Process for improving activity of beta-fructosyl transferase

Abstract

The invention relates to the technical field of enzyme activity, in particular to a process for improving the activity of beta-fructosyl transferase. The invention provides a process for improving the activity of beta-fructosyl transferase. The process is characterized by comprising the following steps: activating strains, sucking lactobacillus casei, lactobacillus fermentum and bifidobacterium animalis, and respectively putting the lactobacillus casei, the lactobacillus fermentum and the bifidobacterium animalis into different culture mediums for culturing; preparing a seed solution, namely selecting bacterial colonies obtained by activating three strains, namely lactobacillus casei, lactobacillus fermentum and bifidobacterium animalis, to compound the seed solution; preparing a fermentation culture medium, namely mixing one or more of a carbon source, a nitrogen source and inorganic salt with the basic components and distilled water, fixing the volume to the use amount, and inoculating the compound seed solution. The beta-fructosyl transferase aims at solving the technical problem that in the prior art, due to insufficient activity of beta-fructosyl transferase, too much sugar is taken in a human body, and body

Description

Technical Field

[0001] This invention relates to the technical field of enzyme activity, and particularly to a process for improving the activity of β-fructose transferase. Background Technology

[0002] Fructose oligosaccharides (FOS) are a novel type of health food. They are utilized by probiotics to provide health benefits to the human body without being absorbed by the body, making them one of the most representative functional oligosaccharides. The bio-enzymatic method for producing FOS is the safest, most efficient, and environmentally friendly solution. The principle behind the bio-enzymatic production of FOS is that β-fructosyltransferase (β-FT) acts on the β-(2,1) glycosidic bond of sucrose, causing it to connect 1-3 fructose groups to form a series of fructose oligomers, mainly including fructotriose (GF2), fructotetraose (GF3), and fructopentose (GF4). In daily life, the sugars ingested by the human body are primarily sucrose. Sucrose is a disaccharide and cannot be directly absorbed by the body. Instead, it is rapidly hydrolyzed into two monosaccharides: glucose and fructose. These monosaccharides are quickly absorbed by the body and enter the metabolic cycle to maintain normal bodily functions. However, excessive sugar intake can lead to many health problems, such as elevated blood sugar, reduced fat burning, impaired calcium absorption leading to osteoporosis, accelerated skin aging, and tooth decay.

[0003] Therefore, it is necessary to propose a method to enhance the activity of β-fructosyltransferase, and to use β-fructosyltransferase to convert as many monosaccharides and disaccharides as possible into polymers, thereby reducing the absorption of excessive sugars by the human body and achieving the goal of reducing harm to the body. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a process for improving the activity of β-fructose transferase, which aims to solve the technical problem that insufficient β-fructose transferase activity in the prior art leads to excessive sugar intake in the human body, affecting health.

[0005] To achieve the above objectives, this invention proposes a process for improving the activity of β-fructosyltransferase, characterized by comprising the following steps: a. Activation of bacterial strains: Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis were cultured separately in different culture media. b. Seed liquid preparation: Select the activated colonies of three strains, Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis, to prepare a seed liquid. c. Preparation of fermentation medium: Mix one or more carbon source, nitrogen source, and inorganic salt with the basic components and distilled water to the required volume, then inoculate with the prepared seed culture.

[0006] As a preferred method, during the strain activation process, equal amounts of Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis were taken and placed in MRS agar plates, spread evenly, and incubated in an anaerobic incubator at 30-35 ℃ for 16-24 h. The colony growth status was observed, and after the formation of single colonies with uniform morphology and clear edges, single colonies were picked and cultured in Erlenmeyer flasks.

[0007] As a preferred method, during the seed culture preparation process, single colonies of three activated bacterial strains—Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis—were picked and inoculated onto MRS broth medium. The OD value at 600 nm was measured to be 0.7, and the concentration of each of the three strains reached 6×10⁸ cfu / ml. Then, the three strains were mixed in a ratio of 1:2:1 to prepare a compound seed culture with a final concentration of approximately 2×10⁹ cfu / ml for later use.

