Strain for producing L-arabinose isomerase, method for producing tagatose and application of strain

Abstract

The invention provides a strain for producing L-arabinose isomerase, a method for producing tagatose and application of the strain, and belongs to the technical field of biological fermentation. The bacterial strain disclosed by the invention is bacillus subtilis BLCY-015, and the preservation number of the bacterial strain is CCTCC (China Center For Type Culture Collection) NO: M 20242748. The bacillus subtilis BLCY-015 is fermented to obtain L-arabinose isomerase, and galactose liquid is converted by the L-arabinose isomerase to obtain tagatose. The enzyme obtained by the method does not depend on metal ions, and has high conversion rate on galactose. Meanwhile, glucose and galactose generated in the tagatose production process can be recycled, the utilization rate of the raw materials is increased, no organic solvent or ion is added in the process, and no pollution is caused to the environment.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and specifically relates to a strain producing L-arabinose isomerase and a method for producing tagatose and applications thereof. Background Art

[0002] D-Tagose is an isomer of D-galactose and a diastereomer of D-fructose. It is a rare six-carbon ketose in nature. The physical properties of D-Tagose are similar to those of sucrose, and its sweetness is 92% of sucrose, but its calorie content is 1.5Kcal / g, which is only one-third of sucrose. D-Tagose does not cause an increase in blood sugar and insulin levels in the human body, and has an auxiliary therapeutic effect on patients with type 2 diabetes. It can be selectively fermented by beneficial microorganisms in the colon to produce short-chain fatty acids, maintain intestinal health, and effectively prevent tooth decay and tooth enamel corrosion.

[0003] D-tagatose is rare in nature and cannot be directly obtained. The chemical synthesis of tagatose causes pollution and produces impurities such as fructose and mannose, which reduces the yield of D-tagatose. Therefore, biotransformation is a better way to industrially produce D-tagatose. However, the current biotransformation method uses galactose as a raw material to produce tagatose, which has problems such as low raw material utilization, imperfect industrial production methods, high dependence on metal ions in the conversion process, and environmental unfriendliness. Therefore, studying a complete industrial production method for preparing D-tagatose with high raw material utilization and high conversion rate is a technical problem that needs to be solved at this stage. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a strain producing L-arabinose isomerase and a method for producing tagatose and its application. The L-arabinose isomerase obtained by fermentation of the strain has a high conversion rate for galactose, and the glucose and galactose produced in the process of producing tagatose can be recycled, thereby improving the utilization rate of raw materials.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a strain producing L-arabinose isomerase. The strain is Bacillus subtilis BLCY-015, and the deposit number is CCTCCNO: M 20242748.

[0007] The invention provides application of the strain in producing L-arabinose isomerase.

[0008] The invention provides a bacterial liquid for producing L-arabinose isomerase. The Bacillus subtilis BLCY-015 is inoculated into a secondary seed culture medium for culture to obtain a bacterial liquid with an OD value of 7-9. The formula of the secondary seed culture medium comprises: 9-11 g / L peptone, 1.8-2.4 g / L yeast extract powder, 9-11 g / L sodium chloride, 9-11 g / L glucose, and the rest water, with a pH value of 7.0-7.2.

[0009] The present invention provides application of the strain or the bacterial liquid in producing D-tagatose.

[0010] The present invention provides a method for producing D-tagatose, comprising the following steps: inoculating the bacterial liquid into a fermentation medium for fermentation, and starting to feed after dissolved oxygen rebounds; fermenting until OD 600 =38-42, start continuous flow addition of lactose induction; when the enzyme activity reaches the highest, stop feeding; raise the temperature to exhaust the nutrients in the fermentation system, and release the tank when the dissolved oxygen and pH rebound to obtain a fermentation liquid containing L-arabinose isomerase; mix galactose with the fermentation liquid containing L-arabinose isomerase to react to obtain D-tagatose; the fermentation medium formula includes: corn steep liquor powder 48-52g / L, glucose 18-22g / L, potassium dihydrogen phosphate 11.5-15.5g / L, magnesium sulfate heptahydrate 0.8-1.2g / L, diammonium hydrogen phosphate 2-4g / L, citric acid monohydrate 1-2g / L, balance water, pH 6.8-7.2; the feed medium formula includes: glucose 450-550g / L, diammonium hydrogen phosphate 50-70g / L, corn steep liquor powder 6-10g / L, magnesium sulfate heptahydrate 15-19g / L, balance water.

[0011] Preferably, the inoculation amount of the bacterial liquid is 8-12 v / v%; the fermentation temperature when the bacterial liquid is inoculated into the fermentation medium is 34-37° C. and the pH is 6.6-7.0.

