Method for reducing fructose content in fructose-containing beverage

Abstract

The invention discloses a method for reducing the content of fructose in a fructose-containing beverage. The method comprises the following steps: mixing the fructose-containing beverage with D-psicose 3-epimerase, and carrying out a reaction, the D-psicose 3-epimerase is the D-psicose 3-epimerase which is derived from Rumiclostridium papyrosolvens, and the D-psicose 3-epimerase is the D-psicose 3-epimerase which is derived from Rumiclostridium papyrosolvens. The D-psicose 3-epimerase disclosed by the invention has high enzyme activity which can reach 489.78 U / mL, and the enzyme still keeps a quite high psicose conversion rate under a low pH condition, so that the D-psicose 3-epimerase can be directly used for reducing sugar in fruit juice, and the change of flavor and nutritional ingredients caused by adding a pH regulator such as baking soda is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for reducing the fructose content in fructose-containing beverages. Background Art

[0002] Juice is a liquid naturally derived from fruits, which is extracted by mechanically squeezing or macerating fruits. It is usually consumed as a beverage or used as an ingredient or flavoring in foods. In addition to having a unique flavor, juice also contains a large amount of healthy functional elements such as vitamins, minerals, and dietary fiber, so it is widely popular among the public. However, juice presents a high sugar concentration, and the sugars found in natural juice usually include sucrose, fructose, and glucose. Therefore, in recent years, influenced by the increasing awareness of healthy eating, the juice market has gradually developed towards low-calorie and low-sugar directions.

[0003] Currently, low-sugar juice products on the market are usually mixtures of juice and other beverages (i.e., coconut water), or mixtures diluted with water and added with low-calorie sweeteners, so they do not have the same sensory and nutritional characteristics as natural juice. As one of the main sugars in natural juice, reducing the fructose content is also one of the strategies for the current juice market to develop towards low-calorie and low-sugar directions. D-allulose is an epimer of D-fructose at the C-3 position. Its sweetness is 70% of that of sucrose, and its taste and characteristics are very similar to those of sucrose, but its calorie content is only 0.4 kcal / g. At the same time, it has special physiological functions such as improving lipid metabolism, reducing postprandial blood glucose, anti-diabetes, and anti-obesity. Related studies have also shown that D-allulose can competitively inhibit the influx and efflux of transporters, reduce the absorption of dietary fructose and glucose by the body, and activate the afferent signals of the vagus nerve to limit food intake and hyperglycemia by inducing the release of glucagon-like peptide GLP-1. The US Food and Drug Administration (FDA) certified D-allulose as GRAS (Generally recognized as safe) in 2011 and issued a notice in 2019 to exclude it from the added sugar and total sugar ingredient lists. D-allulose can be used as a sugar substitute for obese and diabetic patients and as an ideal sweetener for healthy people, and has broad application prospects in the fields of food, health products, etc.

[0004] Patent CN113249287B discloses a food safety-grade strain that efficiently secretes and expresses D-allulose 3-epimerase, and its crude fermentation enzyme solution or immobilized enzyme can be used to catalyze the conversion of fructose. In the disclosed examples, it is necessary to use baking soda to adjust the pH of the fruit juice to not less than 5.0, then add 1% (v / v) of the crude enzyme solution, and react at 60 °C for 4 h. The results show that the conversion rate of fructose in fruit juices such as apple juice, orange juice, grape juice, and red date juice can reach 30% - 31%. It can be seen that this D-allulose 3-epimerase needs to be used under the conditions of a pH not less than 5 and a reaction temperature of 60 °C to achieve the described catalytic effect, while the pH of pure fruit juices such as pure orange juice and pure apple juice without other additives is lower than 5. Therefore, this enzyme is not suitable for processing pure fruit juices. And in the prior art, there is no disclosure of a D-allulose 3-epimerase that is resistant to low pH conditions, nor is there a disclosure of a D-allulose 3-epimerase that can be directly applied to pure orange juice and apple juice. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the defect in the prior art that there is a lack of D-allulose 3-epimerase that can reduce the fructose content in fruit juice without additional addition of a pH regulator, and a method for reducing the fructose content in fructose-containing beverages is provided. The D-allulose 3-epimerase used in the method still retains a relatively high allulose conversion rate at low pH and can be directly used for fructose reduction in fruit juice, avoiding changes in flavor and nutritional components caused by the addition of pH regulators such as baking soda.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides a method for reducing the fructose content in a fructose-containing beverage, which comprises mixing the fructose-containing beverage with D-allulose 3-epimerase (DPE) for reaction;

