CN101691597B - Methods for preparing L-tagatose - Google Patents

Abstract

The invention relates to methods for preparing L-tagatose, which belongs to the field of biotechnology. A method for preparing the L-tagatose comprises the following steps: preparing a culture medium; culturing acid-producing bacteria to obtain primary seed liquid; obtaining secondary seed liquid; inoculating secondary seed liquid to a fermentation culture medium to obtain fermentation liquor; obtaining primary yeast seed liquid and secondary yeast seed liquid and centrifuging the primary yeast seed liquid and the secondary yeast seed liquid to obtain yeast cells; culturing the yeast cells to obtain clear fermentation liquor; concentrating, decolorizing and filtering the clear fermentation liquor to obtain solution; and passing the solution through an ion exchange resin, performing crystallization, washing crystals and drying the crystals to obtain the L-tagatose. A method for preparing the L-tagatose by converting D-dulcitol by using resting cells comprises the following steps: preparing a culture medium; culturing bacteria; suspending somatic cells of the bacteria to obtain solution; inoculating candida ssp. CGMCC No.3268 to obtain fermentation liquor; and subjecting the fermentation liquor to decolorizing and desalting processing to obtain aqueous solution, concentrating the aqueous, performing crystallization, washing crystals and drying the crystals to obtain the L-tagatose. The methods of the invention can be used for preparing pharmaceutical L-tagatose.

Description

The method for preparing the L-tagatose

Technical field

That the present invention relates to is a kind of preparation method of biological technical field, is specifically related to a kind of method of the L-of preparation tagatose.

Background technology

Tagatose belongs to a kind of rare hexose, is divided into D-tagatose and L-tagatose, though they exist at nature, amount is very rare, and wherein the amount of L-tagatose is very rare especially.The D-tagatose is just used by drugs approved by FDA as far back as calendar year 2001 as a kind of safety sugar low in calories, and immeasurable restriction, and its clinical medicine as prevent diabetes has also got into clinical III phase experimental stage.

Literature search through to prior art finds that the main method of synthetic D-tagatose has at present: come isomerizing D-semi-lactosi to generate D-tagatose (U.S. Pat 5002612, open day: 1991.3.26 with metal hydroxides or sodium aluminate as catalyzer; Chinese patent CN200610123316.5, open day: 2007.6.27; Chemistry world, 187～188 pages of the third phases in 2009); Come isomery D-semi-lactosi to generate D-tagatose (U.S. Pat 6933138B2, open day: 2005.8.23 with the L-arabinose isomerase as catalyzer; U.S. Pat 2008/0124770 A1, open day: 2008.5.29; Chinese patent CN200810037303.5, open day: 2008.9.17; LeeDong-Woo etc., ApplEnviron Microbiol, 2004, vol.70, No.3,1397～1404), generate D-tagatose (Chinese patent CN200610085922.2, open day: 2006.5.26) with the microorganism cells fermentation.

In recent years, biological chemistry industry and carbohydrate chemistry scientific development are rapid, and increasing rare sugar is used to the midbody or the precursor of synthetic other medicines, and this just requires can cheap synthetic various rare sugar, comprise the L-tagatose.The L-tagatose can be used as the precursor of more synthetic medicines; Such as synthetic 1-deoxy-1-(indol-3-yl)-L-tagatose and analogue (Carbohydr Res thereof; 2003; 338:143-152), 1-(1-Butylinol-3-yl)-1-deoxy-L-tagatose and analogue thereof (NucleosidesNucleotidesNucleicAcids, 2004,23:281～289).Yet a large amount of cheap preparation of L-tagatose does not also have practical way at present, has limited the exploitation of L-tagatose greatly and has used.Therefore, press for the preparation method of the L-tagatose of a kind of efficient cheapness of exploitation, to satisfy biological chemistry industry and carbohydrate chemistry industry use to the L-tagatose.