[0012] Preferably, the dissolved oxygen is maintained at 25-35% during the feeding process.

[0013] Preferably, the temperature for inducing the lactose addition is 22-27° C.; and the final concentration of lactose in the fermentation system is 0.3-0.7 g / L.

[0014] Preferably, in the mixed reaction, the concentration of galactose is 360-450 g / L; the amount of L-arabinose isomerase fermentation broth added is 0.6-1.0 wt% of galactose; the temperature of the mixed reaction is 58-62° C., the pH is 7.3-7.7, and the time is 21-25 h.

[0015] The present invention provides application of D-tagatose obtained by the method in preparing food or medicine.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a Bacillus subtilis BLCY-015, which is fermented to obtain L-arabinose isomerase. The L-arabinose isomerase obtained by the present invention is mixed with galactose to improve the galactose conversion rate and obtain a higher yield of D-tagatose. The L-arabinose isomerase obtained by fermentation of the strain of the present invention is independent of metal ions, and the conversion rate can reach more than 55%.

[0018] Furthermore, the present invention provides a complete industrial production method for preparing D-tagatose, and the processes from strain expansion, enzyme induction and galactose conversion to D-tagatose can all be industrially realized; the utilization rate of raw materials in this method is high, the glucose produced in the production process can be used as a fermentation carbon source again, and the produced galactose is recycled as a substrate for L-arabinose isomerase conversion, thereby improving the utilization rate of raw materials. After one month of continuous operation, one ton of lactose raw material can obtain 0.35 tons of D-tagatose finished product. No organic solvent or ion is added in the production process, and there is no pollution to the environment.

[0019] Biological deposit information

[0020] Bacillus subtilis BLCY-015, classified as Bacillus subtilis, with a Latin name of Bacillus subtilis, was deposited in the China Center for Type Culture Collection on December 6, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M20242748. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the production flow chart of D-tagatose. DETAILED DESCRIPTION

[0022] The invention provides a strain producing L-arabinose isomerase. The strain is Bacillus subtilis BLCY-015, and the deposit number is CCTCCNO: M 20242748.

[0023] The present invention also provides the use of the Bacillus subtilis BLCY-015 in producing L-arabinose isomerase. The L-arabinose isomerase obtained by fermenting the Bacillus subtilis BLCY-015 of the present invention can convert galactose into D-tagatose.

[0024] The present invention also provides a bacterial solution for producing L-arabinose isomerase, wherein the Bacillus subtilis BLCY-015 is inoculated into a secondary seed culture medium for culture, and a bacterial solution with an OD value of 7-9 is obtained. The formula of the secondary seed culture medium of the present invention includes: peptone 9-11 g / L, yeast extract powder 1.8-2.4 g / L, sodium chloride 9-11 g / L, glucose 9-11 g / L, and the rest is water, pH 7.0-7.2; preferably, the formula of the secondary seed culture medium includes: peptone 10 g / L, yeast extract powder 2.1 g / L, sodium chloride 10 g / L, glucose 10 g / L, and the rest is water, pH 7.0-7.2. The OD value of the bacterial solution of the present invention is preferably 8.

[0025] In the present invention, the inoculation amount of the Bacillus subtilis BLCY-015 inoculated into the secondary seed culture medium is 0.3-0.7 v / v%, preferably 0.5 v / v%. The temperature of the Bacillus subtilis BLCY-015 inoculated into the secondary seed culture medium is 30-37°C and the rotation speed is 200-240 rpm, preferably the temperature is 33°C and the rotation speed is 220 rpm.

[0026] In the present invention, before inoculating the Bacillus subtilis BLCY-015 into the secondary seed culture medium, the Bacillus subtilis BLCY-015 needs to be streaked on the LB culture medium, and then the cultured single colony is inoculated into the primary seed culture medium (LB liquid culture medium) for shaking flask culture, and then inoculated into the secondary seed culture medium for culture to obtain a bacterial liquid. The temperature of the streaking culture in the LB culture medium of the present invention is 31-35°C, preferably 33°C, and the time is 9-12h, preferably 10h. The method of culturing in the primary seed culture medium of the present invention includes: shaking culture at 200-240rpm at 31-35°C for 7-10h; preferably shaking culture at 220rpm at 33°C for 8h. The LB medium formula of the present invention includes: 8-12g / L of tryptone, 3-7g / L of yeast extract, 8-12g / L of sodium chloride, 13-17g / L of agar powder, and the rest is water; preferably, the LB medium formula includes: 10g / L of tryptone, 5g / L of yeast extract, 10g / L of sodium chloride, 15g / L of agar powder, and the rest is water. The primary seed culture medium (LB liquid culture medium) of the present invention is to remove agar powder on the basis of LB culture medium.