[0008] The D-allulose 3-epimerase is the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens.

[0009] In the present invention, the fructose-containing beverage refers to a liquid for human consumption that contains fructose in its components.

[0010] In some preferred embodiments, the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens comprises the amino acid sequence shown in SEQ ID NO:2.

[0011] In some embodiments, the fructose-containing beverage is pure fruit juice, for example, orange juice, pear juice, apple juice, grape juice, peach juice, and mango juice.

[0012] In some embodiments, the D-psicose 3-epimerase is an immobilized enzyme.

[0013] In some preferred embodiments, the reaction satisfies one or more of the following conditions:

[0014] The addition amount of the immobilized enzyme is 15-20%, where % is the mass ratio of the immobilized enzyme to the fructose-containing beverage;

[0015] The pH of the reaction is 3-7;

[0016] The temperature of the reaction is 35-37 °C;

[0017] The reaction time is 2-5 h, for example 4 h; and,

[0018] The reaction is carried out under shaking conditions at a rotation speed of 150-250 rpm.

[0019] In some embodiments, the immobilized enzyme is prepared by the following steps:

[0020] (1) Mix the crude enzyme solution of D-psicose 3-epimerase with an aqueous sodium alginate solution and stir evenly to obtain a mixed solution;

[0021] (2) Add a CaCl2 solution dropwise to the mixed solution obtained in step (1) for immobilization to obtain the immobilized enzyme.

[0022] In some preferred embodiments, the concentration of the sodium alginate is 2.5% (g / mL).

[0023] In some preferred embodiments, the volume ratio of the crude enzyme solution to the aqueous sodium alginate solution is 1:10.

[0024] In some embodiments, the crude enzyme solution in step (1) is prepared by the following steps:

[0025] (a) Culture the engineering bacterium B. subtilis 168 expressing D-psicose 3-epimerase in an LB medium to obtain a seed solution;

[0026] (b) Inoculate the seed solution obtained in step (a) into a TB medium for culture and take the supernatant to obtain the crude enzyme solution.

[0027] The second aspect of the present invention provides a low-fructose beverage, which is prepared by the method described in the first aspect; the low-fructose beverage is a low-fructose fruit juice.

[0028] In some preferred embodiments, the low-fructose beverage contains allulose, and the content of allulose is 5-20 g / L.

[0029] In some preferred embodiments, the low-fructose juice is prepared from pure fruit juice, such as orange juice, pear juice, apple juice, grape juice, peach juice, and mango juice.

[0030] The third aspect of the present invention provides a low-fructose orange juice prepared by the method as described in the first aspect.

[0031] In some preferred embodiments, the low-fructose orange juice contains allulose and fructose, the content of allulose is 5 - 7 g / L, and the content of fructose is 18 - 20 g / L.

[0032] The fourth aspect of the present invention provides a genetically engineered bacterium, which is B. subtilis 168 expressing D-allulose 3-epimerase;

[0033] The D-allulose 3-epimerase is the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens.

[0034] In some preferred embodiments, the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens contains the amino acid sequence shown in SEQ ID NO:2.

[0035] The fifth aspect of the present invention provides a method for culturing the genetically engineered bacterium as described in the fourth aspect, the method includes culturing the genetically engineered bacterium in a seed medium and then transferring it to a fermentation medium for fermentation.

