recombinant type enzyme, be subordinated to the saccharase that the host cell beyond the microorganism of bacillus obtains and also can utilize in the present invention.Have again, the DNA of disclosed coding saccharase in the same communique is used variant enzyme that the skill and technique of protein engineering obtains need only the transferance of expectation is not disappeared, also can utilize in the present invention.

The non-usability yeast of the said in the present invention sucrose of what is called means, can utilize monose, but can not utilize and/or can not decompose the yeast of the above oligosaccharides of disaccharides.The non-usability yeast of the sucrose that adopts among the present invention, so long as can utilize glucose and fructose, but can not utilize and/or can not decompose: the yeast of sucrose, lactose and lactosucrose, its kind be restriction especially just.For example, make from natural isolating yeast with various mutagenss and to undergo mutation, make the saccharase disappearance, perhaps also can use sucrose to utilize Disability, the yeast of cultivating according to purpose.In addition, as saccharase disappearance yeast, known known bacterium yeast saccharomyces cerevisiae ATCC56741 or yeast saccharomyces cerevisiae ATCC56742 etc. can both be advantageously used in the present invention.

Become the sucrose and the lactose of the raw material in the manufacture method of the present invention, its preparation method or form be restriction especially not, for example, comprise commercially available product, by natural isolating concoction, enzyme method or chemical process preparation and even synthetic concoction, and then, enzyme reaction in carrying out manufacture method of the present invention or in of the utilization of the non-usability yeast of sucrose to monose, or in the utilization of the resultant that produces by this manufacture method, in the scope that does not produce obstruction, also can be to contain such carbohydrate concoction that is mingled with material in addition, or make up the composition of forming as above concoction.

Use saccharase and the non-usability yeast of sucrose simultaneously from the microorganism that belongs to bacillus, if comprise sucrose and lactose (below, refer to any or both of sucrose and lactose, be sometimes referred to as " substrate ") the aqueous solution in, except the pH with reaction solution is controlled at 4.0 to 5.5, under according to the character of the zymetology of used saccharase and condition that the physiological character of the non-usability zymic of sucrose is selected aptly, have an effect, just can access to form and contain more than or equal to 70% lactosucrose as sugar, and by product 1-kestose and fructosyl lactosucrose content are formed as sugar and are low to moderate total less than 3% reactant.The lactosucrose content of the reactant that obtains with this lactosucrose formation reaction only, forming as sugar usually is more than or equal to 70%, less than 80%, do not reach 80% or more than.Condition as the lactosucrose formation reaction, for example be used to when the spy who belongs to the same Patent applicant opens the saccharase of the microorganism of disclosed bacillus in the flat 9-224665 communique and the non-usability yeast saccharomyces cerevisiae of sucrose ATCC56741, the incomplete inactivation of this enzyme, and this yeast can utilize the condition of glucose and lactose, promptly, temperature is about 0 to 40 ℃ scope usually, suitable is to wish about 15 to 35 ℃ scope, pH, 4.0 to 5.5 the scope of being controlled at usually is desirable.Concentration of substrate in the reaction mixture,, desired response obtains reactant so long as being carried out, contain more than or equal to 70% lactosucrose as the sugar composition, and as the sugar composition, the content of by product 1-kestose and fructosyl lactosucrose adds up to less than 3%, just not restriction especially, for example, usually making sucrose and lactose is respectively the scope of 0.1 to 40 quality %, wishes the scope of preferred 1 to 30 quality %, preferably both amounts ratio be 1: 0.65 to 1.3.

About the usage quantity of saccharase, with respect to every 1g dry mass of the sucrose in the reaction raw materials (substrate), the scope of 0.1 to 50 unit normally, suitable is the scope of wishing to reach 0.5 to 10 unit.Moreover, active 1 unit of said saccharase means in so-called this specification sheets, spy according to the same Patent applicant opens the method for putting down in writing in the flat 9-224665 communique, when pH6.0,40 ℃ react as substrate with sucrose, made the enzyme amount of the amount of the D-glucose that the reducing power increase is equivalent to 2 μ mol in per 1 minute.

About the non-usability zymic of sucrose usage quantity, with respect to every 1g dry mass of the sucrose in the reaction raw materials (substrate), usually, in the scope of wet quality 0.01 to 1g, suitable is to wish to reach 0.05 to 0.2g scope.