[0027] The present invention also provides the use of the Bacillus subtilis BLCY-015 or the bacterial liquid in producing D-tagatose.

[0028] The present invention also provides a method for producing D-tagatose, comprising the following steps: inoculating the bacterial liquid into a fermentation medium for fermentation, and starting to feed after dissolved oxygen rebounds; fermenting until OD 600=38-42, start continuous flow addition of lactose for induction; when the enzyme activity reaches the highest, stop feeding; raise the temperature to exhaust the nutrients in the fermentation system, and when the dissolved oxygen and pH rebound, release the tank to obtain a fermentation broth containing L-arabinose isomerase; mix galactose with the fermentation broth containing L-arabinose isomerase to react, and obtain a fermentation broth containing D-tagatose. The fermentation medium formula of the present invention comprises: 48-52 g / L corn steep liquor powder, 18-22 g / L glucose, 11.5-15.5 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L magnesium sulfate heptahydrate, 2-4 g / L diammonium hydrogen phosphate, 1-2 g / L citric acid monohydrate, and the balance water, pH 6.8-7.2; preferably, the fermentation medium formula comprises: 50 g / L corn steep liquor powder, 20 g / L glucose, 13.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 3.0 g / L diammonium hydrogen phosphate, 1.5 g / L citric acid monohydrate, and the balance water, pH 6.8-7.2. The feed medium formula of the present invention comprises: 450-550 g / L of glucose, 50-70 g / L of diammonium phosphate, 6-10 g / L of corn steep liquor powder, 15-19 g / L of magnesium sulfate heptahydrate, and the balance is water; preferably, the feed medium formula comprises: 500 g / L of glucose, 60 g / L of diammonium phosphate, 8 g / L of corn steep liquor powder, 17 g / L of magnesium sulfate heptahydrate, and the balance is water.

[0029] In the present invention, the inoculation amount of the bacterial liquid is 8-12 v / v%, preferably 10 v / v%; the fermentation temperature of the bacterial liquid inoculated into the fermentation medium is 34-37°C and the pH is 6.6-7.0, preferably the temperature is 35°C and the pH is 6.8.

[0030] In the present invention, the dissolved oxygen rebound refers to the situation where the oxygen content in the fermentation system decreases after a period of time and then rises rapidly; the dissolved oxygen content is displayed in real time on the display screen of the fermentation tank of the present invention, and when the actual dissolved oxygen value begins to be greater than or equal to 5% of the target dissolved oxygen value (about 30%), it is considered as a dissolved oxygen rebound.

[0031] In the present invention, the dissolved oxygen is maintained at 25-35%, preferably 30% during the feeding process. The addition of the feeding in the present invention provides glucose as the main carbon source for the fermentation system.

[0032] In the present invention, the temperature of lactose induction is 22-27°C, preferably 25°C; the final concentration of lactose in the fermentation system is 0.3-0.7 g / L, preferably 0.5 g / L. The original concentration of lactose in the present invention is 3-7 g / L, preferably 5 g / L.

[0033] In the present invention, the heating temperature is 31-35°C, preferably 33°C.

[0034] In the present invention, in the mixed reaction of galactose and L-arabinose isomerase-containing fermentation broth, the galactose concentration is 360-450 g / L, preferably 400 g / L; the amount of L-arabinose isomerase fermentation broth added is 0.6-1.0 wt% of the mass of galactose, preferably 0.8 wt%; the temperature of the mixed reaction is 58-62°C, the pH is 7.3-7.7 and the time is 21-25h, preferably the temperature is 60°C, the pH is 7.5 and the time is 23h.

[0035] In the present invention, the galactose is obtained by lactase catalyzing lactose. The steps of preparing galactose in the present invention include: adding lactase according to 0.5-1.5wt% of the dry matter weight of lactose to a 30-50wt% lactose solution, reacting at 38-42°C and pH 6-7 for 8-12h, killing the enzyme to obtain a galactose crude liquid; the galactose crude liquid is subjected to decolorization, ion exchange, and concentration to obtain a first refined concentrated liquid; the first refined concentrated liquid is subjected to continuous simulated mobile chromatography to obtain a galactose liquid and a glucose liquid, and the glucose liquid can be used in the process of preparing D-tagatose by fermenting a bacterial liquid producing L-arabinose isomerase.