[0036] In some preferred embodiments, the culturing satisfies one or more of the following conditions:

[0037] The seed medium is LB medium,

[0038] The temperature of the culturing is 35 - 40 °C, for example, 37 °C,

[0039] The culturing is carried out with shaking, and the rotation speed of the shaking is 180 - 220 rpm, for example, 200 rpm,

[0040] The time of the culturing is 14 - 18 h, for example, 14 - 16 h.

[0041] In some preferred embodiments, the fermentation satisfies one or more of the following conditions:

[0042] The fermentation medium includes tryptone, yeast extract, KH2PO4, K2HPO4, and glycerol,

[0043] The temperature of the fermentation is 35 - 40 °C, for example, 37 °C,

[0044] and the fermentation time is 20 - 26 h, for example, 24 h.

[0045] The sixth aspect of the present invention provides an application of the genetically engineered bacterium as described in the fourth aspect in the preparation of D - allulose 3 - epimerase or in reducing the content of allulose in fructose - containing beverages.

[0046] The seventh aspect of the present invention provides an immobilized enzyme, which uses sodium alginate - CaCl₂ as a carrier to immobilize D - allulose 3 - epimerase, and the D - allulose 3 - epimerase contains the amino acid sequence shown in SEQ ID NO:2.

[0047] In some preferred embodiments, the immobilized enzyme is prepared by the following steps:

[0048] (1) Mix the crude enzyme solution of the D - allulose 3 - epimerase with an aqueous sodium alginate solution, and stir evenly to obtain a mixed solution;

[0049] (2) Add a CaCl₂ solution dropwise to the mixed solution obtained in step (1) for immobilization to obtain the immobilized enzyme.

[0050] In some more preferred embodiments, the concentration of the sodium alginate is 2.5% (g / mL).

[0051] In some more preferred embodiments, the volume ratio of the crude enzyme solution to the aqueous sodium alginate solution is 1:10.

[0052] In some specific embodiments, the crude enzyme solution in step (1) is prepared by the following steps:

[0053] (a) Cultivate the engineering bacterium B.subtilis 168 expressing D - allulose 3 - epimerase in an LB medium to obtain a seed solution;

[0054] (b) Inoculate the seed solution obtained in step (a) into a TB medium for cultivation, and take the supernatant to obtain the crude enzyme solution.

[0055] The eighth aspect of the present invention provides an application of D - allulose 3 - epimerase in reducing the content of allulose in fructose - containing beverages;

[0056] The D - allulose 3 - epimerase is the D - allulose 3 - epimerase derived from Ruminiclostridium papyrosolvens.

[0057] In some preferred embodiments, the D-psicose 3-epimerase comprises the amino acid sequence shown in SEQ ID NO:2.

[0058] Based on common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0059] The reagents and raw materials used in the present invention are all commercially available.

[0060] The positive and progressive effects of the present invention are as follows:

[0061] The D-psicose 3-epimerase of the present invention has high enzyme activity and still retains a relatively high conversion rate of psicose at low pH; the genetically engineered bacterium capable of efficiently secreting this enzyme can be directly used for reducing sugar in fruit juice, avoiding changes in flavor and nutritional components caused by adding pH regulators such as baking soda. Detailed implementation mode

[0062] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0063] The pMA5 plasmid was purchased from Biofeng.

[0064] High-fidelity enzyme Flash KOD Dye Mix was purchased from Tianlu Diagnostics; IIOne Step Cloning Kit was purchased from Novizan; DpnI endonuclease was purchased from Thermo Fisher Scientific; UE DNA Gel Extraction Kit was purchased from Youyiland; Plasmid Mini Extraction Kit was purchased from Tianlu Diagnostics.

[0065] Antibiotic usage concentration: The usage concentration of KanR (kanamycin) is 50 μg / mL; the usage concentration of Amp (ampicillin) is 100 μg / mL.

[0066] TB medium: 10 g of tryptone, 18 g of yeast extract, 2.31 g of KH2PO4 (potassium dihydrogen phosphate), 16.43 g of K2HPO4 (dipotassium hydrogen phosphate) and 4 mL of glycerol, made up to 1000 mL with deionized water.