Use the non-usability yeast method of saccharase and sucrose simultaneously, so long as make resulting reactant, contain more than or equal to 70% lactosucrose as the sugar composition, and the content of by product 1-kestose and fructosyl lactosucrose adds up to less than 3%, just not restriction especially, for example, can be in the aqueous solution that contains sucrose and lactose add saccharase constantly and yeast is had an effect it simultaneously or different, perhaps in reaction, append saccharase and zymic any or both aptly it is had an effect.Have again, in reaction vessel separately, utilize saccharase reaction and the non-usability zymic of sucrose to handle, circulate each other by the reaction solution that makes each and also can react continuously, under such a situation, use immobilization saccharase and the non-usability zymic of immobilization sucrose bio-reactor advantageously to utilize.

The time of using saccharase and non-usability yeast to react simultaneously, can select aptly according to the degree of using saccharase and non-usability yeast method or reaction to carry out simultaneously, 2 to 200 hours scope normally, suitable is the scope of wishing 10 to 80 hours.

The non-usability yeast of saccharase that uses in the reaction and/or sucrose as required, utilizes methods such as centrifugation, membrane filtration to separate, reclaim reactant, also can advantageously implement to use in this reaction again.

By adopting manufacture method as above sugar with high content of lactosucrose of the present invention, as sugar with high content of lactosucrose, can access as the sugar composition and contain more than or equal to 70%, and the content of by product 1-kestose and fructosyl lactosucrose is formed as sugar and is low to moderate total less than 3% reactant.Like this contain sugar with high content of lactosucrose, can be with intactly state use, in addition, by alkaline purification remaining lactose partly is isomerizated into lactulose, and lactose-content is lowered, after becoming the sugar with high content of lactosucrose that the crystallization of the lactose that inhibition contains separates out, also can arbitrarily use.Have again, use customary way, the for example decolouring that causes by activated carbon treatment etc., by ion exchange resin treatment etc. cause desalination, make that impurity is insoluble, make lactose remove, use the filtration of auxiliary agents such as diatomite, use concentrating of vaporizer etc. by the carbonic acid dipping by crystallization, as required, utilize the purifying goods that are refined to desired level from the suitable technology of selections such as spraying drying again, can be used in various uses.

In addition, also can sugar with high content of lactosucrose of the present invention as raw material, relending to help makes with extra care the chromatography of spent ion exchange resin, the fraction that contains more than or equal to 90% lactosucrose is formed in recovery as sugar, concentrate, form syrupy shape high purity lactosucrose, perhaps re-use the suitable drying process of from spraying drying, vacuum-drying, lyophilize etc., selecting and make it form the powder or the particle product of powdered, be used for various uses.

In addition, also can use above-mentioned sugar with high content of lactosucrose or high purity lactosucrose, form the supersaturated aqueous solution of lactosucrose, then, make crystallization lactosucrose crystallization, advantageously implement to extract this crystallization lactosucrose.As crystallization condition, so long as the supersaturated aqueous solution of lactosucrose is separated out just the crystallization lactosucrose, usually, lactosucrose concentration is suitable more than or equal to 75% the aqueous solution.If specifically describe, for example with purity approximately more than or equal to 75%, more wish about lactosucrose of 80 to 95% form about 75 to the 95 quality % of concentration or concentration, the temperature of its solution, so long as solution does not freeze, and below the crystalline fusing point, be not easy to cause the brown stain of lactosucrose, the temperature of decomposition, for example make the solution of about 5 to the 25 quality % of moisture carry out crystallization just at 20 to 60 ℃.In addition, when carrying out crystallization, for the degree of supersaturation of adjusting solution, viscosity etc., it also is random for example making coexistences such as methyl alcohol, ethanol, acetone.

Crystallization method, the solution that makes the higher temperatures that contains supersaturation lactosucrose solution usually wish to make kind of brilliant coexistence 0.1 to 20 a quality %, Yi Bian Yi Bian stir slow cooling at leisure, promote crystallization, and become massecuite (マ ス キ Star ト) just in helping crystallizer.

From crystallization massecuite make the method for crystallization lactosucrose, as long as can obtain the crystallization lactosucrose, for example, can utilize known method such as purging method, piece breaking method, fluidization granulation method, spray-drying process.