[0008] As a preferred option, peptone, beef extract, yeast extract, glucose, sodium acetate, ammonium citrate, Tween 80, K2HPO4, MgSO4, and MnSO4 were selected as the basic components of the culture medium.

[0009] Preferably, glucose is selected as the carbon source of the culture medium, a combination of peptone and yeast extract is selected as the nitrogen source of the culture medium, and a combination of K2HPO4, sodium acetate, and ammonium citrate is selected as the inorganic salt of the culture medium.

[0010] Preferably, glucose accounts for 1.5% to 3.5% of the mass percentage of the basic components; the peptone and yeast powder combination accounts for 2% to 6% of the mass percentage of the basic components; and the K2HPO4, sodium acetate, and ammonium citrate combination accounts for 0.75% to 1.75% of the mass percentage of the basic components.

[0011] As a preferred method, the basic components, carbon source, nitrogen source, and inorganic salts are mixed and then diluted to the required volume with distilled water. The initial pH of the culture medium is adjusted to 6.1-6.7 with ammonia water, and then sterilized at 105-125℃.

[0012] As a preferred method, the proportion of the compound seed solution is selected based on the amount used; and it is cultured at 30-35℃ for 24-48 hours.

[0013] Preferably, the weight percentages of peptone, beef extract, yeast extract, glucose, sodium acetate, ammonium citrate, Tween, K2HPO4, MgSO4, and MnSO4 are 1%, 1%, 0.5%, 2%, 0.5%, 0.2%, 0.1%, 0.2%, 0.02%, and 0.005%, respectively.

[0014] Preferably, the mass ratio of peptone and yeast powder composition is 1:1, and the mass ratio of K2HPO4, sodium acetate, and ammonium citrate composition is 1:3:1.

[0015] Compared with the prior art, the beneficial effects of the process for improving β-fructose transferase activity provided by the present invention are as follows: Fermentation was carried out using a compound probiotic strain of Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis in a 1:2:1 ratio. The resulting bacterial concentration was 2×10⁹ CFU / ml, with an inoculum size of 5% (v / v). The optimal culture medium composition was 2.5% glucose, 1.5% peptone, 1.5% yeast extract, 0.25% K₂HPO₄, 0.75% sodium acetate, and 0.25% ammonium citrate. The pH was 6.4, the temperature was 32 ℃, and the fermentation time was 48 h. Under these conditions, the enzyme activity reached 32.28 U / ml, which was 64.1% higher than that of the unoptimized control group.

[0016] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0017] Figure 1 This is a comparison result of the carbon source selection in the embodiments of the present invention.

[0018] Figure 2 This is a comparison result of the nitrogen source selection in the embodiments of the present invention.

[0019] Figure 3 This is a comparison result of the selection of inorganic salts in the embodiments of the present invention.

[0020] Figure 4 This invention illustrates the effect of different glucose concentrations on the fermentation OD value in various embodiments.

[0021] Figure 5 This invention illustrates the effect of different nitrogen source composition concentrations on fermentation OD values ​​in various embodiments.

[0022] Figure 6 This invention relates to the effect of different concentrations of K2HPO4, sodium acetate, and ammonium citrate in the embodiments on the fermentation OD value. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0024] In the description of this invention, it should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly on or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to or indirectly connected to the other element.

[0025] In the description of this invention, it should be noted that the terms "center," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this invention is in use. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. "Several" means one or more, unless otherwise explicitly specified.

[0026] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0027] First, the materials were selected. Strains selected: *Lactobacillus casei*, *Lactobacillus fermentum*, and *Bifidobacterium animalis*. Reagents selected: MRS broth, glucose (AR), peptone, yeast extract, fructose (AR), fructotriose (AR), K₂HPO₄ (AR), sodium acetate (AR), ammonium citrate (AR), sucrose (AR), and β-fructose transferase standard. Instruments and equipment: Thermo LM17 high-speed centrifuge, UV-2102 UV-Vis spectrophotometer, H2050R refrigerated centrifuge, MEMMET anaerobic incubator, HealForce clean bench, LX-B50c autoclave, and high-performance liquid chromatography (HPLC).