[0036] In the present invention, the fermentation broth containing D-tagatose is subjected to decolorization, ion exchange, vacuum concentration, continuous simulated moving bed chromatography separation, and secondary vacuum concentration treatment to obtain a tagatose concentrate; the tagatose concentrate is crystallized, centrifuged, and dried to obtain high-purity crystalline D-tagatose. The unconverted galactose solution separated by chromatography in the present invention can be recycled and continued to be used as a substrate for L-arabinose isomerase conversion.

[0037] The present invention also provides application of the D-tagatose obtained by the method in preparing food or medicine.

[0038] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0039] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The culture medium and feed formula used in the following examples are as follows:

[0041] The LB medium formula is: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L.

[0042] The formula of the primary seed culture medium (LB liquid culture medium) is: trypsin peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0043] The formula of secondary seed culture medium is: peptone 10g / L, yeast extract powder 2.1g / L, sodium chloride 10g / L, glucose 10g / L, the rest is water, pH 7.0-7.2

[0044] The fermentation medium formula is: corn steep liquor powder 50g / L, glucose 20g / L, potassium dihydrogen phosphate 13.5g / L, magnesium sulfate heptahydrate 1.0g / L, diammonium hydrogen phosphate 3.0g / L, citric acid monohydrate 1.5g / L, balance water, pH 6.8-7.2.

[0045] The feed formula is: glucose 500g / L, diammonium phosphate 60g / L, corn steep liquor powder 8g / L, magnesium sulfate heptahydrate 17g / L, and the balance water.

[0046] The HPLC detection conditions involved in the following examples are: Waters ion column (WAT085188), column temperature of 85° C., detector temperature of 40° C., mobile phase of ultrapure water, and flow rate of 0.5 mL / min.

[0047] Example 1

[0048] 1. Obtaining Bacillus subtilis BLCY-015 bacterial liquid

[0049] (1) Streak Bacillus subtilis BLCY-015 (CCTCC NO: M 20242748) on an LB medium plate and invert and culture at 33°C for 10 h;

[0050] (2) picking a single colony cultured in step (1) and inoculating it into a shake flask containing a primary seed culture medium, shaking and culturing at 220 rpm for 8 h at a culture temperature of 33° C. to obtain a primary seed culture solution;

[0051] (3) The first-level seed culture solution of step (2) was inoculated into the seed tank containing the second-level seed culture medium at an inoculation rate of 0.5 v / v% (first-level seed culture solution / volume of the seed tank containing the second-level seed culture medium) at a temperature of 33° C. and a rotation speed of 220 rpm. When the OD of the second-level seed culture solution reached 8, the culture was stopped to obtain a Bacillus subtilis BLCY-015 bacterial solution.

[0052] 2. Obtaining fermentation broth containing L-arabinose isomerase

[0053] (1) Inoculating the Bacillus subtilis BLCY-015 bacterial solution obtained in step 1 into a fermentation tank containing a fermentation medium at an inoculation rate of 10 v / v%, culturing and fermenting at pH 6.8 and 35° C., and starting feeding when dissolved oxygen rebounds during the fermentation process (the fermentation tank display screen displays the dissolved oxygen content in real time, and when the actual dissolved oxygen value begins to be greater than or equal to 5% of the target dissolved oxygen value (about 30%)), the dissolved oxygen rebound is considered. The amount of feed added is controlled in real time by controlling the glucose content in the fermentation tank to 10 g / L, and the dissolved oxygen is maintained at 30% during the feeding process (the dissolved oxygen content is controlled by controlling the pure oxygen ventilation rate); OD 600 The value rises to 10 and starts to cool down gradually. 600 When the temperature is 40°C, 5 g / L lactose is added for induction, and then the temperature is lowered to 25°C as the induction temperature to continue fermentation, and the final concentration of lactose in the fermentation liquid is 0.5 g / L; when the enzyme activity reaches the highest during the fermentation process, the feeding and lactose are stopped to obtain the fermentation liquid;

[0054] The enzyme activity in the fermentation broth was detected by setting the galactose substrate concentration to 100 g / L, adding the fermentation broth at an addition amount of 10% (V / V) of the system, reacting for 3 hours at 60°C and pH 8.0, and detecting the content of D-tagatose and galactose in the fermentation broth by high performance liquid chromatography after inactivating the enzyme. When the enzyme activity was the highest, the conversion rate of galactose was 56.37%;

[0055] (2) After stopping the feeding, the temperature is raised to 33° C. to exhaust the nutrients in the fermentation system. When the dissolved oxygen and / or pH rebound (a rebound is considered when the dissolved oxygen monitored on the fermentation tank display is above 60% or the pH is above 7.3), the tank is released to obtain a fermentation broth containing L-arabinose isomerase.