[0067] Competent cell preparation medium for Bacillus subtilis:

[0068] Preparation of SPI-A solution: Weigh 0.4 g of ammonium sulfate ((NH4)2SO4), 1.2 g of potassium dihydrogen phosphate (KH2PO4), 2.8 g of dipotassium hydrogen phosphate anhydrous (K2HPO4), and 0.2 g of sodium citrate trihydrate (C6H5Na3O7·3H2O), add deionized water and make up the volume to 100 mL.

[0069] Preparation of SPI-B solution: Weigh 0.04 g of magnesium sulfate heptahydrate (MgSO4·7H2O), add deionized water and make up the volume to 100 mL.

[0070] Preparation of 100×CAYE solution: Weigh 0.2 g of casein hydrolysate and 1 g of yeast extract, inject deionized water and make up the volume to 10 mL.

[0071] Preparation of SPI solution: Take 98 mL of SPI-A solution, 98 mL of SPI-B solution, 2 mL of 50% glucose solution, and 2 mL of 100×CAYE solution, and mix well.

[0072] Preparation of SPII solution: Take 98 mL of SPI, 1 mL of 50 mmol·L -1 calcium chloride (CaCl2) solution, and 1 mL of 250 mmol·L -1 magnesium chloride hexahydrate (MgCl2·6H2O) solution, and mix well.

[0073] Preparation of 100×EGTA (ethylene glycol bis(α-aminoethyl ether) tetraacetic acid): Weigh 0.38 g of EGTA, dissolve it in deionized water, adjust the pH to 8.0, and make up the volume to 10 mL. All culture media need to be autoclaved at 121 °C for 15 - 20 min.

[0074] Example 1: Screening and enzyme activity determination of D-allulose 3-epimerase

[0075] 1. Construction of plasmid expressing D-allulose 3-epimerase

[0076] D-allulose 3-epimerase DPE001 is derived from Ruminiclostridium papyrosolvens (NCBI accession number: WP_020816257.1), and its nucleotide sequence is SEQ ID NO:1; D-allulose 3-epimerase DPE002 is derived from Synergistaceae bacterium (NCBI accession number: MBQ9564196.1), and its nucleotide sequence is SEQID NO:3.

[0077] Table 1 Primer table for plasmid construction

[0078]

[0079]

[0080] The primers designed according to Table 1 (synthesized by Tsingke) were used to specifically amplify each fragment with the template plasmid, and the high-fidelity enzyme of Tianluo Diagnostic Company was used. Flash KOD Dye Mix was used for the PCR reaction, and the reaction system was as follows (Table 2):

[0081] Table 2 PCR amplification reaction system

[0082] Reagent PCR Reaction System (50 μL) cDNA 1 μL Primer F 1 μL Primer R 1 μL PCR Mix 25 μL <![CDATA[ddH2O]]> 22 μL

[0083] The PCR amplification program was as follows (Table 3):

[0084] Table 3 PCR reaction program

[0085]

[0086] 5 μL of the amplification product was taken for 1% agar electrophoresis to detect the amplification result.

[0087] (2) Multi-fragment recombination technology was used to construct pMA5-PHpall-pylb-DPE001 and pMA5-PHpall-pylb-DPE002 plasmids. The constructed plasmids pMA5-PHpall-pylb-DPE001 and pMA5-PHpall-pylb-DPE002 were respectively introduced into the competent cell B. subtilis 168 (purchased from biobw) to obtain B. subtilis 168 / pMA5-PHpall-pylb-DPE001 (named strain BSD001) and B. subtilis 168 / pMA5-PHpall-pylb-DPE002 (named strain BSD002).

[0088] 2. Fermentation and enzyme activity determination of strains BSD001 and BSD002

[0089] The above strains BSD001, BSD002 and 1A751-DPE (the source patent number of this strain: CN 116057183A patent, obtained as a gift from Anhui Jinhe Industry Co., Ltd.) were inoculated into LB seed medium and cultured at 37 °C and 200 rpm for 14 - 16 h, and then inoculated into TB fermentation medium at an inoculation amount of 2 - 5% and cultured at 37 °C and 200 rpm for 24 h. Samples were taken to measure the enzyme activity of D-allulose 3-epimerase in the fermentation broth.