For example, the purging method typically uses basket centrifuge, makes the isolating method of crystallization lactosucrose and molasses, as required, a spot of cold water of spraying in this crystallization, it also is easy cleaning, and is suitable to the crystallization lactosucrose of making higher degree.Other three kinds of methods are not separated molasses, though thereby in the crystallization goods that obtain, fail to make purity to rise, have the many features of harvest product amount.Therefore, under the situation of these goods, in the crystallization goods, beyond lactosucrose, contain carbohydrate, for example sucrose, lactose etc. usually from raw material or manufacturing process.

Under the situation of spray drying process, usually about 20 to 50% the massecuite of percent crystallization in massecuite is sprayed from nozzle with high-pressure pump, in crystalline powder fused temperature not, for example use about 40 to 65 ℃ warm braw drying, then, if carry out dry slaking, just can easily make non-hygroscopic or the difficult hygroscopic crystalline powder that contains with warm wind.

In addition, the piece comminuting method leaves standstill about 10 to 60% the massecuite of percent crystallization in massecuite about 0.5 to 5 day usually, make all crystallizations be solidified into bulk, use methods such as pulverizing or cutting with its fragmentation again, carry out drying, just can easily make and contain non-hygroscopic, difficult hygroscopic crystalline powder.

In addition, according to ordinary method, the aqueous solution heating that will contain lactosucrose is concentrated into moisture and is lower than 5 quality %, formation is the supersaturated aqueous solution of the lactosucrose of molten state, in the temperature of being less than or equal to the crystalline fusing point, in this aqueous solution, mix and pinch kind of a crystalline substance, make it directly form various states, for example powder, particle, bar-shaped, tabular, cubes etc. are made non-hygroscopic or the difficult hygroscopic crystalline solid that contains also is random.

The sugar with high content of lactosucrose and the high purity lactosucrose that obtain by manufacture method of the present invention, also has the crystallization lactosucrose, with known lactosucrose in the past and contain in the identical purposes of lactosucrose carbohydrate, for example prevent agent, medicines for relieving intestinal disorders, mineral absorbefacient etc., can advantageously utilize in various fields such as the diet product field that comprises heath food, beverage, cosmetic field, field of medicaments, field of fodder as sweeting agent, indigestibility sweeting agent, low cariogenicity sweeting agent, wetting Agent for Printing Inks, age of starch.Especially, the crystallization lactosucrose is except above-mentioned various functions, coming down to non-hygroscopicly, is mobile, is not easy to adhere to, set, handle easily, have the feature that the thing that needs and people's funds can be cut down significantly in the management of its packing, transportation, storage etc.

In addition, crystallization lactosucrose of the present invention is non-hygroscopic and even difficult hygroscopic powder in fact, the thermotolerance height, stability is also good, so extremely difficult in the past various solid-state compositions that form of conduct, powder-mixed sweeting agent for example, chocolate, chewing gum, instant fruit juice, fast food soup, particle, the fixedly inhibitor of tablet etc., vehicle, extender, powder matrix etc. wish to make to contain more than or equal to 1 quality % crystallization lactosucrose, even for example make the diet product, makeup, medicine, various solid-state compositions such as shaping thing, and then as reagent, chemical industry raw material etc. also can advantageously utilize.

In addition, lactosucrose of the present invention, with other crystalline sugars, for example maltose, lactose, maltose alcohol, trehalose, palatinose, sucrose etc., wish the quality of relative crystallization lactosucrose, with the proportioning and the usefulness of 0.01 to 100 times of amount, can implement advantageously also that water absorbability is low, the manufacturing of the various solid-state compositions of processing ease.

Have again, high purity lactosucrose powder product or the crystallization product making as described above and obtain, with suitable water-soluble polymer, for example pullulan, water-soluble pullulan ether, water-soluble pullulan ester, gelatin, Sudan Gum-arabic, carboxymethyl cellulose, Sargassum polysaccharides class etc. are as wedding agent, utilize the ordinary method granulation, form mobile good, the easy granulation product of processing, also can advantageously be implemented in the various uses and utilize.

Below, illustrate in greater detail the present invention based on embodiment.

