A preparation method of D-tagatose

Abstract

The present invention discloses a method for preparing D-tagatose, comprising the following steps: mixing red algae powder with an organic acid for reaction, and centrifuging to remove solids after the reaction to obtain a red algae acidolysis solution rich in D-galactose; the red algae includes agar-rich red algae of the genus Gelidium or Gracilaria; separating the red algae acidolysis solution through a macroporous adsorption resin, adding solid calcium oxide, and centrifuging to remove solids; adding boric acid, and then adding a sodium hydroxide solution to adjust the pH to weakly alkaline; adding a recombinant bacterium expressing L-arabinose isomerase and MnCl2, and obtaining a solution containing D-tagatose after the reaction. The present invention uses renewable biomass red algae as a raw material to produce D-tagatose, which conforms to the advanced concept of green bio-manufacturing, has low cost and is environmentally friendly; compared with the methods for producing D-tagatose using D-galactose or other raw materials, the D-tagatose production method of the present invention has a higher yield of D-tagatose.

Description

Technical Field

[0001] The invention relates to a method for preparing D-tagatose, and belongs to the field of D-tagatose preparation. Background Art

[0002] D-tagatose is an isomer of D-galactose, a rare ketohexose, and a new functional sweetener. D-tagatose has 92% of the sweetness of sucrose, but only 30% of the calories of sucrose, and a very low glycemic index. It can be used in food, health products, diabetes drugs and other industries. In recent years, the market demand for D-tagatose is strong and the development trend is good.

[0003] The production of D-tagatose mostly uses D-galactose as a substrate, which is isomerized to D-tagatose under the catalysis of chemical catalysts or L-arabinose isomerase (L-AI, EC 5.3.1.4) or recombinant cells containing L-AI. However, no matter which method is used, it faces the problem of low yield of D-tagatose, which increases the production cost of D-tagatose and limits the industrial production process of D-tagatose. In order to reduce costs, relatively cheap raw materials can be used while increasing the yield of D-tagatose. For example, the patent (application number: 2018116104494) prepares D-galactose by hydrolyzing lactose or whey, and the yield of D-tagatose is about 50% based on D-galactose.

[0004] my country has vast sea areas and abundant seaweed resources. Seaweed grows rapidly, has a high carbohydrate content, and is easy to handle. In recent years, it has become a potential resource for the production of biofuels and chemicals. At present, there are reports of using red algae as raw materials for bioethanol production. However, there are no reports on methods for producing D-tagatose using red algae as raw materials while achieving a high yield of D-tagatose. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing high-yield D-tagatose using red algae, a renewable biomass with abundant production in nature, as raw materials.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides a method for producing D-tagatose, comprising the following steps:

[0007] (1) Chemical catalysis: red algae are mixed with organic acids for reaction, and after the reaction is completed, the solids are removed by centrifugation to obtain a red algae acid hydrolyzate rich in D-galactose;

[0008] (2) Substrate preparation: The red algae acid hydrolysate is separated by macroporous adsorption resin to remove byproducts in the acid hydrolysate that affect biocatalysis; solid calcium oxide is added to the acid hydrolysate, and solids are removed by centrifugation after the reaction; boric acid is then added, and then sodium hydroxide solution is added to adjust the pH of the acid hydrolysate, which is used as the substrate for the next step of biocatalysis;

[0009] (3) Biocatalysis: recombinant Escherichia coli BL21 (DE3) expressing L-arabinose isomerase was cultured as a biocatalyst, the substrate prepared in step (2) was mixed with the biocatalyst, and MnCl2 was added. After the reaction was completed, the supernatant was collected by solid-liquid separation to obtain a reaction solution containing D-tagatose.

[0010] In step (1), the red algae is a commercially available red algae rich in agar of the genus Gelidium or Gracilaria, and after being washed and dried, the red algae is crushed to 20-80 mesh; the organic acid is oxalic acid with a mass concentration of 1% to 6%.

[0011] Among them, the red algae of the genus Gelidium is preferably Gelidium amansii, and the red algae of the genus Gracilaria is preferably Gracilaria lemaneiformis.

[0012] Among them, the mass concentration of oxalic acid is preferably 1.5% to 3%.