[0090] The measurement method is as follows: In a 1 mL reaction system, the final concentration of fructose is 100 g / L, the buffer is phosphate buffer (10 mM, pH 6), 10 μL of appropriately diluted crude fermentation enzyme solution is added, and Mn with a final concentration of 1 mM is added. 2+ It is incubated at 35 °C for 10 min, and then boiled for 5 min to terminate the enzyme reaction. The amount of D - psicose produced is detected by HPLC, and the enzyme activity is calculated. The results are shown in Table 4.

[0091] Definition of unit enzyme activity: 1 U enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of D - psicose per minute.

[0092] Table 4 Enzyme activities of enzymes produced by strains BSD001, BSD002 and 1A751 - DPE

[0093] Strain Name DPE Enzyme Number Enzyme Activity (U / mL) BSD001 DPE001 489.78 BSD002 DPE002 351.37 1A751-DPE DPE003 320.58

[0094] The enzyme activities of D - psicose 3 - epimerase expressed in Bacillus subtilis from different sources are different, and among them, the enzyme activity of D - psicose 3 - epimerase in the fermentation broth of strain BSD001 is the highest.

[0095] Using the same method, when the pH of the reaction system is 4, the activity of the above - mentioned D - psicose 3 - epimerase is measured. The detection results show that the enzyme activity of DPE001 is 272.21 U / mL, while the enzyme activities of DPE002 and DPE003 are almost 0. Thus, it can be seen that the D - psicose 3 - epimerase prepared by fermenting strain BSD001 has strong acid resistance.

[0096] Example 2: Immobilized D - psicose 3 - epimerase

[0097] The fermentation broth prepared in Example 1 is centrifuged at 2000 rpm for 20 min, and the supernatant is taken to obtain the D - psicose 3 - epimerase enzyme solution. It is added to a 2.5% (g / mL) sodium alginate aqueous solution according to a volume ratio of 1:10 and stirred evenly. Stand still or use ultrasound to remove the bubbles in the mixed solution. Using a 50 mL syringe with a needle and a LongerPump device, the above - mentioned mixed solution is dropped into 3 times the volume of 4% (g / mL) CaCl2 solution. At the same time, a small aeration pump or regular stirring of the immobilized beads in the CaCl2 solution is carried out to make the beads fully contact with the CaCl2 solution. It is placed overnight at 4 °C, and then filtered to obtain the immobilized D - psicose 3 - epimerase.

[0098] Example 3: Determination of the pH range for using immobilized D - psicose 3 - epimerase

[0099] The immobilized D - psicose 3 - epimerase DPE001 prepared by the method of Example 2 was placed in reaction systems with different pH values (adjusted by citrate buffer), and its enzyme activity was measured.

[0100] Measurement method: In a 1 - mL reaction system with different pH values, the final concentration of fructose was 100 g / L. 1 g of immobilized D - psicose 3 - epimerase was added, and Mn with a final concentration of 1 mM was added. 2+ It was incubated at 35 °C for 10 min, and then boiled for 5 min to terminate the enzyme reaction. The amount of D - psicose produced was detected by HPLC, and the enzyme activity was calculated. The enzyme activity was the highest when the pH was 7. Taking this enzyme activity as 100%, the relative enzyme activities under other pH conditions were calculated according to the enzyme activity. The results are shown in Table 5.

[0101] The definition of enzyme activity is: 1 U of enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of D - psicose per minute.

[0102] From the reaction results of fructose and immobilized D - psicose 3 - epimerase under different pH conditions, it can be concluded that the immobilized D - psicose 3 - epimerase has relatively high activity under the conditions of pH 5 - 7; when the pH is 4, the enzyme activity decreases significantly, but still retains more than 77.2% of the relative enzyme activity; when the pH is reduced to 2, the enzyme activity is only 15.6% of the enzyme activity under the condition of pH 7. This shows that the immobilized DPE001 can maintain relatively high enzyme activity within a relatively wide pH range.

