Bacillus subtilis b1b-24-C and its application in fermentation and co-production of D-psicose and isomaltooligosaccharide

Abstract

The present invention relates to the technical field of polysaccharide production, and in particular to a Bacillus subtilis b1b-24-C and its application in fermentation and co-production of D-psicose and isomaltooligosaccharide. Bacillus subtilis )b1b‑24‑C is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a deposit number of CGMCC NO.32594 and a deposit date of November 13, 2024. The address of the deposit institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The D‑psicose 3‑epimerase produced by fermentation of Bacillus subtilis of the present invention has a high enzyme activity. At the same time, the enzyme immobilization can effectively improve the enzyme stability, conversion efficiency and utilization efficiency, improve production efficiency, and significantly reduce production costs. At the same time, the present invention can also co-produce isomaltooligosaccharide products.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide production, and in particular to a Bacillus subtilis b1b-24-C and application thereof in fermentation and co-production of D-psicose and isomaltooligosaccharide. Background Art

[0002] Sugar brings taste enjoyment to people, and normal metabolism of living organisms also requires sugar. However, obesity and diabetes caused by high sugar intake are harmful to human health. Therefore, new low-calorie sweeteners have become a research hotspot in the health food industry. They do not produce energy and will not cause new metabolic problems. New low-calorie sweeteners that do not metabolize or produce energy in the human body have gradually become one of the research hotspots in the health care industry.

[0003] D-allulose belongs to ketohexose and is the C-3 diastereomer of D-fructose. Its texture is similar to that of sucrose, and its calories are only 10% of sucrose, but its sweetness is 70% of sucrose. In addition, compared with other low-calorie sweeteners such as erythritol, the pure sweetness and taste of D-allulose are closer to sucrose, and it can undergo Maillard reaction with proteins or amino acids during baking. Therefore, D-allulose can effectively enhance the flavor of food. Due to its higher sweetness and lower energy, as well as its unique physiological functions and potential health benefits, D-allulose is considered to be a new sweetener with great potential and has become a research hotspot in the field of rare sugar biosynthesis worldwide.

[0004] However, D-psicose is extremely rare in nature. Traditional chemical synthesis methods require complex reaction steps and produce a large number of by-products, so it is difficult to obtain a single-configuration product. On the contrary, the biotransformation method has the advantages of mild reaction conditions, few by-products, simple purification and environmental friendliness, so it has become the main method for synthesizing D-psicose. However, the D-psicose-3-epimerase produced by the microorganisms (Bacillus subtilis and yeast) reported so far has low enzyme activity and poor ability to convert fructose, which increases the efficiency and difficulty of industrial production of D-psicose. At the same time, in the fermentation production of D-psicose, the by-products produced are difficult to be effectively utilized. Summary of the invention

[0005] In view of the technical problems in the prior art that the D-psicose-3-epimerase produced by microbial fermentation has low enzyme activity, resulting in poor fructose conversion ability, the present invention provides a Bacillus subtilis b1b-24-C and an application thereof in the fermentation and co-production of D-psicose and isomaltooligosaccharide. The D-psicose 3-epimerase produced by the Bacillus subtilis fermentation has high enzyme activity. At the same time, the enzyme immobilization can effectively improve the enzyme stability, conversion efficiency and utilization efficiency, improve the production efficiency, and significantly reduce the production cost. At the same time, the present invention can also co-produce isomaltooligosaccharide products.

[0006] In a first aspect, the present invention provides a Bacillus subtilis b1b-24-C, wherein the Bacillus subtilis ( Bacillus subtilis )b1b-24-C is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.32594, the deposit date is November 13, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In a second aspect, the present invention provides a use of Bacillus subtilis b1b-24-C in the fermentation and co-production of D-psicose and isomaltooligosaccharide.