Embodiment 1

The manufacturing of sugar with high content of lactosucrose

Embodiment 1-1: the preparation of saccharase

Spy according to the same Patent applicant opens the method for putting down in writing in the flat 9-224665 communique, and the cultivation scale with 40L in the substratum that contains sucrose as carbon source is cultivated genus bacillus kind V230 (FERM BP-5054).Continuation is according to the method for putting down in writing in the above-mentioned communique, and the centrifugation culture extracts its supernatant liquor of about 38L, carries out the determination of activity of saccharase, has confirmed this culture supernatant demonstration saccharase activity.Above-mentioned culture supernatant is carried out the UF membrane concentration, obtain the active crude enzyme liquid 360ml of saccharase that every 1ml has about 360 units.Continuation is according to the method for putting down in writing in the above-mentioned communique, this crude enzyme liquid is supplied with ion exchange chromatography, the hydrophobic chromatography of use " Block チ Le-ト ヨ パ-Le " gel (TOSOH Co., Ltd's system) and the ion exchange chromatography that resupplies use " DEAE-ト ヨ パ-Le " gel that uses " DEAE-ト ヨ パ-Le " gel (TOSOH Co., Ltd's system), in 7.5% (w/v) polyacrylamide gel electrophoresis, obtain showing the refining standard substance of the saccharase of single band.The specific activity of the refining standard substance that obtain is about 205 units of every 1mg protein.

Embodiment 1-2: the non-usability zymic preparation of sucrose

In the trilateral flask of 500ml capacity, put into the liquid nutrient medium that 200ml is made of 2.0% (w/v) glucose, 2.0% (w/v) yeast extract and water, in autoclave, carry out sterilization in 121 ℃, 20 minutes, cooling then, inoculation yeast saccharomyces cerevisiae ATCC56741, with at 27 ℃, carry out the nutrient solution of rotational oscillation cultivation in 24 hours as kind of a cultivation with 230r/min.In the fermentation container of capacity 30L, put into and plant the substratum of cultivating same composition, carry out heat sterilization, cooling, reach 27 ℃ of temperature after, inoculation 1% (capacity/capacity) planted nutrient solution, keep pH4.0 to 7.0 for 27 ℃ in temperature on one side, cultivate Yi Bian carry out aeration-agitation in 48 hours.After the cultivation, centrifugation (8000rpm, 20 minutes) nutrient solution, this zymic thalline reclaims about 680g by wet quality.

Embodiment 1-3: the formation reaction of lactosucrose

Be adjusted to pH6.0 with containing the sucrose of 20 quality % and the aqueous solution of lactose respectively, with every 1g sucrose 1 unit and with every solids quality is the yeast for preparing among the refining standard substance of saccharase that prepare in wherein adding embodiment 1-1 respectively of 6.9% ratio and the embodiment 1-2, sodium hydroxide solution with 1N is controlled to pH4.0 to 5.5 with this reaction solution on one side, on one side 30 ℃ of reactions 24 hours and 42 hours.After the reaction, form with the sugar of high performance liquid chromatography (HPLC) assaying reaction liquid.HPLC uses " TSK-GEL AMIDE-80 " post (TOSOH Co., Ltd's system), 35 ℃ of column temperatures, use acetonitrile: carry out under the condition of water (71: 29), flow velocity 1.0ml/min, use differential refractometer " RI-8012 " (TOSOH Co., Ltd's system) to detect as elutriant.The results are shown in the table 1 of the analysis that sugar is formed.Moreover, experiment as a comparison, with rattan Tian Xiaohui etc., Agric Biol.Chem., 54 volumes, the method for record in the 913-919 page or leaf (1990), refining saccharase from genus arthrobacter kind K-1, add 10 unit purified saccharases with every 1g sucrose, except reaction solution being controlled to pH5.5 to 6.5, carry out identical operation.What obtain the results are shown in the table 1.

Table 1

The zymic source	Reaction times (hour)	Sugar is formed (%)
										Gly	DPl	Suc	Lac	Kes	LS	F-LS	UN
		V230 (the present invention)	24	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 (contrast)	24	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 in the table, DPl, Suc, Lac, Kes, LS, F-LS and UN mean monose, sucrose, lactose, 1-kestose, lactosucrose, fructosyl lactosucrose and other the unknown carbohydrate that glycerine, glucose and fructose are collectively referred to as respectively.