[0028] Methods for determining the activity of β-fructosyltransferase (β-FT): A 10% (w / v) sucrose solution was prepared using a pH 5.5, 50 mmol / L Na2HP04-NaH2PO4 buffer solution as the substrate. An appropriate amount of enzyme solution was added (to control the final amount of fructotriose to be less than 10%). The reaction was carried out in a 40 °C water bath with shaking for 40 min, and then immediately placed in a 100 °C boiling water bath for 15 min to terminate the reaction.

[0029] The activity of β-fructosyltransferase is defined as the amount of enzyme required to produce 1 μmol GF2 per minute under the above reaction conditions, which is denoted as U.

[0030] High-performance liquid chromatography (HPLC) was used for determination. An Aglient NH2 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was acetonitrile-water (70:30), the flow rate was 1.5 ml / min, and the reference standards were GF2 and fructose. The sugar content was calculated using the external standard method.

[0031] Preparation of seed culture for strain activator: 1. Activation method of bacterial strains: Take 100 μL of each of the three bacterial strains stored in glycerol tubes at -20 ℃ and place them on MRS agar plates. Spread them evenly and incubate them in an anaerobic incubator at 32 ℃ for 16 h-24 h. Observe the growth status of the colonies. When a single colony with uniform morphology and clear edge is produced, the single colony can be picked for liquid culture in Erlenmeyer flasks.

[0032] 2. Seed culture preparation: Single colonies of activated Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis were picked and inoculated into 50 ml of MRS broth medium. The OD value at 600 nm was measured to be 0.7, and the concentration of each of the three strains reached 6×10⁸ cfu / ml. Then, the three strains were mixed in a ratio of 1:2:1 to prepare a compound seed culture with a final concentration of approximately 2×10⁹ cfu / ml for later use.

[0033] Selection of single-factor components for fermentation culture medium: 1. Basic fermentation medium formulation and preparation: peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, sodium acetate 0.5%, ammonium citrate 0.2%, Tween 80 0.1%, K2HPO4 0.2%, MgSO4 0.02%, MnSO4 0.005%, all of the above components are mass percentages; distilled water was brought to the required volume, the initial pH of the medium was adjusted to 6.4 with ammonia water, and sterilized at 115 ℃ for 30 minutes.

[0034] Carbon source selection: Sucrose, glucose, glycerol, and soluble starch (all at a concentration of 2%) were used as carbon sources, while other components were the same as those in the basic fermentation medium. After preparation according to the method of the basic fermentation medium, the compound seed culture was inoculated at a ratio of 5% (v / v), cultured at 32 ℃ for 24 h, and the OD value of the fermentation broth was measured at 600 nm to determine the optimal carbon source.

[0035] according to Figure 2 The carbon source selection results show that when glucose is used as the sole carbon source, the cell volume in the fermentation broth is higher than with other carbon sources, making it the most suitable for cell growth. Therefore, glucose as a sole carbon source is more beneficial for cell utilization. Glucose can be directly utilized by the cells, reducing the saccharification process required for other carbon sources.

[0036] 2. Nitrogen source selection: Under optimal carbon source conditions, 2% peptone, 2% yeast extract, 2% ammonium sulfate, and a combination of 1% peptone and 1% yeast extract were used as nitrogen sources, respectively. Other components were the same as those in conventional fermentation medium. After mixing, the mixture was prepared according to the method of basal fermentation medium and inoculated with the compound seed culture at a ratio of 5% (v / v). The culture was carried out at 32 ℃ for 24 h, and the OD value of the fermentation broth was measured at 600 nm to determine the optimal nitrogen source.