[0008] Furthermore, the method for co-producing D-psicose and isomaltooligosaccharide by fermentation comprises the following steps:

[0009] (1) inoculating Bacillus subtilis b1b-24-C into a seed culture medium for cultivation to obtain a seed solution;

[0010] (2) inoculating the seed liquid into a fermentation medium for fermentation to obtain a fermentation liquid;

[0011] (3) After centrifuging the fermentation liquid, the fermentation bacteria are obtained;

[0012] (4) The fermented bacteria are homogenized and centrifuged to obtain an enzyme solution, and the enzyme solution is immobilized to obtain an immobilized enzyme;

[0013] (5) D-fructose solution enters the immobilized enzyme for conversion at the feed end, and samples are taken for testing at the discharge end;

[0014] (6) When the test is qualified, the output material is subjected to a primary chromatographic separation to obtain a D-psicose-containing component and a primary separation liquid;

[0015] (7) Decolorizing, ion exchanging, and concentrating the D-psicose component in step (6) to obtain a D-psicose product;

[0016] (8) The primary separation liquid in step (6) is subjected to secondary chromatographic separation to obtain a D-fructose-containing component and a secondary separation liquid. The secondary separation liquid is concentrated, enzymatically converted, decolorized, ion exchanged, and concentrated to obtain isomaltooligosaccharide.

[0017] Furthermore, in step (1), the culturing method is to inoculate Bacillus subtilis b1b-24-C into a seed culture medium at a culturing temperature of 36-38°C; the seed culture medium comprises the following components in weight percentages: 1% peptone, 0.5% yeast extract powder, 1% sodium chloride, and the remainder water; and the pH of the seed culture medium is 6.8-7.2.

[0018] Furthermore, in step (2), the fermentation method is as follows: the fermentation temperature is 31-33°C; the fermentation medium comprises the following components in weight percentage: 2%-3% peptone, 1%-2% corn steep liquor, 0.5%-1% glycerol, 0.05% anhydrous magnesium sulfate, 0.05% disodium hydrogen phosphate, 0.4% sodium dihydrogen phosphate, and the balance is water; the fermentation medium has a pH of 6.5-7.0.

[0019] Furthermore, in step (5), the sugar concentration in the D-fructose solution is 55% to 65%, and the pH is 5.5 to 7.5.

[0020] Furthermore, in step (6), when the discharge conductivity is ≤50 μs / cm, the transmittance is ≥97%, and the sugar concentration is 55%-65%, it is determined to be qualified.

[0021] Furthermore, in step (8), the secondary separation liquid is subjected to enzymatic conversion using D-glucosyltransferase.

[0022] Furthermore, in step (8), the D-fructose-containing component is returned to step (5) and combined with the D-fructose solution for use.

[0023] Furthermore, both the primary chromatographic separation and the secondary chromatographic separation adopt a sequential intermittent operation process, and the chromatographic columns are all calcium-type or potassium-type chromatographic resins, and the number is all six columns.

[0024] The beneficial effects of the present invention are:

[0025] (1) The D-psicose 3-epimerase activity of the Bacillus subtilis b1b-24-C provided by the present invention in the fermentation broth is ≥2000 U / mL, which greatly improves the efficiency of enzyme conversion and significantly reduces the production cost.

[0026] (2) The present invention immobilizes the D-psicose 3-epimerase produced by fermentation. Compared with the free enzyme, the immobilized enzyme has significantly improved pH and temperature stability, the enzyme conversion process can react continuously, and the output has high light transmittance and low conductivity, and can directly enter chromatographic purification.

[0027] (3) The present invention uses two sets of chromatographic separations in combination to purify the product, thereby producing D-psicose with a purity of ≥98.5% and simultaneously producing an isomaltooligosaccharide product.

[0028] (4) The entire D-psicose production process of the present invention has no extra waste, does not require high-temperature enzyme inactivation, saves energy, produces less pigment in the slurry, and realizes a clean production process.

[0029] (5) The present invention does not require decolorization, ion exchange and other processes before chromatographic separation, thus saving a lot of costs, reducing environmental pressure and achieving clean production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 It is a process flow chart of a specific implementation mode of the present invention.

[0032] Figure 2 It is a schematic diagram of the structure of an enzyme immobilization device according to a specific embodiment of the present invention.

[0033] In the figure, 1-plate heat exchanger, 2-liquid distributor, 3-fixed column, 4-sieve plate. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0035] The method for determining the enzyme activity of D-psicose-3-epimerase on D-fructose in the embodiment of the present invention is as follows: 800 μL of reaction substrate solution and 200 μL of enzyme solution are added to 1 mL of the reaction system, wherein the reaction substrate solution is prepared by dissolving D-fructose in 50 mL of phosphate buffer (pH 7.0), and the D-fructose concentration in the prepared reaction substrate solution is 100 g / L. The reaction is carried out at a temperature of 55° C. for 10 min, and then boiled for 10 min to terminate the enzyme reaction.