L-tagatose and L-semi-lactosi are epimer, and the L-semi-lactosi changes the L-tagatose into after can passing through isomerizing.U.S. Pat 5002612 (open day: 1991.3.26) invented a kind of method that generates the L-tagatose by the L-semi-lactosi with calcium hydroxide and calcium chloride as the catalyzer isomery.This method adds the 0.5gL-semi-lactosi in the 25ml reaction flask, 5ml water adds 0.2g calcium hydroxide and 22mg calcium acetate in this reactor drum then; Reaction certain hour (such as 2 hours); Form the thickness mixture of L-tagatose and calcium hydroxide, remove by filter insolubles, feed carbonic acid gas and make the formation ZG 301; The L-tagatose is then free in solution from this thickness mixture; Remove scavenging process such as salt ion through IX again, obtain 0.22 gram L-tagatose, transformation efficiency is 44%.The raw material L-semi-lactosi itself that this method adopted also is a kind of rare sugar, and it is very limited to originate, and price is also very expensive.Next adopts metal hydroxides to come isomerizing L-semi-lactosi yield also lower, also produces a certain amount of other unknown product except that the L-tagatose, and its follow-up separation and purification process is also comparatively complicated.Owing to contain a large amount of salt ions in the reaction solution, at the process need of purifying salt ion is removed, can produce a large amount of waste water.Therefore the L-tagatose that adopts this method to produce also will be very expensive, be difficult to accepted, and make applying of L-tagatose receive great restriction.

U.S. Pat 5811271 (open day: 1998.9.22) invented a kind of method for transformation of enzyme that adopts and prepared the L-tagatose.This method adopts microbe-derived D-tagatose isomerase to make the isomerizing of L-sorbose prepare the L-tagatose.This method is culturing micro-organisms cell (Pseudomonas cichorii ST-24) at first, D-tagatose isomerase is extracted from cell again, and immobilized enzyme transforms 4% the L-sorbose aqueous solution then, transforms 90h at 45 ℃, and transformation efficiency is 20%.Through calcium type resin chromatography L-tagatose and unconverted L-sorbose are separated again.The employed raw material L-sorbose of this method itself also is a kind of rare sugar, and it is also very limited to originate, and price is also very expensive.Add that its transformation efficiency is also lower, has only 20%.And at first need prepare immobilized enzyme, this itself is exactly a very expensive process.Also need pass through the resin chromatography after the enzymatic conversion separates product.Raw material that these are expensive and process have determined that the product L-tagatose of preparation also will be very expensive thus.

The D-melampyrum is that Xylitol prepares a kind of by product in the process, in the process that purifies Xylitol, must the D-melampyrum be removed.Therefore in the process of preparation Xylitol, can obtain a large amount of D-melampyrums.The 5th carbon hydroxyl of D-melampyrum is oxidized to ketone group promptly becomes the L-tagatose.Therefore utilizing cheap D-melampyrum to prepare the L-tagatose is a kind of very feasible method.People (the Journal of Fermentation andBioengineering such as Tsuyoshi Shimonishi of Japan Kagawa Univ.; 1995; Vol.79; No.6,620～622) utilize Cray uncle streptococcus pneumoniae (Klebsiellapneumoniae strain 40b) conversion D-melampyrum to generate the L-tagatose, but the glycerine that needs to add 1% (mass percent) just can reach the transformation efficiency more than 60%.And the concentration of initial D-melampyrum is lower, is up to 2.0%, the rare cost that increases later separation of strength of solution.And employed bacterial strain is the pathogenic microorganism streptococcus pneumoniae, big limitations its use in actual production.The people such as AlexanderHuwig (CarbohydrateResearch, 1998, vol.305,337～339) of Germany are used to galactitol dehydrogenase from red bacterium (Rhodobacter sphaeroides D), and to transform melampyrum be the L-tagatose.At first cultivate red bacterium Rhodobacter sphaeroides D, smudge cells obtains containing the enzyme liquid of galactitol dehydrogenase, again with enzyme liquid and melampyrum reaction, transforms and generates the L-tagatose.Though total transformation efficiency can reach 78%, this process need prepares enzyme liquid, and enzyme reaction needs coenzyme NAD, and this coenzyme need be regenerated reaction is constantly gone on.This method realizes the regeneration of coenzyme NAD through serum lactic dehydrogenase, makes this regenerative process very expensive.Need preparation enzyme liquid and coenzyme to regenerate and make that preparing the L-tagatose through this method becomes unrealistic.People (Journal of Bioscience and Bioengineering such as the Devendar Rao of nearest Japanese Kagawa Univ.; 2008; Vol.106; No.5,473～480) be substrate with L-than Lip river ketose (L-psicose), adopting a kind of enterobacteria (Enterobacteraerogenes 230S) to transform L-is the L-tagatose than Lip river ketose.At first L-is reduced to L-talitol than Lip river ketose under the condition of the little oxygen of cell, L-talitol is oxidized to the L-tagatose again under the aerobic condition of cell.The raw material L-that this method adopted, costs an arm and a leg also for a kind of very rare rare sugar than Lip river ketose itself, but also will have the regeneration problem of cofactors equally successively through the process of reduction with oxidation.Make this method of employing prepare the L-tagatose and become unrealistic.