[0013] In step (1), the solid-liquid ratio of red algae to organic acid is controlled to be 1:5-1:15 g / mL, the reaction temperature is 110-140° C., and the reaction time is 20-50 minutes.

[0014] The reaction temperature is preferably 115 to 125° C., and the reaction time is preferably 30 to 40 minutes.

[0015] In step (2), the macroporous adsorption resin is XAD16N; the amount of solid calcium oxide added is 2:1 to 15:1 g / L acid hydrolyzate; the ratio of the molar concentration of boric acid to the molar concentration of D-galactose is 0.3:1 to 1.8:1; and the substrate pH is 7.0 to 8.5.

[0016] The preferred amount of boric acid added is such that the ratio of its molar concentration to the molar concentration of D-galactose is 0.5:1 to 1.2:1.

[0017] In step (3), the nucleotide sequence of the L-arabinose isomerase is shown in SEQ ID NO.1; the construction method of the recombinant Escherichia coli BL21 (DE3) is as follows: the nucleotide sequence of the L-arabinose isomerase is cloned into an expression plasmid, the expression plasmid is pETDuet-1, to obtain a recombinant plasmid, and the recombinant plasmid is transformed into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli BL21 (DE3).

[0018] In step (3), the concentration of D-galactose in the substrate is 20-60 g / L, the amount of the recombinant bacteria added is 4-16 g dry weight / L, the amount of MnCl2 added is 0.2-2 mM, and the reaction temperature is 45-60°C.

[0019] Among them, the added amount of MnCl2 is preferably 0.5 to 1 mM.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The production of D-tagatose using renewable biomass red algae as raw material is in line with the advanced concept of green biomanufacturing, with low cost and environmental friendliness; 2. Compared with the method of producing D-tagatose using D-galactose or other raw materials, the D-tagatose production method of the present invention has a higher yield of D-tagatose. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the catalytic reaction process described in Example 13. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0023] The detection methods involved in the following embodiments include:

[0024] The method for detecting the components of red algae acid hydrolysate is as follows: Agilent 1200 liquid chromatograph is used for isocratic elution, equipped with a Bio-Rad Aminex HPX-87H (7.8×300mm, Agilent) separation column, column temperature of 55°C, mobile phase of 5mM H2SO4, flow rate of 0.6mL / min, injection volume of 10μL, and a differential refractometer.

[0025] In the biocatalytic experiment, the method for detecting the content of D-galactose and D-tagatose is as follows: Agilent 1200 liquid chromatograph is used for isocratic elution, equipped with Sugar-pak1 (6.5×300mm, Waters) separation column, column temperature 80°C, mobile phase is deionized water, flow rate 0.4mL / min, injection volume 10μL, differential refractometer is used, and the detector temperature is controlled at 35°C.

[0026] The calculation formula of D-tagatose yield is:

[0027] D-tagatose yield (%) = D-tagatose at the end of reaction (g / L) / D-galactose at the start of reaction (g / L).

[0028] Example 1: Preparation of red algae acid hydrolysate using agar-agar chemical catalysis

[0029] Purchase commercially available Gelidium amansii, wash it and dry it naturally, crush it with a grinder, and sieve 20-80 mesh as the acid hydrolysis raw material. Weigh 10g of Gelidium amansii with an absolute dry weight and mix it with 100mL of 2.5% oxalic acid, and keep it at 120°C for 30 minutes. Centrifuge to remove solids to obtain D-galactose-rich red algae acid hydrolyzate. The main substances in the acid hydrolyzate are as follows: D-galactose 37g / L; 5-hydroxymethylfurfural 8.1g / L; formic acid 1.7g / L; levulinic acid 1.4g / L.

[0030] Example 2: Preparation of red algae acid hydrolysate using Gracilaria lemaneiformis chemical catalysis

[0031] Gracilaria lemaneiformis was purchased from the market, washed and dried naturally, crushed with a grinder, and sieved to 20-80 mesh as the acid hydrolysis raw material. 10 g of Gracilaria lemaneiformis with an absolute dry weight was weighed and mixed with 80 mL of 2% oxalic acid, and kept at 120°C for 35 minutes. The solids were removed by centrifugation to obtain a red algae acid hydrolyzate rich in D-galactose. The main substances in the acid hydrolyzate were as follows: D-galactose 45 g / L; 5-hydroxymethylfurfural 11.2 g / L; formic acid 2.1 g / L; levulinic acid 2.2 g / L.