As shown in table 1, clear, the saccharase of the bacillus kind V230 that uses among the present invention, form as sugar, generate the lactosucrose in 71.0% (24 hours reaction times) to 73.6% (42 hours reaction times), more than or equal to 70%, and by product 1-kestose and fructosyl lactosucrose add up to and only generated for 0.1% (24 hours reaction times) to 0.3% (42 hours reaction times), less than 1%.On the other hand, it is the saccharase of correlated genus arthrobacter kind K-1, form the lactosucrose that only generates 63.3% (24 hours reaction times) to 63.0% (42 hours reaction times) as sugar, less than 65%, and it is clear, by product 1-kestose and fructosyl lactosucrose add up to 5.9% (the 24 hours reaction times) of generation to 12.4% (42 hours reaction times), significantly generate.

Embodiment 2

The manufacturing of sugar with high content of lactosucrose powder

The aqueous solution that will contain 24 quality % sucrose and 16 quality % lactose is adjusted to pH6.0, with every 1g sucrose 1 unit and with the wet quality of every raw material solids is the non-usability yeast of sucrose that is prepared among the saccharase crude enzyme liquid that is prepared in wherein adding embodiment 1-1 respectively of 7.5% ratio and the embodiment 1-2, sodium hydroxide solution with 1N is adjusted to pH4.0 to 5.5 with this reaction solution on one side, on one side 30 ℃ of reactions 45 hours.After the reaction, form with the sugar of HPLC assaying reaction liquid, contain 2.0% glycerine, add up to 2.1% glucose and fructose, 5.4% sucrose, 14.7% lactose, 1.4%1-kestose, 73.0% lactosucrose, 0.1% fructosyl lactosucrose and 1.3% other unknown carbohydrate.The reaction solution that obtains 90 ℃ the heating 30 minutes, make enzyme deactivation after, according to the ordinary method activated carbon filtration.After the filtrate that obtains adjusted to 60 ℃, every solids quality added slaked lime 1.5%, is adjusted to pH11, keeps being blown into carbonic acid gas after 15 minutes, becomes pH8, carries out carbonation.According to ordinary method it is filtered, the spent ion exchange resin desalination, after concentrating, carry out spraying drying, be made as sugar form contain 1.9% glycerine, add up to 2.2% glucose and fructose, 5.5% sucrose, 10.8% lactose, 3.3% lactulose, 1.3%1-kestose, 73.5% lactosucrose, 0.1% fructosyl lactosucrose and 1.4% other the sugar with high content of lactosucrose powder of unknown carbohydrate.

This product prevents agent, medicines for relieving intestinal disorders etc. as sweeting agent, indigestibility sweeting agent, low cariogenicity sweeting agent, wetting Agent for Printing Inks, age of starch, can be advantageously used in the diet product field that comprises heath food, beverage, cosmetic field, various fields such as field of medicaments, field of fodder.

Embodiment 3

The manufacturing of sugar with high content of lactosucrose slurry

The aqueous solution that will contain 18 quality % sucrose and 22 quality % lactose is adjusted to pH6.0, with every 1g sucrose 1 unit and with the wet quality of every solids is the non-usability yeast of sucrose that is prepared among the saccharase crude enzyme liquid that is prepared in wherein adding embodiment 1-1 respectively of 5.0% ratio and the embodiment 1-2, sodium hydroxide solution with 1N is adjusted to pH4.0 to 5.5 with this reaction solution on one side, on one side 32 ℃ of reactions 42 hours.After the reaction, form with the sugar of HPLC assaying reaction liquid, contain 1.9% glycerine, add up to 0.1% glucose and fructose, 1.2% sucrose, 22.6% lactose, 0.3%1-kestose, 72.4% lactosucrose, 0.1% fructosyl lactosucrose and 1.4% other unknown carbohydrate.According to ordinary method, the reaction solution that obtains with the SF membrane filtration and remove yeast is again according to ordinary method, use the UF membrane filtration, remove polymer compositions such as saccharase, then with behind the activated carbon filtration, carry out drying under reduced pressure, obtain the concentrated solution of the about 80 quality % of solids.After this concentrated solution is adjusted to about 25 ℃ of temperature, add the crystallization lactose powder of about 3 quality % of the lactose that is added, make the lactose partial crystallization.According to ordinary method by the lactose of filter removing crystallization and going out after, the spent ion exchange resin desalination, concentrate, be made as sugar form contain 2.2% glycerine, add up to 0.1% glucose and fructose, 1.4% sucrose, 10.1% lactose, 0.4%1-kestose, 84.1% lactosucrose, 0.1% fructosyl lactosucrose and 1.6% other the sugar with high content of lactosucrose slurry of unknown carbohydrate.