Summary of the invention

The objective of the invention is to overcome the deficiency of prior art, a kind of method of the L-of preparation tagatose is provided.Method green safety of the present invention, transformation efficiency are high, are fit to be used for preparing the L-tagatose of pharmaceutical grade.

The present invention realizes through following technical scheme, the present invention includes following steps:

Step 1, the substratum of acid formers is cultivated in preparation, and the composition of substratum is: the 1L substratum is composed of the following components: D-galactitol 2～20 grams; Polyvalent alcohol 1～10 gram, glucose 5～50 grams, yeast powder 10 grams; Anhydrous magnesium sulfate 0.5 gram, lime carbonate 10 grams, surplus is a water;

Step 2 is seeded in acid formers on the step 1 gained substratum, and 25～35 ℃ of shaking culture get primary seed solution;

Step 3 is inoculated into primary seed solution in the step 1 gained substratum, and 25～35 ℃ of shaking culture get secondary seed solution;

Step 4 is that 5～20% inoculum size is inoculated in the fermention medium with secondary seed solution according to volume ratio, cultivates, and is centrifugal, obtains fermented liquid;

The composition of said fermention medium is that the 1L fermention medium is composed of the following components: D-galactitol 10～100 grams, and polyvalent alcohol 1～10 gram, glucose 5～50 grams, yeast powder 5～50 grams, sal epsom 0.5 gram, potassium hydrogenphosphate 2 grams, surplus is a water;

Step 5, preparation are used for the substratum of culturing yeast, and medium component is: the 1L substratum is composed of the following components: glucose 15 grams, and yeast powder 10 grams, anhydrous magnesium sulfate 0.5 gram, surplus is a water;

Step 6, in step 5 gained substratum, 25～35 ℃ of shaking culture obtain the yeast primary seed solution with yeast-inoculated;

Step 7 is inoculated into the yeast primary seed solution in the step 5 gained substratum, and 25～35 ℃ of shaking culture obtain secondary yeast starter liquid, and are centrifugal, gets yeast cell;

Step 8 is inoculated into yeast cell in the step 4 gained fermented liquid, and 25～35 ℃ of shaking culture 5～40 hours are centrifugal, clarifying fermented liquid;

Step 9 is concentrated into 1/10 of original volume with step 8 gained fermented liquid, adds mass percent and be 2% powdery gac, and under 80 ℃, decolouring is 120 minutes under the 100rpm condition, crosses diameter and be 0.22～0.45 micron millipore filtration, solution;

Step 10 is passed through D001 Zeo-karb and D201 anionite-exchange resin successively with step 9 gained solution, until specific conductivity≤20 μ s/cm; Reconcentration is＞60% to refractive index, adds the absolute ethyl alcohol of 2～8 times of volumes, mixing; 4～30 ℃ of following crystallizations 20～30 hours, centrifugal, crystal; The volume percent that adds 2 times of quality is 95% washing with alcohol 2～3 times, and drying promptly gets the L-tagatose.

In step 1 and the step 4, said polyvalent alcohol is one or more the mixing in glycerine, erythritol, Xylitol, ribitol, arabitol, sorbyl alcohol and the N.F,USP MANNITOL.

In the step 2, said acid formers is that acetobacter (Acetobacter), bacillus of oxidizing glucose belong to the arbitrary bacterium in (Gluconobacter) or the gluconic acid bacterium genus (Gluconacetobacter).

In the step 2, said acid formers is weak oxidized acetic acid bacteria (Acetobacter suboxydans), the inferior bacillus aceticus of the Chinese (Acetobacter hansenii) or Pasteur's bacillus aceticus (Acetobacter pasteurianus).

In the step 2, said acid formers is weak oxidized acetic acid bacteria (Acetobacter suboxydans).

In the step 2, said acid formers is gluconobacter oxydans (Gluconobacter oxydans), weak glucose oxidation bacteria (Gluconobacter suboxydans) or Gluconobacter frateurii.

In the step 2, said acid formers is the inferior glucose vinegar acidfast bacilli of the Chinese (Gluconacetobacter hansenii).

In the step 6, said yeast is candiyeast CGMCC NO:3268.