[0036] The production of D-psicose in the system was detected by HPLC, and the enzyme activity was calculated. The enzyme activity unit (U) was the amount of enzyme required to catalyze the production of 1 μmol of D-psicose per minute.

[0037] Example 1

[0038] 1. Mutation of strains

[0039] Plate culture medium: composed of the following ingredients in percentage by weight: 1% tryptone, 0.5% yeast extract powder, 1% sodium chloride, 2% agar powder, and the balance deionized water.

[0040] Slant culture medium: composed of the following components in percentage by weight: 1% tryptone, 0.5% yeast extract powder, 1% sodium chloride, 2% agar powder, and the balance deionized water.

[0041] Shake flask medium: composed of the following ingredients in percentage by weight: 2.5% peptone, 1% corn steep liquor, 1% glycerol, 0.05% anhydrous magnesium sulfate, 0.05% disodium hydrogen phosphate, 0.4% sodium dihydrogen phosphate, and the balance is deionized water, pH 6.8.

[0042] The original strain is a strain producing D-psicose 3-epimerase preserved by Baolingbao Biotechnology Co., Ltd. The enzyme activity of D-psicose 3-epimerase on D-fructose was measured in the fermentation broth of the original strain, and the enzyme activity was 500U / mL under pH 6.5. The strain was mutated by ARTP (normal pressure room temperature plasma source) mutagenesis system.

[0043] Preparation of mutagenic bacterial solution: transfer the activated bacterial solution to the shake flask medium and culture until OD 600 To about 0.8, as the mutagenic bacterial solution.

[0044] Mutagenesis operation process: This operation is carried out in an ultra-clean workbench. The metal sheet with the mutagenic solution is burned on the flame of an alcohol lamp and then placed in a sterilized glass culture dish to cool. After sufficient cooling, 10 μL of the prepared mutagenic solution is evenly coated on the surface of the metal sheet; the glass culture dish containing the metal sheet is carefully transferred to the operation room of the ARTP mutagenesis breeding instrument, and the metal sheet coated with the mutagenic solution is placed in the groove of the metal sheet in turn with tweezers wiped with alcohol; the instrument power, mutagenesis time, helium gas volume used for mutagenesis and other parameters are set; click to start processing samples; after all samples are processed, use tweezers to place the metal sheet in the correspondingly marked EP tube in turn, and add 1 mL of sterilized saline to the EP tube to elute the mutagenic solution; the eluate is gradient diluted, 200 μL of the dilution is evenly coated on the plate, and inverted in a 37°C incubator for culture.

[0045] 2. Determination of lethality

[0046] In the clean bench, 10 μL of the prepared mutagenic bacterial solution was evenly coated on the surface of the metal sheet, and the mutagenic treatment time was set to 0s, 20s, 40s, 60s, 80s, 100s, 120s, and 140s. The treated metal sheet was transferred to an EP tube pre-added with 900 μL of physiological saline and fully shaken for elution. The eluate was diluted by 10 -3 , 10 -4 , 10 -5 Spread on the plate, invert and culture at 30℃. Each treatment is paralleled 3 times, select the plate with the appropriate dilution multiple to count and calculate the lethality. The formula for calculating the lethality is as follows:

[0047]

[0048] Select the treatment time with a lethality rate of more than 90% for mutagenesis, at which the mutation rate is high and the possibility of recovery is small. Pick the colonies with better growth on the resistance plate and inoculate them in a 250mL conical flask containing 50mL shake flask medium, and shake and culture them at 37℃ and 200rpm for 10h; 5% inoculation amount is inoculated in a 250mL conical flask with 50mL shake flask medium, and cultured in a constant temperature shaker at 30℃ and 200rpm for 24h; the fermentation broth is tested for the enzyme activity of D-psicose 3-epimerase on D-fructose. Repeat the mutagenesis operation several times until a strain producing high enzyme activity D-psicose 3-epimerase is screened and named b1b-24-C.

[0049] The selected strains were entrusted to the Institute of Microbiology, Chinese Academy of Sciences for microbial identification, and the identification report number is: Micro-inspection No. 2024JB355. The cell morphology and physical and chemical test results are shown in Table 1.

[0050] Table 1 Cell morphology and physicochemical test results

[0051]

[0052] In the table, “+” represents positive and “-” represents negative.