[0032] Example 3: Preparation of red algae acid hydrolysate using agar-agar chemical catalysis

[0033] Purchase commercially available Gelidium amansii, wash it and dry it naturally, crush it with a grinder, and sieve 20-80 mesh as the acid hydrolysis raw material. Weigh 10g of Gelidium amansii with an absolute dry weight and mix it with 50mL of 1% oxalic acid, and keep it at 140℃ for 50 minutes. Centrifuge to remove solids to obtain red algae acid hydrolyzate rich in D-galactose. The main substances in the acid hydrolyzate are as follows: D-galactose 65g / L; 5-hydroxymethylfurfural 12.2g / L; formic acid 2.7g / L; levulinic acid 2.3g / L.

[0034] Example 4: Preparation of red algae acid hydrolysate using Gracilaria lemaneiformis chemical catalysis

[0035] Gracilaria lemaneiformis was purchased from the market, washed and dried naturally, crushed with a grinder, and sieved to 20-80 mesh as the acid hydrolysis raw material. 10 g of Gracilaria lemaneiformis with an absolute dry weight was weighed and mixed with 150 mL of 6% oxalic acid, and kept at 110°C for 20 minutes. The solids were removed by centrifugation to obtain a red algae acid hydrolyzate rich in D-galactose. The main substances in the acid hydrolyzate were as follows: D-galactose 25 g / L; 5-hydroxymethylfurfural 5.7 g / L; formic acid 1.0 g / L; levulinic acid 0.9 g / L.

[0036] Example 5: Preparation of substrate based on Example 1

[0037] The red algae acid hydrolysate in Example 1 was separated using the macroporous adsorption resin XAD16N. First, a glass chromatography column was filled with XAD16N resin, and the red algae acid hydrolysate in Example 1 was pumped into the chromatography column. The speed of the constant flow pump was controlled to be 4 rpm. The D-galactose in the acid hydrolysate had a weak adsorption capacity with the resin, while other components in the acid hydrolysate could be adsorbed to varying degrees. After the red algae acid hydrolysate passed through the resin, the main substance contents were as follows: D-galactose 32 g / L; 5-hydroxymethylfurfural 0 g / L; formic acid 0.3 g / L; levulinic acid 0.05 g / L. Among them, 5-hydroxymethylfurfural, which has side effects on biocatalysis, could not be detected.

[0038] Solid calcium oxide was added to the above acid hydrolyzate according to the mass volume ratio of solid calcium oxide to the acid hydrolyzate after resin treatment of 4:1 g / L, and the reaction was allowed to stand for 2 hours, and the solids were removed by centrifugation at 10,000 rpm for 10 minutes. Boric acid was then added to make its molar concentration the same as that of D-galactose, and sodium hydroxide solution was added to adjust the pH of the acid hydrolyzate to 7.5, which was used as a substrate for the biocatalytic reaction.

[0039] Example 6: Preparation of substrate based on Example 1

[0040] The red algae acid hydrolysate in Example 1 was separated using the macroporous adsorption resin XAD16N. First, a glass chromatography column was filled with XAD16N resin, and the red algae acid hydrolysate in Example 1 was pumped into the chromatography column. The speed of the constant flow pump was controlled to be 4 rpm. The D-galactose in the acid hydrolysate had a weak adsorption capacity with the resin, while other components in the acid hydrolysate could be adsorbed to varying degrees. After the red algae acid hydrolysate passed through the resin, the main substance contents were as follows: D-galactose 32 g / L; 5-hydroxymethylfurfural 0 g / L; formic acid 0.3 g / L; levulinic acid 0.05 g / L. Among them, 5-hydroxymethylfurfural, which has side effects on biocatalysis, could not be detected.