The invention still further relates to a kind of method of the L-of preparation tagatose, this method is to adopt resting cell to transform the D-galactitol to prepare the L-tagatose simultaneously, comprises the steps:

Step 1, the preparation substratum, the composition of substratum is: the 1L substratum is composed of the following components, D-galactitol 5 grams, sorbyl alcohol 5 grams, glucose 20 grams, yeast powder 15 grams, sal epsom 0.5 gram, potassium hydrogenphosphate 2 grams, lime carbonate 5 grams, surplus is a water; Arbitrary microbionation in acetobacter (Acetobacter), bacillus of oxidizing glucose genus (Gluconobacter) or the gluconic acid bacterium genus (Gluconacetobacter) in substratum, is cultivated;

Step 2, centrifugal after cultivate finishing, somatic cells, suspended bacteria somatocyte, 25～35 ℃, 100～300rpm transform 24～96 hours down, solution;

The solution that said suspension is used is that the component of solution is: 1%～10% D-galactitol, and 0.1～0.5% yeast powder, surplus is a water, said percentage ratio is massfraction;

Step 3 inserts candiyeast (Candida ssp.) CGMCC NO:3268 in the step 2 gained solution, cultivates 10～48 hours for 25～35 ℃, gets fermented liquid;

Step 4 successively through decolouring and desalting treatment, obtains clear aqueous solution with step 3 gained fermented liquid; The aqueous solution is concentrated to refractive index＞60%, adds the absolute ethyl alcohol of 2～8 times of volumes, mixing; 4～30 ℃ of following crystallizations 20～30 hours, centrifugal, crystal; The volume percent that adds 2 times of quality is 95% washing with alcohol 2～3 times, and dried crystals promptly gets the L-tagatose.

The acid formers that the present invention adopted is generally regarded as safe mikrobe, and is all open, belongs to prior art, at Chinese common micro-organisms preservation center or Chinese industrial microbial preservation center all can buy.

The yeast that is adopted among the present invention is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation; The title of this bacterial strain: candiyeast (Candida ssp.); Preserving number: CGMCC NO:3268; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preservation date is on September 11st, 2009.

This yeast strain is cell oval or long oval, gemmation, and individual cells or 2～4 cells are connected in a string.Bacterium colony is circular, and edge spination, white or pearl, surface drying are protruding slightly; Can contain glucose, semi-lactosi, wood sugar, Xylitol, sorbyl alcohol, N.F,USP MANNITOL, the D-melampyrum, L-arabinose alcohol, the D-arabitol is as well growth on the minimum medium of sole carbon source.Can utilize D-tagatose and L-tagatose.It is carried out repeatedly chemomorphosis with EMS and NTG, and forfeiture utilizes the ability of L-tagatose.

Compared with prior art, the present invention has following beneficial effect: method of the present invention can be from D-galactitol efficient production L-tagatose, has green safety, advantage that transformation efficiency is high, is fit to be used for preparing pharmaceutical grade L-tagatose.

Description of drawings

The HPLC collection of illustrative plates of Fig. 1 for adopting weak oxidized acetic acid bacteria in the fermention medium that contains the 1%D-melampyrum, to ferment 24 hours;

Fig. 2 is for adopting weak oxidized acetic acid bacteria HPLC collection of illustrative plates of 48 hours of conversion of resting cells in the solution that contains the 4%D-melampyrum;

Fig. 3 adopts weak oxidized acetic acid bacteria HPLC collection of illustrative plates of 48 hours of conversion of resting cells in the solution that contains the 8%D-melampyrum;

Fig. 4 is for adopting gluconobacter oxydans HPLC collection of illustrative plates of 48 hours of conversion of resting cells in the solution that contains the 4%D-melampyrum;

Fig. 5 is for adopting Pasteur bacillus aceticus bacillus HPLC collection of illustrative plates of 48 hours of conversion of resting cells in the solution that contains the 3%D-melampyrum;

Fig. 6 is the HPLC collection of illustrative plates for adopting weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 4%D-melampyrum to adopt candiyeast (Candida ssp.) CGMCC NO:3268 to ferment after 48 hours again 10 hours;

Fig. 7 adopts 10 hours HPLC collection of illustrative plates of candiyeast (Candida ssp.) CGMCC NO:3268 fermentation again for adopting gluconobacter oxydans conversion of resting cells in the solution that contains the 4%D-melampyrum after 48 hours;

Fig. 8 adopts 10 hours HPLC collection of illustrative plates of candiyeast (Candida ssp.) CGMCC NO:3268 fermentation again for adopting Pasteur's bacillus aceticus conversion of resting cells in the solution that contains the 3%D-melampyrum after 48 hours;

Fig. 9 is the HPLC collection of illustrative plates of crystalline L-tagatose.