This product prevents agent, medicines for relieving intestinal disorders etc. as sweeting agent, indigestibility sweeting agent, low cariogenicity sweeting agent, wetting Agent for Printing Inks, age of starch, can be advantageously used in the diet product field that comprises heath food, beverage, cosmetic field, various fields such as field of medicaments, field of fodder.

Embodiment 4

The manufacturing of high purity lactosucrose powder

The aqueous solution that will contain 20 quality % sucrose and 20 quality % lactose is adjusted to pH6.0, with every 1g sucrose 1 unit and with the wet quality of every solids is the non-usability yeast of sucrose that is prepared among the saccharase crude enzyme liquid that is prepared in wherein adding embodiment 1-1 respectively of 5.0% ratio and the embodiment 1-2, sodium hydroxide solution with 1N is adjusted to pH4.0 to 5.5 with this reaction solution on one side, on one side 30 ℃ of reactions 30 hours.After the reaction, form with the sugar of HPLC assaying reaction liquid, contain 2.0% glycerine, add up to 0.2% glucose and fructose, 1.6% sucrose, 19.2% lactose, 0.6%1-kestose, 74.7% lactosucrose, 0.1% fructosyl lactosucrose and 1.7% other unknown carbohydrate.According to ordinary method, the reaction solution that obtains with the SF membrane filtration and remove yeast is again according to ordinary method, use the UF membrane filtration, remove polymer compositions such as saccharase, then with behind the activated carbon filtration, carry out drying under reduced pressure, obtain the concentrated solution of the about 60 quality % of solids.As former syrup,, use storng-acid cation exchange resin (trade(brand)name " ア Application バ-ラ イ ト CR-1310 " (Na with this sugar with high content of lactosucrose slurry in order to improve the purity of lactosucrose ⁺Type), オ Le ガ ノ Co., Ltd. system), implement column chromatography.That is, overlap at the strap clamp of 10 internal diameter 12.5cm and to fill above-mentioned resin in the stainless steel post, these posts are connected in series, make the resin layer total length reach 16m.Make the interior temperature of post maintain 40 ℃ on one side, amount of resin adds the above-mentioned syrup of 1.5% (v/v) relatively on one side, with SV (air speed) 0.2 40 ℃ warm water being flow through therein carries out, when forming, separate lactosucrose high-content fraction with the sugar of HPLC method monitoring elutriant, filter this fraction according to ordinary method, make spent ion exchange resin, gac carries out desalination, decolorizing and refining, after concentrating, carry out spraying drying, be made as the sugar composition and contain 0.1% sucrose, 2.3% lactose, the 1.1%1-kestose, 96.0% lactosucrose, 0.2% fructosyl lactosucrose and 0.3% other the high purity lactosucrose powder of unknown carbohydrate.

This product prevents agent, medicines for relieving intestinal disorders etc. as sweeting agent, indigestibility sweeting agent, low cariogenicity sweeting agent, wetting Agent for Printing Inks, age of starch, can be advantageously used in the diet product field that comprises heath food, beverage, cosmetic field, various fields such as field of medicaments, field of fodder.

Embodiment 5

The manufacturing of high purity lactosucrose granulation product

The high purity lactosucrose powder that 100 mass parts embodiment 4 are obtained, becoming about 3% (by weight/volume) with ultimate density makes pullulan (trade(brand)name " Pullulan PF-20 ", the former business of Co., Ltd. woods is sold) be dissolved in the deionized water and about 6 mass parts of the pullulan aqueous solution that obtains are sprayed according to ordinary method and carried out granulation in advance.The granulation product that obtain obtain high purity lactosucrose granulation product with the screen sizing of screen-aperture 1.7mm.Measure the size-grade distribution of these granulation product, when comparing with the size-grade distribution of raw material high purity lactosucrose powder, account for more than or equal to 85% opposite with particle that particle diameter in the raw material high purity lactosucrose powder is less than or equal to 106 μ m, particle diameter accounts for more than or equal to 70% more than or equal to the particle of 150 μ m in the high purity lactosucrose granulation product.