[0103] Table 5 Relative enzyme activities of immobilized D - psicose 3 - epimerase under different pH conditions

[0104]

[0105] Example 4: Application of immobilized D - psicose 3 - epimerase in reducing sugar content of orange juice

[0106] The immobilized D - psicose 3 - epimerase DPE001 prepared by the method of Example 2 was added to 100 g of pure orange juice (pH 3.5) at different addition amounts (mass ratio, g / g) respectively, placed in a shaker at 35 °C, and shaken and reacted for 4 h at a rotation speed of 200 rpm. After the reaction product was diluted by an appropriate multiple, the enzyme was removed in a metal bath at 98 °C for 7 min. After filtration, the contents of fructose and D - psicose before and after the reaction were detected by HPLC, and the D - fructose conversion rate was calculated. The content and conversion rate of D - psicose in pure orange juice after the reaction of immobilized D - psicose 3 - epimerase with different addition amounts are shown in Table 6.

[0107] In addition, DPE002 and DPE003 were immobilized using the immobilization method described in Example 2, and added to pure orange juice (pH 3.5) at an addition amount of 20% (mass ratio, g / g). Other conditions were the same as above. After the reaction ended, no D - allulose was detected.

[0108] Table 6 Conversion of fructose to D - allulose after the reaction of immobilized DPE001 with pure orange juice at different addition amounts

[0109]

[0110]

[0111] Example 5: Application of immobilized D - allulose 3 - epimerase in different fruit juices

[0112] The immobilized D - allulose 3 - epimerase DPE001 prepared by the method described in Example 2 was added to 100 g of different fruit juices at an addition amount of 18% (mass ratio, g / g) respectively, placed in a shaker at 35 °C, and oscillated and reacted for 4 h under the condition of a rotation speed of 200 rpm. After the reaction product was diluted by an appropriate multiple, the enzyme was removed in a metal bath at 98 °C for 7 min. After filtration, the contents of fructose and D - allulose before and after the reaction were detected by HPLC, and the D - fructose conversion rate was calculated. The conversion reactions were carried out with pear juice, apple juice, grape juice, peach juice and mango juice respectively. The contents of D - allulose and the conversion rates in the fruit juices after the reaction are shown in Table 7.

[0113] Table 7 Conversion of fructose to D - allulose in different fruit juices

[0114]

[0115] As can be seen from the results, the immobilized D - allulose 3 - epimerase can effectively convert fructose in different fruit juices into D - allulose to obtain low - fructose and healthy fruit juice products.

[0116] The sequences used in the present invention are shown in Table 8.

[0117] Table 8 Sequences used in the present invention

[0118]

[0119]

[0120]

Claims

1. A method for reducing the fructose content in a fructose-containing beverage, characterized in that, Mix the fructose-containing beverage with D-allulose 3-epimerase for reaction; The D-allulose 3-epimerase is the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens; Preferably, the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens comprises the amino acid sequence shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein The fructose-containing beverage is pure fruit juice, for example, orange juice, pear juice, apple juice, grape juice, peach juice and mango juice; And / or, the D-allulose 3-epimerase is an immobilized enzyme; Preferably, the reaction satisfies one or more of the following conditions: The addition amount of the immobilized enzyme is 15-20%, and % is the mass ratio of the immobilized enzyme to the fructose-containing beverage; The pH of the reaction is 3-7; The temperature of the reaction is 35-37 °C; The reaction time is 2-5 h, for example 4 h; and, The reaction is carried out under shaking conditions, and the rotation speed is 150-250 rpm.

3. The method according to claim 2, characterized in that The immobilized enzyme is prepared by the following steps: (1) Mix the crude enzyme solution of D-allulose 3-epimerase with an aqueous sodium alginate solution, and stir evenly to obtain a mixed solution; (2) Dropwise add a CaCl2 solution to the mixed solution obtained in step (1) for immobilization to obtain the immobilized enzyme; Preferably, the concentration of the sodium alginate is 2.5% (g / mL), and / or the volume ratio of the crude enzyme solution to the aqueous sodium alginate solution is 1:

10.