Embodiment

Below embodiments of the invention are elaborated: present embodiment provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition.

Embodiment 1

Adopt weak oxidized acetic acid bacteria in the fermention medium that contains the 1%D-melampyrum, to cultivate

Weak oxidized acetic acid bacteria (Acetobacter suboxydans) cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.Every liter of seed culture medium contains: D-melampyrum (also claiming galactitol or sweet and pure) 2 grams; Sorbyl alcohol 5 grams; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 5 grams, water is added 1 liter, pH6.0.This seed liquor 10ml is inoculated in the fermention medium of 90ml, at 32 ℃, 300rpm cultivated 24 hours again.The composition of every liter of fermention medium contains: D-melampyrum 10 grams; Sorbyl alcohol 1 gram; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 5 grams, water is added 1 liter, pH6.0.Cultivate and finish the content that back HPLC analyzes transformation efficiency and tagatose; Result such as Fig. 1; Through integral and calculating, the content of D-tagatose and L-tagatose is respectively 1.16g/L and 3.73g/L, and residual D-galactitol is 5.22g/L; The total conversion rate that is converted into L-tagatose and D-tagatose by the D-galactitol is 48.36%, and productive rate is 0.204g/Lh.

Embodiment 2

Adopt weak oxidized acetic acid bacteria in the fermention medium that contains the 4%D-melampyrum, to cultivate

Weak oxidized acetic acid bacteria cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.Every liter of seed culture medium contains: D-melampyrum 4 grams; Glycerine 2 grams; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 10 grams, water is added 1 liter, pH6.0.This seed liquor 10ml is inoculated in the fermention medium of 90ml, at 32 ℃, 300rpm cultivated 24 hours again.The composition of every liter of fermention medium contains: D-melampyrum 40 grams; Sorbyl alcohol 2 grams; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 5 grams, water is added 1 liter, pH6.0.Cultivate and finish the content that back HPLC analyzes transformation efficiency and tagatose; Through integral and calculating; The content of L-tagatose and D-tagatose is respectively 4.46g/L and 2.73g/L; Residual D-galactitol is 30.34g/L, and the transformation efficiency that is converted into the L-tagatose by the D-galactitol is 11.15%, and productive rate is 0.093g/Lh.

Embodiment 3

Adopt weak oxidized acetic acid bacteria in the fermention medium that contains the 10%D-melampyrum, to cultivate

Weak oxidized acetic acid bacteria cultivated in 25 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.Every liter of seed culture medium contains: D-melampyrum 4 grams; Xylitol 2 grams; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 10 grams, water is added 1 liter, pH6.0.This seed liquor 10ml is inoculated in the fermention medium of 90ml, at 32 ℃, 300rpm cultivated 48 hours again.The composition of every liter of fermention medium contains: D-melampyrum 100 grams; Xylitol 5 grams; Glucose 10 grams; Yeast powder 10 grams; Potassium hydrogenphosphate 2 grams; Lime carbonate 5 grams, water is added 1 liter, pH6.0.Cultivate and finish the content that back HPLC analyzes transformation efficiency and tagatose; Through integral and calculating; The content of L-tagatose and D-tagatose is respectively 26.46g/L and 6.73g/L; Residual D-galactitol is 60.51g/L, and the transformation efficiency that is converted into the L-tagatose by the D-galactitol is 26.46%, and productive rate is 0.551g/Lh.

Embodiment 4

Adopt weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 4%D-melampyrum

Weak oxidized acetic acid bacteria (Acetobacter suboxydans) cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours, the seed culture based component is with embodiment 1.Cultivate the centrifugal supernatant of abandoning in end back and obtain thalline, add the 20ml 4%D-galactitol aqueous solution, cell density OD600 is 10.30 ℃, 300rpm are cultivated 48h, detect the transformation efficiency and the content of tagatose.Result such as Fig. 2, through integral and calculating, the content of L-tagatose and D-tagatose is respectively 7.68g/L, 5.68g/L, and residual D-galactitol is 23.58g/L, and the total conversion rate that is converted into the L-tagatose by the D-galactitol is 37.8%, and productive rate is 0.30g/Lh.