This product is because be granulation product, thus good fluidity, handle easily, and, have and when dissolving, be difficult for the feature that generation still is exactly a powder.This product prevents agent, medicines for relieving intestinal disorders etc. as sweeting agent, indigestibility sweeting agent, low cariogenicity sweeting agent, wetting Agent for Printing Inks, age of starch, can be advantageously used in the diet product field that comprises heath food, beverage, cosmetic field, various fields such as field of medicaments, field of fodder.

Embodiment 6

The crystallization lactosucrose

Filter the lactosucrose high-content fraction that the method with embodiment 4 obtains according to ordinary method, spent ion exchange resin, gac desalination, decolouring, make with extra care, be evaporated to the about 25 quality % of moisture, utilization helps crystallizer, the limit adds 1 quality % kind crystal edge stirs at leisure, with extremely about 30 ℃ of slow cooling in 24 hours.Place it on the separating centrifuge, obtain containing 5 water crystallization lactosucroses with yield about 40%.This product is a purity more than or equal to about 98% crystallization lactosucrose, even under the hot and humid environment in summer, do not show water absorbability in fact yet, it is easy to handle, as sweeting agent, indigestibility sweeting agent, medicines for relieving intestinal disorders etc., can be advantageously used in various fields such as diet product, makeup, medicine, especially can be advantageously used in the purposes of the solid-state composition that contains the crystallization lactosucrose.

Embodiment 7

Sweeting agent

In the crystallization lactosucrose that 1 mass parts obtains with the method for embodiment 6, mix 0.05 mass parts alpha-glucosyl stevioside (Japan refined sugar Co., Ltd. sells, trade(brand)name " α G-Sweet ") equably, obtain granular sweetener with granulating machine.This product sweet taste best in quality has the sweet taste of about 2 times granulated sugar, and the heat when sugariness is suitable is low to moderate about 1/2 of granulated sugar.This sweeting agent as low calorie sweetener, is suitable to the low-calorie diet product acquisition sweet taste for the obese person of caloric restriction picked-up, diabetics etc.In addition, this sweeting agent is because to bring out the generation of microbial acid by carious tooth few, and the generation of insoluble glucan is few, is suitable to the acquisition sweet tastes such as diet product that suppress carious tooth.This sweeting agent also is suitable as the medicines for relieving intestinal disorders of adjusting the flora in the intestines.

Embodiment 8

The shaping sweeting agent

Mix 1 mass parts crystallization lactosucrose and 2 mass parts granulated sugar that the method with embodiment 6 obtains equably, to an amount of water of wherein spraying, according to the ordinary method shaping and obtain the sweeting agent that is shaped.This product can effectively utilize in the acquisition sweet taste that makes coffee, black tea etc. as the shaping sweeting agent with whole intestines function of adjusting the flora in the intestines.

Embodiment 9

Chocolate

The 30 mass parts crystallization lactosucroses that mix 40 mass parts cocoa benevolence, 10 mass parts theobroma oils, 20 mass parts granulated sugar, obtain with the method for embodiment 6, in refining mill (レ Off ア イ Na-), pass through, after granularity is reduced, put into コ Application チ エ, carried out 2 refinings round the clock at 50 ℃.Add 0.5 mass parts Yelkin TTS therebetween, and make its blending dispersion fully.Then, be adjusted to 31 ℃, before theobroma oil is about to solidify, inject mould, outgas, pass under 10 ℃ the cooling air channel and made its curing in 20 minutes with bobbing machine with the temperature regulation machine.With its demoulding, pack and obtain goods.This product does not have water absorbability, and color, gloss are all good, and interior tissue is also good, successfully dissolves in mouth, has top grade sweet taste and satisfied local flavor.In addition, this product also is suitable as the chocolate with whole intestines effect.

Embodiment 10

Chewing gum

3 mass parts glue nurse base heating and meltings are become the softish degree, (the former business of Co., Ltd. woods is sold to wherein adding 4 mass parts anhydrous crystalline maltitols, registered trademark " マ PVC Star ト ") and the 3 mass parts crystallization lactosucroses that obtain with the method for embodiment 6, spices that remix is an amount of and colouring matter, mix with roller according to ordinary method, form, pack and obtain goods.This product is all good chewing gum of structure, local flavor.In addition, this product also is suitable as the chewing gum with whole intestines effect.