[0037] according to Figure 3 The results of nitrogen source selection showed that when peptone and yeast powder were used as a combined nitrogen source, the cell volume of the fermentation broth was higher than when other nitrogen sources were used, making it the most suitable for cell growth. However, when ammonium sulfate was used as a single nitrogen source, cell growth was the worst. This indicates that organic nitrogen sources can be hydrolyzed into smaller molecules such as peptides and amino acids that are more easily utilized by cells. Therefore, organic nitrogen sources are more beneficial to mixed-strain fermentation than inorganic nitrogen sources, promoting cell growth and increasing cell volume.

[0038] 3. Selection of inorganic salts: Under optimal carbon and nitrogen source conditions, 1% K2HPO4, 1% sodium acetate, 1% ammonium citrate, and a combination of 0.2% K2HPO4 + 0.6% sodium acetate + 0.2% ammonium citrate were selected as inorganic salts, respectively. Other components were the same as those in the basic fermentation medium. After mixing, the mixture was prepared according to the method of the basic fermentation medium and inoculated into the compound seed culture at a ratio of 5% (v / v). The culture was carried out at 32℃ for 24 h, and the OD value of the fermentation broth was measured at 600 nm to determine the optimal inorganic salts.

[0039] Inorganic salts are essential trace substances for microbial growth, participating in the synthesis of various components of microbial cells and regulating osmotic pressure, pH, and redox potential. Through screening and combining inorganic salts, as shown in Figure 4, the combination of K₂HPO₄, sodium acetate, and ammonium citrate exhibited the best fermentation effect for the probiotics selected in this experiment. This combination provided all the necessary trace substances for the fermentation of the compound probiotics, and the concentration of inorganic salts was not excessively high, thus not inhibiting cell growth.

[0040] Optimization of the concentrations of each component in the fermentation medium: 1. Glucose concentration optimization: The concentrations of peptone, yeast extract, K2HPO4, sodium acetate, and ammonium citrate were fixed. Glucose was added at concentrations of 1.5%, 2%, 2.5%, 3%, and 3.5%, respectively. After mixing, the mixture was prepared according to the method for basal fermentation medium. The mixed seed culture was inoculated at a ratio of 5% (v / v) and cultured at 32 ℃ for 24 h. The OD value of the fermentation broth was measured at 600 nm to determine the optimal glucose concentration.

[0041] 2. Optimization of peptone and yeast extract concentrations: With fixed concentrations of glucose, K2HPO4, sodium acetate, and ammonium citrate, 2%, 3%, 4%, 5%, and 6% of the peptone and yeast extract composition (mass ratio of 1:1) were added respectively. After mixing, the mixture was prepared according to the method of basal fermentation medium. The mixed seed culture was inoculated at a ratio of 5% (v / v) and cultured at 32 ℃ for 24 h. The OD value of the fermentation broth was measured at 600 nm to determine the optimal concentration of the peptone and yeast extract composition.

[0042] 3. Optimization of K2HPO4 + sodium acetate + ammonium citrate concentration: With the concentrations of glucose, peptone, and yeast extract fixed, 0.75%, 1%, 1.25%, 1.5%, and 1.75% of the K2HPO4 + sodium acetate + ammonium citrate composition (mass ratio of 1:3:1) were added respectively. After mixing, the mixture was prepared according to the method of basal fermentation medium, inoculated at a ratio of 5% (v / v), and cultured at an initial pH of 6.4 and a temperature of 32 ℃ for 24 h. The OD value of the fermentation broth was measured at 600 nm to determine the optimal concentration of K2HPO4, sodium acetate, and ammonium citrate composition.

[0043] The orthogonal experiment was used to optimize the fermentation process: Based on the results of the single-factor experiment and the optimization experiment of each component concentration, the culture time was increased to 24 h, 36 h and 48 h, the culture temperature was 32 ℃, and the OD value of the fermentation broth was measured at 600 nm. The orthogonal experiment was carried out according to the L9(34) orthogonal table. The four factors and three levels selected are shown in Table 1.