Embodiment 5

Adopt weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 8%D-melampyrum

Weak oxidized acetic acid bacteria (Acetobacter suboxydans) cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.The seed culture based component is with embodiment 1.Cultivate the centrifugal supernatant of abandoning in end back and obtain thalline, add the 20ml 8%D-galactitol aqueous solution, cell density OD600 is 10.Cultivate 48h at 30 ℃, 300rpm, detect the transformation efficiency and the content of tagatose.Result such as Fig. 3, through integral and calculating, the content of L-tagatose and D-tagatose is respectively 23.58g/L and 14.36g/L, and residual D-galactitol is 46.14g/L, and the total conversion rate that is converted into tagatose by the D-galactitol is 36.7%, and productive rate is 0.56g/Lh.

Embodiment 6

Adopt gluconobacter oxydans conversion of resting cells in the solution that contains the 4%D-melampyrum

Gluconobacter oxydans (Gluconobacter oxydans) cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.The seed culture based component is with embodiment 1.Cultivate the centrifugal supernatant of abandoning in end back and obtain thalline, add the 20ml4%D-galactitol aqueous solution, cell density OD600 is 10.Cultivate 48h at 30 ℃, 300rpm, detect the transformation efficiency and the content of tagatose.Result such as Fig. 4, through integral and calculating, the content of L-tagatose and D-tagatose is respectively 5.72g/L and 1.85g/L, and residual D-galactitol is 33.94g/L, and the total conversion rate that is converted into tagatose by the D-galactitol is 18.9%, and productive rate is 0.16g/Lh.

Embodiment 7

Adopt Pasteur bacillus aceticus bacillus conversion of resting cells in the solution that contains the 4%D-melampyrum

Pasteur's bacillus aceticus (Acetobacter pasteurianus) cultivated in 30 ℃, 250rpm in seed culture medium obtained kind of a daughter bacteria liquid in 48 hours.The seed culture based component is with embodiment 1.Cultivate the centrifugal supernatant of abandoning in end back and obtain thalline, add the 20ml 3%D-galactitol aqueous solution, cell density OD600 is 10.Cultivate 48h at 30 ℃, 300rpm, detect the transformation efficiency and the content of tagatose.Result such as Fig. 5, through integral and calculating, the content of D-tagatose and L-tagatose is respectively 1.41g/L and 3.18g/L, and residual D-galactitol is 25.5g/L, and the total conversion rate that is converted into tagatose by the D-galactitol is 15.3%, and productive rate is 0.094g/Lh.

Embodiment 8

Adopt weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 4%D-melampyrum to adopt false silk candiyeast (Candida ssp.) CGMCC NO:3268 fermentation after 48 hours again

The enforcement of weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 4%D-melampyrum is with embodiment 4.In conversion fluid, be that 10% to insert candiyeast (Candidassp.) CGMCC NO:3268 and add mass percent be 0.5% yeast powder (degerming) then, continue to cultivate 10 hours at 32 ℃ according to volume ratio.The spinning thalline obtains the supernatant of the clarifying L-of containing tagatose.HPLC analyzes and should clarify supernatant, and result such as Fig. 6 are visible by Fig. 6, and candiyeast (Candida ssp.) CGMCC NO:3268 is complete with whole D-tagatoses and most residual D-melampyrum consumption, and the purity of L-tagatose reaches more than 90%.

Embodiment 9

Adopt weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 10%D-melampyrum to adopt false silk candiyeast (Candida ssp.) CGMCC NO:3268 fermentation after 48 hours again

The enforcement of weak oxidized acetic acid bacteria conversion of resting cells in the solution that contains the 10%D-melampyrum is with embodiment 4.In conversion fluid, be that 20% to insert candiyeast (Candidassp.) CGMCC NO:3268 and add mass percent be 0.5% yeast powder (degerming) then, continue to cultivate 20 hours at 35 ℃ according to volume ratio.The spinning thalline obtains the supernatant of the clarifying L-of containing tagatose.HPLC analyzes and should clarify supernatant, and candiyeast (Candida ssp.) CGMCC NO:3268 is complete with whole D-tagatoses and most residual D-melampyrum consumption, and the purity of L-tagatose reaches more than 90%.

Embodiment 10

Adopt gluconobacter oxydans conversion of resting cells in the solution that contains the 10%D-melampyrum to adopt false silk candiyeast (Candida ssp.) CGMCC NO:3268 fermentation after 60 hours again

The enforcement of gluconobacter oxydans conversion of resting cells in the solution that contains the 10%D-melampyrum is with embodiment 4.In conversion fluid, be that 10% to insert candiyeast (Candida ssp.) CGMCC NO:3268 and add mass percent be 0.5% yeast powder (degerming) then, continue to cultivate 24 hours at 32 ℃ according to volume ratio.The spinning thalline obtains the supernatant of the clarifying L-of containing tagatose.HPLC analyzes and should clarify supernatant, and candiyeast (Candida ssp.) CGMCC NO:3268 is complete with whole D-tagatoses and most residual D-melampyrum consumption, and the purity of L-tagatose reaches more than 90%.