[0056] 3. Galactose acquisition

[0057] (1) Preparation of galactose crude liquid: a lactose solution was prepared with a concentration of 41 wt %, and a fermentation liquid containing L-arabinose isomerase was added at 1 wt % of the dry matter weight of lactose, and the mixture was reacted at a temperature of 40° C. and a pH of 6.5 for 10 h. After the enzyme was inactivated, a galactose crude liquid was obtained; the galactose content in the galactose crude liquid was detected by high performance liquid chromatography, and the lactose decomposition rate was 92.35%;

[0058] (2) Decolorization and filtration: The galactose crude liquid described in step (1) is decolorized by a granular activated carbon column, and the feed temperature is 65°C; when the material temperature is 50°C, it is filtered through an Ama filter, and the working pressure of the Ama filter is 0.2 MPa; wherein the granular activated carbon column is prepared by: washing and activating the granular activated carbon with deionized water, filling the activated activated carbon particles into the column, and the feed temperature is 65°C;

[0059] (3) Ion exchange: the decolorized and filtered material is sequentially passed through a cation exchange resin-anion exchange column-cation exchange resin-anion exchange resin-cation exchange resin, wherein the cation resin is a strong acid cation resin and the anion resin is a weak base anion resin. The material temperature of the ion exchange is 35° C. and the material flow rate of the ion exchange is 2 BV / h.

[0060] (4) Vacuum concentration: Use a six-effect evaporator to concentrate the ion exchanged material at a temperature of 65° C. and a pressure of -0.08 MPa until the solid content of the material increases to 60%, thereby obtaining a concentrated refined liquid;

[0061] (5) The concentrated refined liquid is separated by continuous simulated mobile chromatography to obtain galactose solution and glucose solution; the glucose solution is recovered and used in the fermentation medium of step (1) of step 2 of this embodiment for recycling; wherein the continuous simulated mobile chromatography separation material-water ratio is 1:3 (V / V), and the processing capacity is 0.020 TDS / h / m 3 Resin. This step obtains a high-purity galactose solution with a purity of 98.51%.

[0062] 4. Preparation of D-tagatose

[0063] (1) Conversion of galactose to D-tagatose: The concentration of galactose prepared in step 3 was 400 g / L, and the fermentation broth containing L-arabinose isomerase obtained in step 2 was added in an amount of 0.8 wt% of the mass of galactose, and the reaction was carried out at a temperature of 60° C. and a pH of 7.5 for 23 h, and the enzyme was inactivated to obtain a tagatose conversion liquid; the tagatose content was detected by high performance liquid chromatography, and the tagatose conversion rate was calculated to be 56.74%;

[0064] (2) Decolorization: The obtained tagatose conversion liquid is decolorized by using a granular activated carbon column, and the feed temperature is 55°C; when the material temperature is 50°C, it is filtered through an Ama filter, and the working pressure of the Ama filter is 0.2Mpa; the preparation of the granular activated carbon column is as follows: the granular activated carbon is washed and activated with deionized water, and the activated activated carbon particles are filled into the column, and the feed temperature is 65°C. ；

[0065] (3) Ion exchange: the decolorized material is sequentially passed through a cation exchange resin-anion exchange resin-cation exchange resin-anion exchange resin-cation exchange resin, wherein the cation resin is a strong acid cation resin and the anion resin is a weak base anion resin, the material temperature is 35° C., and the flow rate is 2 BV / h;

[0066] (4) First vacuum concentration: Use a six-effect evaporator to concentrate the material after ion exchange for the first time, at a temperature of 65°C and a pressure of -0.06 MPa, to increase the solid content of the material to 60%, thereby obtaining the first concentrated refined liquid;

[0067] (5) The first concentrated refined liquid is separated by continuous simulated mobile chromatography to obtain galactose liquid and tagatose liquid. The material-water ratio of the continuous simulated mobile chromatography separation is 1:3.5 (V / V) and the processing capacity is 0.025 T.DS / h / m 3 Resin; the galactose solution separated by chromatography is recycled and continues to be used as a substrate for L-arabinose isomerase conversion; preparation of continuous simulated moving chromatography column: after the inside and outside of the chromatography column and the column head are rinsed with pure water, the chromatography separation resin LX-1850Ca (Xi'an Lanxiao Technology New Materials Co., Ltd.) is filled into the chromatography separation system, first transported by water and then added with material.