[0044]

[0045] The data obtained in the orthogonal experiment were processed using SPSS Statistics 28.0 software.

[0046] Fermentation of compound probiotics is a dynamic process with complex fermentation conditions and a chain reaction mechanism. The composition and content of the culture medium, as well as the fermentation process methods, all significantly affect the fermentation results. Therefore, it is necessary to scientifically select and optimize conditions such as carbon sources, nitrogen sources, inorganic salts, and fermentation time.

[0047] After determining the optimal carbon source, nitrogen source, and inorganic salt, the concentrations of each component are optimized. After determining glucose as the sole carbon source based on the aforementioned single-factor experiments, different concentrations of glucose were selected as the sole carbon source for fermentation culture. The OD600 of the fermentation broth was then measured to determine the optimal glucose concentration range. (See below) Figure 5 Based on the measurement results, glucose concentrations of 2%, 2.5%, and 3% were selected for subsequent orthogonal experiments.

[0048] After determining the peptone and yeast powder combination as the combined nitrogen source based on the aforementioned single-factor experiments, fermentation culture was carried out with different concentrations of the peptone and yeast powder combined nitrogen source, and the OD600 of the fermentation broth was measured to select the optimal concentration range of the combined nitrogen source. The measurement results are shown in Figure 5. When the combined nitrogen source concentration was 3%, the OD600 of the fermentation broth was the highest. Therefore, the concentrations of the peptone and yeast powder combination were selected as 3%, 4%, and 5% for subsequent orthogonal experiments.

[0049] After determining the K2HPO4, sodium acetate, and ammonium citrate combination as the inorganic salt based on the aforementioned single-factor experiments, different concentrations of the K2HPO4, sodium acetate, and ammonium citrate combination were selected for fermentation culture. The OD600 of the fermentation broth was measured to select the optimal concentration range of the K2HPO4, sodium acetate, and ammonium citrate combination inorganic salt. Based on the measurement results in Figure 6, the concentrations of K2HPO4, sodium acetate, and ammonium citrate combination were selected as 1%, 1.25%, and 1.5%, respectively, for subsequent orthogonal experimental analysis.

[0050] Finally, the optimal culture medium ratio was determined through orthogonal experiments: Four-factor, three-level experiments were conducted according to Table 1, and the experimental data and results are shown in Table 2. The range R0 indicates that, using the fermentation broth OD600 as the test index, the order of influence of each factor on cell growth is: glucose concentration (A) > fermentation time (D) > nitrogen source mixture concentration (B) > inorganic salt concentration (C). The optimal culture conditions for compound probiotic fermentation were finally determined as A2B1C2D3, namely: glucose 2.5%, peptone 1.5%, yeast extract 1.5%, K2HPO4 0.25%, sodium acetate 0.75%, ammonium citrate 0.25%, and fermentation time 48 h. Under these culture conditions, three fermentation experiments were conducted, and the measured OD600 values ​​were 3.4, 3.7, and 3.5, respectively, with an average OD600 of 3.53, higher than the experimental data in the orthogonal experiment table. This indicates that the optimized fermentation process has good representativeness and repeatability, and that using these culture conditions in subsequent industrial-scale production is feasible.

[0051] Enzyme activity assay results: The enzyme activity of the compound probiotic fermentation was compared between the optimized fermentation process and the conventional fermentation process. The bacterial cells were collected and the enzyme activity of the products was determined. The experimental data and results are shown in Table 2.

[0052]

[0053] As shown in Table 3, the enzyme activity of β-fructose transferase produced by compound probiotic fermentation was significantly improved under the optimized new fermentation process. The new fermentation process increased the enzyme activity by 64.1% compared with the conventional fermentation process.