Embodiment 11

Adopt gluconobacter oxydans conversion of resting cells in the solution that contains the 4%D-melampyrum to adopt candiyeast (Candida ssp.) CGMCC NO:3268 fermentation after 48 hours again

The enforcement of gluconobacter oxydans conversion of resting cells in the solution that contains the 4%D-melampyrum is with embodiment 6.In this conversion fluid, be that 10% to insert candiyeast (Candidassp.) CGMCC NO:3268 and add mass percent be 0.5% yeast powder (after the degerming) then, continue to cultivate 10 hours at 32 ℃ according to volume ratio.The spinning thalline obtains the supernatant of the clarifying L-of containing tagatose.HPLC analyzes and should clarify supernatant, and result such as Fig. 7 are visible by Fig. 7, and candiyeast (Candida ssp.) CGMCC NO:3268 is complete with whole D-tagatoses and most residual D-melampyrum consumption, and the purity of L-tagatose reaches more than 95%.

Embodiment 12

Adopt Pasteur's bacillus aceticus conversion of resting cells in the solution that contains the 3%D-melampyrum to adopt candiyeast (Candida ssp.) CGMCC NO:3268 fermentation after 48 hours again

The enforcement of Pasteur's bacillus aceticus conversion of resting cells in the solution that contains the 3%D-melampyrum is with embodiment 7.In this conversion fluid, be that 10% to insert candiyeast (Candidassp.) CGMCC NO:3268 and add mass percent be 0.5% yeast powder (after the degerming) then, continue to cultivate 10 hours at 32 ℃ according to volume ratio.The spinning thalline obtains the supernatant of the clarifying L-of containing tagatose.HPLC analyzes and should clarify supernatant, and result such as Fig. 8 are visible by Fig. 8, and candiyeast (Candidassp.) CGMCC NO:3268 is complete with whole D-tagatoses and most residual D-melampyrum consumption, and the purity of L-tagatose reaches more than 95%.

Embodiment 13

Purification separation L-tagatose from weak oxidized acetic acid bacteria conversion of resting cells liquid and yeast fermentation broth

Weak oxidized acetic acid bacteria conversion of resting cells liquid and yeast fermentation are removed the enforcement of D-tagatose and residual D-melampyrum with embodiment 4 and embodiment 8.In the 250ml clarified broth, add Powdered Activated Carbon 5 grams, 80 ℃ decoloured 60 minutes, and rotating speed is 150 rev/mins, and 0.45 micron filtering with microporous membrane is removed gac and obtained colourless fermented liquid.Colourless fermented liquid carries out ion exchange treatment again: successively through 001*7 type cationic exchange coloum, and 201*7 type anion-exchange column and 001*7 type cationic exchange coloum (Shanghai Resin Factory Co., Ltd.'s production).Detect the specific conductivity of effluent, when the specific conductivity of effluent is lower than 20 μ s/cm, stop IX.Fermented liquid after the desalination is concentrated up to refractive index greater than 60% through rotary evaporation; The absolute ethyl alcohol mixing that adds 4 times of volumes stirs gently, places 24 hours sufficient crystallisings for 4 ℃; The centrifugal white crystal that obtains; Use 95% washing with alcohol 2 times again, dry then, obtain 5.5 gram exsiccant L-tagatose white powder crystal.Analyze through HPLC, in this crystal the purity of L-tagatose more than 99%, as shown in Figure 9.

Claims (5)

1. a method for preparing the L-tagatose is characterized in that, comprises the steps:

Step 1, the substratum of acid formers is cultivated in preparation, and the composition of substratum is: the 1L substratum is composed of the following components: D-galactitol 2～20 grams; Polyvalent alcohol 1～10 gram, glucose 5～50 grams, yeast powder 10 grams; Anhydrous magnesium sulfate 0.5 gram, lime carbonate 10 grams, surplus is a water; Said acid formers is the arbitrary bacterium during acetobacter, bacillus of oxidizing glucose belong to;

Step 2 is seeded in acid formers on the step 1 gained substratum, and 25～35 ℃ of shaking culture get primary seed solution;