[0068] (6) Second vacuum concentration: Use a six-effect evaporator to concentrate the chromatographically separated galactose solution for the second time at a temperature of 65° C. and a pressure of -0.06 MPa to increase the solid content of the material to 60%, thereby obtaining a tagatose concentrate;

[0069] (7) A method of combining boiling sugar crystallization with cooling crystallization: the solid content of tagatose concentrate is controlled to 65%, seed crystals are added in an amount equal to 0.5 wt% of the solid content of tagatose concentrate, and sugar is boiled at 41°C for 10 h; after boiling sugar crystallization, the sugar concentration of the material is increased to 75%, and the temperature is controlled at 55°C; then the temperature is cooled to 28°C at 0.5°C / h;

[0070] Centrifugation: Use a vertical scraper centrifuge, centrifuge at 500 rpm for 30 minutes to obtain the precipitate, during which the amount of water used for washing is 1% (V / V) of the volume of the feed liquid;

[0071] Drying: The precipitate was dried in a fluidized bed with the air inlet temperature set at 60°C, the cooling temperature set at 25°C, and the drying time set at 20 min to obtain crystalline D-tagatose with a purity of 99.66%.

[0072] Example 2

[0073] 1. Obtaining Bacillus subtilis BLCY-015 bacterial liquid

[0074] (1) Streak Bacillus subtilis BLCY-015 (CCTCC NO: M 20242748) on an LB medium plate and invert and culture at 33°C for 10 h;

[0075] (2) picking a single colony cultured in step (1) and inoculating it into a shake flask containing a primary seed culture medium, shaking and culturing at 220 rpm for 8 h at a culture temperature of 33° C. to obtain a primary seed culture solution;

[0076] (3) The first-level seed culture solution of step (2) was inoculated into the seed tank containing the second-level seed culture medium at an inoculation rate of 0.5 v / v% (first-level seed culture solution / volume of the seed tank containing the second-level seed culture medium) at a temperature of 33° C. and a rotation speed of 220 rpm. When the OD of the second-level seed culture solution reached 8, the culture was stopped to obtain a Bacillus subtilis BLCY-015 bacterial solution.

[0077] 2. Obtaining fermentation broth containing L-arabinose isomerase

[0078] (1) Inoculating the Bacillus subtilis BLCY-015 bacterial solution obtained in step 1 into a fermentation tank containing a fermentation medium at an inoculation rate of 8 v / v%, culturing and fermenting at pH 6.6 and 34° C., and starting feeding when dissolved oxygen rebounds during the fermentation process (the fermentation tank display screen displays the dissolved oxygen content in real time, and when the actual dissolved oxygen value begins to be greater than or equal to 5% of the target dissolved oxygen value (about 30%)), the feeding amount is controlled in real time by controlling the glucose content in the fermentation tank to 10 g / L, and the dissolved oxygen is maintained at 25% during the feeding process (the dissolved oxygen content is controlled by controlling the pure oxygen ventilation rate); OD during the fermentation process 600 The value rises to 10 and starts to cool down gradually. 600 When the temperature was 40°C, 5 g / L lactose was added for induction, and then the temperature was lowered to 25°C as the induction temperature to continue fermentation, and the final concentration of lactose in the fermentation liquid was 0.3 g / L; when the enzyme activity reached the highest point during the fermentation process, the feeding and lactose addition were stopped to obtain the fermentation liquid;

[0079] The enzyme activity in the fermentation broth was detected in the same manner as in Example 1. When the enzyme activity was the highest, the conversion rate of galactose was 56.88%;

[0080] (2) After stopping the feeding, the temperature is raised to 33°C to exhaust the nutrients in the fermentation system. When the dissolved oxygen and pH rebound (a rebound is considered when the dissolved oxygen monitored on the fermentation tank display is above 60% or the pH is higher than the pH), the tank is released to obtain a fermentation broth containing L-arabinose isomerase.

[0081] 3. The steps of obtaining galactose are the same as those in Example 1.

[0082] 4. Preparation of D-tagatose

[0083] (1) Conversion of galactose to D-tagatose: The concentration of galactose prepared in step 3 was 360 g / L, and the fermentation broth containing L-arabinose isomerase obtained in step 2 was added in an amount of 0.6 wt% of the mass of galactose, and the reaction was carried out at a temperature of 58° C. and a pH of 7.3 for 21 h, and the enzyme was inactivated to obtain a tagatose conversion liquid; the tagatose content was detected by high performance liquid chromatography, and the tagatose conversion rate was calculated to be 56.81%;

[0084] (2) Decolorization, ion exchange, vacuum concentration, sugar boiling crystallization combined with cooling crystallization, centrifugation, and drying: The steps are the same as those in Example 1, and crystalline D-tagatose with a purity of 99.68% is obtained.