Embodiment 11

Beverage

The 50 mass parts high purity lactosucrose powder, 10 mass parts trehaloses, 0.25 mass parts yogurt condiment and the 0.1 mass parts lemon flavour that in water, add 100 mass parts yogurts, obtain with the method for embodiment 4, make and all fit in 1000 mass parts, according to ordinary method, make yoghurt drink.This product local flavor is abundant, and has the whole intestines function of adjusting the flora in the intestines.

Embodiment 12

Enteric nutrient

Be prepared into the title complex that the 10 mass parts crystallization lactosucroses, 10 mass parts trehaloses, 1.1 mass parts glycine, 1 mass parts Sodium Glutamate, 0.4 mass parts calcium lactate, 0.1 mass parts magnesiumcarbonate, 0.01 mass parts VitB1 and the 0.01 mass parts riboflavin that are obtained by the method with embodiment 6 constitute.This title complex of every 24g is filled in the lamination aluminum parcel, seals and obtains goods.This product, 1 bag of amount of dissolving in about water of 33 to 500ml, as nutrient liquid, by through the tube side method, administration such as via intranasal application, stomach and using.This product is not only to the people, and can advantageously utilize as the non-oral feeding bulking liquor to domestic animal etc.In addition, this product has the whole intestines effect of adjusting intestinal flora.

Embodiment 13

Coated tablet

With the plain sheet of quality 150mg as the core agent, in the core agent, use the underlying liquid of the 45 mass parts crystallization lactosucroses, 2 mass parts pullulans (molecular-weight average 200,000), 30 mass parts water, 25 mass parts talcum powder and the 3 mass parts titanium oxide formation that obtain by method with embodiment 6, it is about 230mg that sugar coating makes tablet quality, then, use forms sugar-coat by the upper strata liquid that phase syncrystallization lactosucrose, 1 mass parts pullulan and the 34 mass parts water of 65 mass parts constitute, polish with wax liquid again, obtain the good coated tablet of glossy outward appearance.This product not only operability during sugar coating is good, and shock-resistance is also good, keeps high-quality for a long time.In addition, this product has the whole intestines function of adjusting the flora in the intestines.

Industrial applicibility

As previously discussed, manufacture method according to sugar with high content of lactosucrose of the present invention, even do not use the refining means such as post chromatography, only with the reaction of using simultaneously the non-usability yeast of saccharase and sucrose, just can contain sugar with high content of lactosucrose more than or equal to 70% lactosucrose as the sugar composition from sucrose and lactose manufacturing. In addition, the sugar with high content of lactosucrose that obtains with manufacture method of the present invention, the content of accessory substance GF2 and fructosyl lactosucrose forms total as sugar and is low to moderate less than 3%, if thereby make with extra care with chromatography as raw material with it, just can be easily with the more highly purified lactosucrose of high receipts rate preparation.

Have again, the sugar with high content of lactosucrose patient high-purity lactosucrose that obtains according to the present invention, as sugar form contain more than or equal to 70% or more than or equal to 90% to the effective lactosucrose of performance function, thereby compare with the low lactosucrose carbohydrate that contains of lactosucrose content in the past, just can bring into play difficulty digestion property, the bifidocaterium breeding that lactosucrose had originally with fewer amount and promote the good functions such as property, difficult cariogenicity, moisture retention. Because this when the sugar with high content of lactosucrose that will obtain with the present invention or high-purity lactosucrose cooperate various compositions such as making diet product, cosmetics, medicine, feed, cooperates at the raw material with other, can select the mix proportion of wider scope.

Have again, analyse crystalline substance from the lactosucrose supersaturated aqueous solution that contains sugar with high content of lactosucrose or high-purity lactosucrose, then extract and make contain 5 water crystallization lactosucroses, it is in fact non-hygroscopic, good fluidity, be not easy adhesion, set, process easily, therefore can be advantageously used in various solid-state compositions.

The present invention is the invention of the remarkable action effect that reaches such, is the invention that very large contribution meaning is arranged in this area.

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.