Step 3 is inoculated into primary seed solution in the step 1 gained substratum, and 25～35 ℃ of shaking culture get secondary seed solution;

Step 4 is that 5～20% inoculum size is inoculated in the fermention medium with secondary seed solution according to volume ratio, cultivates, and is centrifugal, obtains fermented liquid;

The composition of said fermention medium is that the 1L fermention medium is composed of the following components: D-galactitol 10～100 grams, and polyvalent alcohol 1～10 gram, glucose 5～50 grams, yeast powder 5～50 grams, sal epsom 0.5 gram, potassium hydrogenphosphate 2 grams, surplus is a water;

Step 5, preparation are used for the substratum of culturing yeast, and medium component is: the 1L substratum is composed of the following components: glucose 15 grams, and yeast powder 10 grams, anhydrous magnesium sulfate 0.5 gram, surplus is a water;

Step 6, in step 5 gained substratum, 25～35 ℃ of shaking culture obtain the yeast primary seed solution with yeast-inoculated; Said yeast is candiyeast CGMCC NO:3268;

Step 7 is inoculated into the yeast primary seed solution in the step 5 gained substratum, and 25～35 ℃ of shaking culture obtain secondary yeast starter liquid, and are centrifugal, gets yeast cell;

Step 8 is inoculated into yeast cell in the step 4 gained fermented liquid, and 25～35 ℃ of shaking culture 5～40 hours are centrifugal, clarifying fermented liquid;

Step 9 is concentrated into 1/10 of original volume with step 8 gained fermented liquid, adds mass percent and be 2% powdery gac, and under 80 ℃, decolouring is 120 minutes under the 100rpm condition, crosses diameter and be 0.22～0.45 micron millipore filtration, solution;

Step 10 is passed through D001 Zeo-karb and D201 anionite-exchange resin successively with step 9 gained solution, until specific conductivity≤20 μ s/cm; Reconcentration is＞60% to refractive index, adds the absolute ethyl alcohol of 2～8 times of volumes, mixing; 4～30 ℃ of following crystallizations 20～30 hours, centrifugal, crystal; The volume percent that adds 2 times of quality is 95% washing with alcohol 2～3 times, and drying promptly gets the L-tagatose;

Wherein, in step 1 and the step 4, said polyvalent alcohol is one or more the mixing in glycerine, erythritol, Xylitol, ribitol, arabitol, sorbyl alcohol and the N.F,USP MANNITOL.

2. the method for preparing the L-tagatose according to claim 1 is characterized in that, in the step 2, said acid formers is weak oxidized acetic acid bacteria or Pasteur's bacillus aceticus.

3. the method for preparing the L-tagatose according to claim 1 is characterized in that, in the step 2, said acid formers is a weak oxidized acetic acid bacteria.

4. the method for preparing the L-tagatose according to claim 1 is characterized in that, in the step 2, the bacterium that said bacillus of oxidizing glucose belongs to is gluconobacter oxydans or weak glucose oxidation bacteria.

5. a method for preparing the L-tagatose is characterized in that, comprises the steps:

Step 1, the preparation substratum, the composition of substratum is: the 1L substratum is composed of the following components, D-galactitol 5 grams, sorbyl alcohol 5 grams, glucose 20 grams, yeast powder 15 grams, sal epsom 0.5 gram, potassium hydrogenphosphate 2 grams, lime carbonate 5 grams, surplus is a water; Arbitrary microbionation in acetobacter, the bacillus of oxidizing glucose genus in substratum, is cultivated;

Step 2, centrifugal after cultivate finishing, somatic cells, suspended bacteria somatocyte, 25～35 ℃, 100～300rpm transform 24～96 hours down, solution;

The solution that said suspension is used is that the component of solution is: 1%～10% D-galactitol, and 0.1～0.5% yeast powder, surplus is a water, said percentage ratio is massfraction;

Step 3 inserts candiyeast CGMCC NO:3268 in the step 2 gained solution, cultivates 10～48 hours for 25～35 ℃, gets fermented liquid;

Step 4 successively through decolouring and desalting treatment, obtains clear aqueous solution with step 3 gained fermented liquid; The aqueous solution is concentrated to refractive index＞60%, adds the absolute ethyl alcohol of 2～8 times of volumes, mixing; 4～30 ℃ of following crystallizations 20～30 hours, centrifugal, crystal; The volume percent that adds 2 times of quality is 95% washing with alcohol 2～3 times, and dried crystals promptly gets the L-tagatose.

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