[0041] Solid calcium oxide was added to the above acid hydrolyzate at a mass volume ratio of 2:1 g / L of solid calcium oxide and acid hydrolyzate after resin treatment, and the reaction was allowed to stand for 2 hours, and the solids were removed by centrifugation at 10,000 rpm for 10 minutes. Boric acid was then added to make the ratio of its molar concentration to that of D-galactose 0.3:1, and sodium hydroxide solution was added to adjust the pH of the acid hydrolyzate to 7.0, which was used as a substrate for the biocatalytic reaction.

[0042] Example 7: Preparation of substrate based on Example 2

[0043] The red algae acid hydrolysate in Example 2 was separated using the macroporous adsorption resin XAD16N. First, the glass chromatography column was filled with XAD16N resin, and the red algae acid hydrolysate in Example 2 was pumped into the chromatography column. The speed of the constant flow pump was controlled to 4 rpm. The D-galactose in the acid hydrolysate had a weak adsorption capacity with the resin, while other components in the acid hydrolysate could be adsorbed to varying degrees. After the red algae acid hydrolysate passed through the resin, the main substance contents were as follows: D-galactose 39 g / L; 5-hydroxymethylfurfural 0 g / L; formic acid 0.9 g / L; levulinic acid 0.1 g / L. Among them, 5-hydroxymethylfurfural, which has side effects on biocatalysis, could not be detected.

[0044] Solid calcium oxide was added to the acid hydrolyzate according to the mass volume ratio of solid calcium oxide to the acid hydrolyzate after resin treatment of 4.5:1 g / L, and the reaction was allowed to stand for 3 hours, and the solids were removed by centrifugation at 10,000 rpm for 10 minutes. Boric acid was then added to make its molar concentration the same as that of D-galactose, and sodium hydroxide solution was added to adjust the pH of the acid hydrolyzate to 7.5, which was used as a substrate for the biocatalytic reaction.

[0045] Example 8: Preparation of substrate based on Example 2

[0046] The red algae acid hydrolysate in Example 2 was separated using the macroporous adsorption resin XAD16N. First, the glass chromatography column was filled with XAD16N resin, and the red algae acid hydrolysate in Example 2 was pumped into the chromatography column. The speed of the constant flow pump was controlled to 4 rpm. The D-galactose in the acid hydrolysate had a weak adsorption capacity with the resin, while other components in the acid hydrolysate could be adsorbed to varying degrees. After the red algae acid hydrolysate passed through the resin, the main substance contents were as follows: D-galactose 39 g / L; 5-hydroxymethylfurfural 0 g / L; formic acid 0.9 g / L; levulinic acid 0.1 g / L. Among them, 5-hydroxymethylfurfural, which has side effects on biocatalysis, could not be detected.

[0047] Solid calcium oxide was added to the acid hydrolyzate at a mass volume ratio of 15:1 g / L between solid calcium oxide and the acid hydrolyzate after resin treatment, and the reaction was allowed to stand for 3 hours, and the solids were removed by centrifugation at 10,000 rpm for 10 minutes. Boric acid was then added to a molar concentration ratio of 1.8:1 to that of D-galactose, and sodium hydroxide solution was added to adjust the pH of the acid hydrolyzate to 8.5, which was used as a substrate for the biocatalytic reaction.

[0048] Example 9: Construction of recombinant Escherichia coli BL21 (DE3) expressing L-arabinose isomerase

[0049] The L-arabinose isomerase encoding gene sequence was chemically synthesized, and its gene nucleotide sequence is shown in SEQ ID NO. 1. A pair of primers was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. for large-scale amplification of the L-arabinose isomerase encoding gene.

[0050] The primer sequences are as follows:

[0051] Upstream primer 5'-CGC GGATCC GATGTTGAAAATAAAAGA-3' (SEQ ID NO. 2), the underline is the BamH I site;

[0052] Downstream primer 5'-CCG GAATTC TCAAATTTTTACAGTTTTAA-3' (SEQ ID NO. 3), the underline indicates the EcoRI site.

[0053] The PCR reaction system was: PrimeSTAR Max Premix 50 μL (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.), upstream primer 2 μL, downstream primer 2 μL, template DNA 2 μL, and sterile water 44 μL.

[0054] The PCR reaction parameters were: 98°C for 5 min; 98°C for 10 s, 60°C for 15 s, and 72°C for 20 s, for a total of 30 cycles; 72°C for 5 min.