4. The method according to claim 3, characterized in that The crude enzyme solution in step (1) is prepared by the following steps: (a) Cultivate the engineering bacterium B.subtilis 168 expressing D-allulose 3-epimerase in LB medium to obtain a seed solution; (b) Inoculate the seed solution in step (a) into TB medium for cultivation, and take the supernatant to obtain the crude enzyme solution.

5. A low-fructose beverage, characterized in that, The low-fructose beverage is prepared by the method described in any one of claims 1-4; the low-fructose beverage is low-fructose fruit juice; Preferably, the low-fructose beverage contains allulose, and the content of allulose is 5-20 g / L; and / or the low-fructose fruit juice is prepared from pure fruit juice, for example, orange juice, pear juice, apple juice, grape juice, peach juice and mango juice.

6. A low-fructose orange juice, characterized in that, The low-fructose orange juice is prepared by the method described in any one of claims 1-4; Preferably, the low-fructose orange juice contains allulose and fructose, the content of allulose is 5-7 g / L, and the content of fructose is 18-20 g / L.

7. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium is B.subtilis 168 expressing D-allulose 3-epimerase; The D-allulose 3-epimerase is the D-allulose 3-epimerase derived from Ruminiclostridium papyrosolvens; Preferably, the D-psicose 3-epimerase derived from Ruminiclostridium papyrosolvens comprises the amino acid sequence shown in SEQ ID NO:

2.

8. A method for culturing the genetically engineered bacterium as described in claim 7, characterized in that, The method includes culturing the genetically engineered bacterium in a seed medium and then transferring it to a fermentation medium for fermentation. Preferably, the culturing satisfies one or more of the following conditions: The seed medium is LB medium. The culturing temperature is 35-40 °C, such as 37 °C. The culturing is carried out with shaking, and the rotation speed of the shaking is 180-220 rpm, such as 200 rpm. The culturing time is 14-18 h, such as 14-16 h; and / or The fermentation satisfies one or more of the following conditions: The fermentation medium includes tryptone, yeast extract, KH2PO4, K2HPO4 and glycerol. The fermentation temperature is 35-40 °C, such as 37 °C. The fermentation time is 20-26 h, such as 24 h.

9. Use of the genetically engineered bacterium according to claim 7 in the preparation of D-psicose 3-epimerase or in reducing the content of psicose in a fructose-containing beverage.

10. An immobilized enzyme, characterized in that, The immobilized enzyme uses sodium alginate-CaCl2 as a carrier to immobilize D-psicose 3-epimerase, and the D-psicose 3-epimerase comprises the amino acid sequence shown in SEQ ID NO:

2. Preferably, the immobilized enzyme is prepared by the following steps: (1) Mix the crude enzyme solution of the D-psicose 3-epimerase with an aqueous sodium alginate solution and stir evenly to obtain a mixed solution. (2) Add a CaCl2 solution dropwise to the mixed solution obtained in step (1) for immobilization to obtain the immobilized enzyme. More preferably, the concentration of sodium alginate is 2.5% (g / mL), and / or the volume ratio of the crude enzyme solution to the aqueous sodium alginate solution is 1:

10. The crude enzyme solution in step (1) is prepared, for example, by the following steps: (a) Culture the engineering bacterium Bacillus subtilis 168 expressing D-psicose 3-epimerase in LB medium to obtain a seed solution. (b) Inoculate the seed solution obtained in step (a) into TB medium for culturing, and take the supernatant to obtain the crude enzyme solution.

11. Use of a D-psicose 3-epimerase in reducing the content of psicose in a fructose-containing beverage. The D-psicose 3-epimerase is the D-psicose 3-epimerase derived from Ruminiclostridium papyrosolvens. Preferably, the D-psicose 3-epimerase comprises the amino acid sequence shown in SEQ ID NO:2.

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