[0053] The strain b1b-24-C screened by the present invention was identified as Bacillus subtilis by the Institute of Microbiology, Chinese Academy of Sciences. (Bacillus subtilis ), named Bacillus subtilis ( Bacillus subtilis ) b1b-24-C, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 13, 2024, and the culture collection number is CGMCC NO.32594.

[0054] Example 2

[0055] Combination Figure 1 Embodiment 2 provides a method for co-producing D-psicose and isomaltooligosaccharide by fermentation, comprising the following steps:

[0056] (1) Take Bacillus subtilis b1b-24-C and inoculate it into a seed culture medium. The seed culture medium includes the following components in weight percentage: 1% peptone, 0.5% yeast extract powder, 1% sodium chloride, and the balance water; the pH of the seed culture medium is 6.8-7.2; and the culture is carried out at 37°C for 10 hours to obtain a seed solution.

[0057] (2) The seed liquid prepared above was inoculated into a fermentation medium at a volume ratio of 5%, wherein the fermentation medium comprises the following components in weight percentage: 2.5% peptone, 1% corn steep liquor, 1% glycerol, 0.05% anhydrous magnesium sulfate, 0.05% disodium hydrogen phosphate, 0.4% sodium dihydrogen phosphate, and the balance is water; the pH value of the fermentation medium is 6.5-7.0; the fermentation was carried out at 32° C., and the fermentation medium was continuously replenished during the fermentation process. The fermentation was carried out for 52 hours to obtain a fermentation liquid, i.e., an enzyme liquid; the enzyme liquid was subjected to an enzyme activity assay of D-psicose 3-epimerase on D-fructose, and the enzyme activity of the bacterial fermentation liquid was determined to be 2047 U / mL, i.e., each mL of the fermentation liquid contained 2047 U (enzyme activity units), which was much higher than 500 U / mL of the original strain.

[0058] (3) Immobilizing the enzyme solution obtained by fermentation, using an enzyme immobilization device such as Figure 2 As shown, the fixed column 3 has a diameter-to-height ratio of 1:2, a feed liquid distributor 2 and a sieve plate 4 are arranged inside the fixed column 3, a carrier is loaded between the feed liquid distributor 2 and the sieve plate 4, and the enzyme liquid contacts the carrier to form an immobilized enzyme.

[0059] (4) Prepare a 60% D-fructose solution as the substrate, enter the plate heat exchanger 1 from the feed end for heating, control the feed flow rate to be 12 times the weight of the immobilized enzyme, keep the temperature in the fixed column 3 at 55°C, and perform continuous conversion of the immobilized enzyme. When the reaction is balanced, take samples at the discharge end to detect the D-psicose concentration and conversion rate. The average conversion rate of D-psicose can reach 30% in the early stage, and the stable continuous conversion reaction time can reach more than 3 months. When the conversion rate of the immobilized enzyme is detected to decrease, reduce the flow rate to ensure a 30% D-psicose conversion rate, and stop the conversion when the flow rate drops to 2 to 3 times the weight of the immobilized enzyme.

[0060] (5) The conductivity of the immobilized enzyme output was detected to be 45.8 μs / cm, the transmittance was 98%, and the sugar concentration was 55.5%. The qualified output was directly subjected to a chromatographic separation and purification. The chromatographic separation adopted a sequential intermittent operation process. The chromatographic column was a calcium-type chromatographic resin, and the number was a six-column mode. A D-psicose component and a primary separation liquid were obtained. The D-psicose component was decolorized, ion exchanged, and concentrated to obtain a D-psicose liquid product with a purity of 99.0%. The D-psicose component can also be decolorized, ion exchanged, concentrated, and crystallized to obtain a D-psicose solid product. The crystallization mother liquor is returned to the chromatographic separation for further separation and purification.

[0061] (6) The D-fructose content in the primary separation liquid of the first chromatographic separation is relatively high, and the primary separation liquid is subjected to secondary chromatographic separation. The chromatographic separation adopts a sequential operation process, and the chromatographic column is a calcium-type chromatographic resin, and the number is a six-column mode. The D-fructose component with a purity of ≥95% is separated, and after concentration, it returns to step (4) and enters the immobilized enzyme for conversion production; the separated secondary separation liquid is concentrated and added with transaminase (D-glucosyltransferase) enzyme conversion, and then decolorized, ion exchanged, and concentrated to produce oligosaccharide products.

[0062] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis b1b-24-C, characterized in that The Bacillus subtilis ( Bacillus subtilis )b1b-24-C is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNO.32594, the deposit date is November 13, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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