[0055] The gene fragment obtained by PCR amplification was cloned into the BamH I and EcoR I restriction sites of the E. coli expression vector pETDuet-1 using conventional molecular cloning methods to obtain a recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) using conventional E. coli transformation methods, and the resulting strain was a recombinant E. coli BL21 (DE3) expressing L-arabinose isomerase.

[0056] Example 10: Preparation of biocatalyst

[0057] The recombinant E. coli in Example 9 was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin at an inoculum size of 1% (v / v), cultured at 37°C with shaking for 12 h, and then transferred to 500 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking until the cell OD 600 When the pH value reaches 0.6, IPTG with a final concentration of 0.5 mM is added to induce the expression of L-arabinose isomerase, and the mixture is shaken and cultured at 25° C. for 8 h. After the induction, the mixture is centrifuged at 10,000 rpm for 5 minutes, the cell precipitate is collected, and the cell precipitate is washed twice with physiological saline to obtain the biocatalyst used in the present invention.

[0058] Example 11: Preparation of D-tagatose using the substrate in Example 5

[0059] The total reaction system was 100 mL. The biocatalyst prepared in Example 10 was added to the substrate solution prepared in Example 5. The biocatalyst concentration was adjusted to 12 g dry weight / L. MnCl2 was added at a final concentration of 0.5 mM. The pH of the reaction system was 7.5. After mixing, the reaction was shaken at 50°C for 18 hours. After the reaction was completed, the reaction mixture was centrifuged and filtered, and the amount of D-tagatose produced was detected by HPLC. The results showed that the yield of D-tagatose was 24.1 g / L, and the yield was 75%.

[0060] Example 12: Preparation of D-tagatose using the substrate in Example 6

[0061] The total reaction system was 100 mL. The biocatalyst prepared in Example 10 was added to the substrate solution prepared in Example 6, the biocatalyst concentration was adjusted to 4 g dry weight / L, MnCl2 was added at a final concentration of 0.2 mM, and the pH of the reaction system was 7.0. After mixing, the reaction was shaken at 55°C for 60 hours. After the reaction was completed, the reaction mixture was centrifuged and filtered, and the amount of D-tagatose produced was detected by HPLC. The results showed that the yield of D-tagatose was 24.2 g / L, and the yield was 75.6%.

[0062] Example 13: Preparation of D-tagatose using the substrate in Example 7

[0063] The total reaction system was 80 mL. The biocatalyst prepared in Example 10 was added to the substrate solution prepared in Example 7. The biocatalyst concentration was adjusted to 12 g dry weight / L. MnCl2 was added to a final concentration of 0.8 mM. The pH of the reaction system was 7.5. After mixing, the mixture was shaken at 50°C for 18 hours. The concentration changes of D-galactose and D-tagatose were detected in real time. The results are as follows: Figure 1 As shown, D-galactose gradually catalyzes the reaction to produce D-tagatose (galactose in the figure represents D-galactose, and tagatose represents D-tagatose). After the reaction is completed, the reaction mixture is centrifuged and filtered, and the amount of D-tagatose produced is detected by HPLC. The results show that the yield of D-tagatose is 29.6 g / L, and the yield is 76%.

[0064] Example 14: Preparation of D-tagatose using the substrate in Example 8

[0065] The total reaction system was 80 mL. The biocatalyst prepared in Example 10 was added to the substrate solution prepared in Example 8, the biocatalyst concentration was adjusted to 16 g dry weight / L, MnCl2 was added at a final concentration of 2 mM, and the pH of the reaction system was 8.5. After mixing, the reaction was shaken at 50°C for 15 hours. After the reaction was completed, the reaction mixture was centrifuged and filtered, and the amount of D-tagatose produced was detected by HPLC. The results showed that the yield of D-tagatose was 29.7 g / L, and the yield was 76%.