[0085] Comparative Example 1

[0086] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that:

[0087] In step 2 (1), the Bacillus subtilis BLCY-015 bacterial solution obtained in step 1 is inoculated into a fermentation tank containing a fermentation medium, and the fermentation liquid is cultured at 40° C. The enzyme activity of the fermentation liquid is detected. When the enzyme activity is the highest, the galactose conversion rate is 21.34%.

[0088] The conversion rate of D-tagatose in step 4 was detected to be 22.58%, and 95.73% of crystalline D-tagatose was finally obtained.

[0089] Comparative Example 2

[0090] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that:

[0091] In step 2 (1), the Bacillus subtilis BLCY-015 bacterial solution obtained in step 1 is inoculated into a fermentation tank containing a fermentation medium, and the fermentation liquid is cultured at 30° C. The enzyme activity of the fermentation liquid is detected. When the enzyme activity is the highest, the galactose conversion rate is 23.62%.

[0092] The tagatose conversion rate in step 4 was detected to be 24.37%, and 95.86% of crystalline D-tagatose was finally obtained.

[0093] Comparative Example 3

[0094] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that:

[0095] In step 2 (1), when the fermentation process OD 600 When the temperature was 40°C, 5 g / L lactose was added for induction, and then the temperature was lowered to 20°C as the induction temperature to continue fermentation. The enzyme activity of the fermentation broth was detected. When the enzyme activity was the highest, the galactose conversion rate was 30.46%.

[0096] The conversion rate of D-tagatose in step 4 was detected to be 31.57%, and 96.35% of crystalline D-tagatose was finally obtained.

[0097] Comparative Example 4

[0098] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that:

[0099] In step 2 (1), when the fermentation process OD600 When the temperature was 40°C, 5 g / L lactose was added for induction, and then the temperature was lowered to 30°C as the induction temperature to continue fermentation. The enzyme activity of the fermentation broth was detected. When the enzyme activity was the highest, the galactose conversion rate was 30.15%.

[0100] The tagatose conversion rate in step 4 was detected to be 30.23%, and 96.57% of crystalline D-tagatose was finally obtained.

[0101] Comparative Example 5

[0102] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that in step 2 (1), the dissolved oxygen was maintained at 25% during the feeding process. The enzyme activity of the fermentation broth was detected, and when the enzyme activity was the highest, the galactose conversion rate was 31.28%.

[0103] The tagatose conversion rate in step 4 was detected to be 32.84%, and 97.32% of crystalline D-tagatose was finally obtained.

[0104] Comparative Example 6

[0105] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that in step 2 (1), the dissolved oxygen was maintained at 40% during the feeding process. The enzyme activity of the fermentation broth was detected, and when the enzyme activity was the highest, the galactose conversion rate was 31.34%.

[0106] The tagatose conversion rate in step 4 was detected to be 32.59%, and 96.18% of crystalline D-tagatose was finally obtained.

[0107] Comparative Example 7

[0108] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that the concentration of galactose prepared in step 3 was set to 350 g / L in step 4 (1).

[0109] The tagatose conversion rate in step 4 was detected to be 36.24%, and 97.48% of crystalline D-tagatose was finally obtained.

[0110] Comparative Example 8

[0111] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that the reaction was carried out at a temperature of 55° C. for 30 h in step 4 (1).

[0112] The tagatose conversion rate in step 4 was detected to be 36.47%, and 97.53% of crystalline D-tagatose was finally obtained.

[0113] Comparative Example 9

[0114] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that the reaction was carried out at a temperature of 75° C. for 20 h in step 4 (1).

[0115] The tagatose conversion rate in step 4 was detected to be 35.42%, and 97.41% of crystalline D-tagatose was finally obtained.

[0116] Comparative Example 10

[0117] In this comparative example, D-tagatose was prepared according to the preparation method of Example 1, except that the reaction was carried out at pH 7.0 in step 4 (1).

[0118] The tagatose conversion rate in step 4 was detected to be 35.24%, and 96.37% of crystalline D-tagatose was finally obtained.

[0119] Comparative Example 11

[0120] On the basis of Example 1, in step 4 (1), the conditions for converting galactose by L-arabinose isomerase were changed, and MnCl2 with a concentration of 5 mmol / L and an addition amount of 1.0 g / L was added. The remaining steps were the same as in Example 1.

[0121] The tagatose conversion rate in step 4 was detected to be 55.98%, and 99.26% of crystalline D-tagatose was finally obtained.

[0122] The data in the above Examples 1-2 and Comparative Examples 1-11 are statistically analyzed, see Table 1.