[0054]

[0055] The culture medium selected in this application uses 2.5% glucose as a single carbon source, 1.5% peptone and 1.5% yeast extract as a combined nitrogen source, and 0.25% K₂HPO₄, 0.75% sodium acetate, and 0.25% ammonium citrate as a combined inorganic salt. This significantly increases the cell yield in the combined fermentation culture of *Lactobacillus casei*, *Lactobacillus fermentum*, and *Bifidobacterium animalis*. Optimization of the combined fermentation process of *Lactobacillus casei*, *Lactobacillus fermentum*, and *Bifidobacterium animalis* yields high-yield β-fructose transferase. Compared to enzyme production by a single high-yield strain, the enzyme activity is significantly improved.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for improving the activity of β-fructosyltransferase, characterized in that: Includes the following steps: a. Activation of bacterial strains: Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis were cultured separately in different culture media. b. Seed liquid preparation: Select the activated colonies of three strains, Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis, to prepare a seed liquid. c. Preparation of fermentation medium: Mix one or more carbon sources, nitrogen sources, and inorganic salts with the basic components and distilled water to the required volume, then inoculate with the prepared seed culture.

2. The process for improving β-fructosyltransferase activity as described in claim 1, characterized in that: During the bacterial activation process, equal amounts of Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis were taken and placed in MRS agar plates, spread evenly, and incubated in an anaerobic incubator at 30-35 ℃ for 16-24 h. The growth status of the colonies was observed. After the formation of single colonies with uniform morphology and clear edges, single colonies were picked and cultured in Erlenmeyer flasks.

3. The process for improving β-fructose transferase activity as described in claim 1, characterized in that: During the preparation of the seed culture, single colonies of three strains—Lactobacillus casei, Lactobacillus fermentum, and Bifidobacterium animalis—were activated and inoculated onto MRS broth medium. The OD value at 600 nm was measured to be 0.7, and the concentration of each of the three strains reached 6×10⁸ CFU / ml. The three strains were then mixed in a ratio of 1:2:1 to prepare a compound seed culture with a final concentration of approximately 2×10⁹ CFU / ml for later use.

4. The process for improving β-fructose transferase activity as described in claim 1, characterized in that: Peptone, beef extract, yeast extract, glucose, sodium acetate, ammonium citrate, Tween 80, K2HPO4, MgSO4, and MnSO4 were selected as the basic components of the culture medium.

5. The process for improving β-fructose transferase activity as described in claim 4, characterized in that: Glucose was selected as the carbon source of the culture medium, a combination of peptone and yeast extract was selected as the nitrogen source of the culture medium, and a combination of K2HPO4, sodium acetate, and ammonium citrate was selected as the inorganic salt of the culture medium.

6. The process for improving β-fructose transferase activity as described in claim 5, characterized in that: The composition of glucose as a basic component is selected as 1.5% to 3.5% by mass; the composition of peptone and yeast powder as a basic component is selected as 2% to 6% by mass; and the composition of K2HPO4, sodium acetate, and ammonium citrate as a basic component is selected as 0.75% to 1.75% by mass.

7. The process for improving β-fructosyltransferase activity as described in claim 6, characterized in that: After mixing the basic components, carbon source, nitrogen source, and inorganic salts, dilute the mixture to the required volume with distilled water. Adjust the initial pH of the culture medium to 6.1-6.7 with ammonia water, and sterilize at 105-125℃.

8. The process for improving β-fructose transferase activity as described in claim 7, characterized in that: Select the appropriate ratio for inoculating the compound seed solution according to the usage amount; culture at 30-35℃ for 24-48 hours.

9. The process for improving β-fructosyltransferase activity as described in claim 4, characterized in that: The weight percentages of peptone, beef extract, yeast extract, glucose, sodium acetate, ammonium citrate, Tween, K2HPO4, MgSO4, and MnSO4 are 1%, 1%, 0.5%, 2%, 0.5%, 0.2%, 0.1%, 0.2%, 0.02%, and 0.005%, respectively.

10. The process for improving β-fructosyltransferase activity as described in claim 5, characterized in that: The mass ratio of peptone and yeast powder composition is 1:1, and the mass ratio of K2HPO4, sodium acetate, and ammonium citrate composition is 1:3:1.

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