[0066] Comparative Example 1

[0067] In an 80mL reaction system, the concentration of commercial D-galactose (Sigma, item number G0750) is 39g / L, boric acid is added to make its molar concentration the same as that of D-galactose, and sodium hydroxide solution is added to adjust the solution pH to 7.5 as a substrate for the biocatalytic reaction. Then, the biocatalyst prepared in Example 10 is added, the concentration of the biocatalyst is adjusted to 12g dry weight / L, MnCl2 is added at a final concentration of 0.8mM, and the pH of the reaction system is controlled to 7.5. After mixing, the reaction is shaken at 50°C for 18 hours. After the reaction is completed, the reaction mixture is centrifuged and filtered, and the amount of D-tagatose generated is detected by HPLC. The results show that the yield of D-tagatose is 24.6g / L, and the yield is 63%.

[0068] Comparative Example 2

[0069] Reference Example 7: The red algae acid hydrolysate in Example 2 was separated using macroporous adsorption resin XAD16N.

[0070] The difference from Example 7 is that calcium oxide is not added to the acid hydrolyzate after passing through the column, boric acid is first added to make its molar concentration the same as the molar concentration of D-galactose, and then sodium hydroxide solution is added to adjust the pH of the acid hydrolyzate to 7.5, which serves as a substrate for the biocatalytic reaction.

[0071] Then, biocatalysis was carried out according to the method described in Example 13, and all conditions were the same as in Example 13. After the reaction was completed, the reaction mixture was centrifuged and filtered, and the amount of D-tagatose produced was detected by HPLC. The results showed that the yield of D-tagatose was 25.7 g / L, and the yield was 66%.

[0072] Comparative Example 3

[0073] Prepare the algae acid hydrolysate according to Example 2. Before passing through the column, solid calcium oxide was added to the algae acid hydrolysate at a mass volume ratio of solid calcium oxide to algae acid hydrolysate of 4.5:1 g / L, and the reaction was allowed to stand for 3 hours, and the solids were removed by centrifugation at 10,000 rpm for 10 minutes. The algae acid hydrolysate was then separated using the macroporous adsorption resin XAD16N. First, a glass chromatography column was filled with XAD16N resin, and the algae acid hydrolysate after the solids were removed was pumped into the chromatography column, and the speed of the constant flow pump was controlled to 4 rpm. After the algae acid hydrolysate passed through the resin, the main substance contents were as follows: D-galactose 9.1 g / L; 5-hydroxymethylfurfural 0 g / L; formic acid 0.2 g / L; levulinic acid 0 g / L. At this time, the loss of D-galactose was serious.

[0074] In summary, the D-tagatose production method provided by the present invention uses renewable biomass as raw material, has a simple preparation method, mild reaction conditions, and a high product yield, and has a good application prospect.

Claims

1. A method for preparing D-tagatose, characterized in that: The following steps are involved: (1) mixing red algae powder and organic acid for reaction, and after the reaction is completed, centrifuging to remove solids to obtain a red algae acid hydrolyzate rich in D-galactose; the red algae is agar-rich red algae of the genus Gelidium or Gracilaria; the organic acid is oxalic acid with a mass concentration of 1% to 6%; the solid-liquid ratio of the red algae powder to the organic acid is 1:5 to 1:15 g / mL; the reaction temperature is 110 to 140° C., and the reaction time is 20 to 50 minutes; (2) The red algae acid hydrolysate in step (1) is separated by a macroporous adsorption resin, solid calcium oxide is added, and solids are removed by centrifugation; boric acid is added, and then sodium hydroxide solution is added to adjust the pH to a weak alkaline state to obtain a biocatalytic reaction substrate solution; the mass volume ratio of the solid calcium oxide to the acid hydrolysate after resin separation is 2:1-15:1 g / L; the molar concentration ratio of the boric acid to the D-galactose in the red algae acid hydrolysate is 0.3:1-1.8:1; and the pH is 7.0-8.5; (3) Adding recombinant bacteria expressing L-arabinose isomerase and MnCl2 to the biocatalytic reaction substrate solution in step (2) to obtain a solution containing D-tagatose after the reaction is completed; the amount of the recombinant bacteria added is 4-16 g dry weight / L; the amount of MnCl2 added is 0.2-2 mM; and the reaction temperature is 45-60°C.

2. The preparation method according to claim 1, characterized in that: The red algae powder in step (1) is 20-80 mesh.

3. The preparation method according to claim 1, characterized in that: The nucleotide sequence encoding the L-arabinose isomerase in step (3) is shown in SEQ ID NO.1.

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