[0123] Table 1 Galactose conversion rate, D-tagatose conversion rate and purity of D-tagatose after drying in each experimental group

[0124]

[0125] As shown in Table 1, in Comparative Examples 1 and 2, the fermentation broth temperature in step 2 (1) was adjusted, and the other variables were the same as those in Example 1. The fermentation broth temperature affected the L-arabinose isomerase activity, galactose and D-tagatose conversion rates in the fermentation broth.

[0126] In Comparative Examples 3 and 4, the induction temperature in step (1) of step 2 was adjusted, and the other variables were the same as those in Example 1. The induction temperature affected the L-arabinose isomerase activity, galactose and D-tagatose conversion rates in the fermentation broth.

[0127] In Comparative Examples 5 and 6, the dissolved oxygen in the feeding process in step 2 (1) was adjusted, and the other variables were the same as those in Example 1. The dissolved oxygen factor affected the L-arabinose isomerase activity, galactose and D-tagatose conversion rates in the fermentation broth.

[0128] In Comparative Example 7, the substrate galactose concentration in step (1) of step 4 was adjusted, and the other variables were the same as those in Example 1. The galactose concentration factor affected the conversion rate of D-tagatose.

[0129] In Comparative Examples 8 and 9, the reaction temperature and time in step 4 (1) were adjusted, and the other variables were the same as those in Example 1. The temperature and time factors affected the conversion rate of D-tagatose.

[0130] Comparative Example 10: The reaction pH in step 4 (1) was adjusted, and the other variables were the same as those in Example 1. The pH factor affected the conversion rate of D-tagatose.

[0131] In Comparative Example 11, manganese ions were added during the conversion of galactose by L-arabinose isomerase in step 4 (1). Comparison with Example 1 proves that the enzyme obtained by the method of the present invention is independent of metal ions.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain producing L-arabinose isomerase, characterized in that: The strain is Bacillus subtilis BLCY-015, and its deposit number is CCTCC NO: M 20242748.

2. Use of the strain according to claim 1 in producing L-arabinose isomerase.

3. A bacterial solution for producing L-arabinose isomerase, characterized in that: Inoculate the strain described in claim 1 into a secondary seed culture medium to obtain a bacterial solution with an OD value of 7-9; The formula of the secondary seed culture medium includes: 9-11 g / L of peptone, 1.8-2.4 g / L of yeast extract powder, 9-11 g / L of sodium chloride, 9-11 g / L of glucose, and the rest is water, with a pH of 7.0-7.

2.

4. Use of the strain according to claim 1 or the bacterial solution according to claim 3 in producing D-tagatose.

5. A method for producing D-tagatose, characterized in that: The steps include: Inoculate the bacterial liquid of claim 3 into the fermentation medium for fermentation, and start feeding after the dissolved oxygen rebounds; ferment until OD 600 =38-42, start continuous flow addition of lactose for induction; when the enzyme activity reaches the highest, stop feeding; raise the temperature to exhaust the nutrients in the fermentation system, and when the dissolved oxygen and pH rebound, release the tank to obtain the fermentation liquid containing L-arabinose isomerase; The galactose is mixed with the fermentation liquid containing L-arabinose isomerase to react and obtain D-tagatose; The fermentation medium formula comprises: 48-52 g / L corn steep liquor powder, 18-22 g / L glucose, 11.5-15.5 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L magnesium sulfate heptahydrate, 2-4 g / L diammonium hydrogen phosphate, 1-2 g / L citric acid monohydrate, and the balance water, with a pH of 6.8-7.2; The feed culture medium formula comprises: 450-550 g / L of glucose, 50-70 g / L of diammonium hydrogen phosphate, 6-10 g / L of corn steep liquor dry powder, 15-19 g / L of magnesium sulfate heptahydrate, and the balance of water.

6. The method according to claim 5, characterized in that The inoculation amount of the bacterial liquid is 8-12 v / v%; the fermentation temperature when the bacterial liquid is inoculated into the fermentation medium is 34-37° C. and the pH is 6.6-7.

0.

7. The method according to claim 5, characterized in that The dissolved oxygen was maintained at 25-35% during the feeding process.

8. The method according to claim 5, characterized in that The temperature for inducing the lactose addition is 22-27° C.; the final concentration of the lactose in the fermentation system is 0.3-0.7 g / L.

9. The method according to claim 5, characterized in that In the mixed reaction, the concentration of galactose is 360-450 g / L; the amount of L-arabinose isomerase fermentation broth added is 0.6-1.0 wt% of galactose; the temperature of the mixed reaction is 58-62° C., the pH is 7.3-7.7, and the time is 21-25 h.

10. Use of D-tagatose obtained by the method according to any one of claims 5 to 9 in the preparation of food or medicine.

Images