Preparation method for agarotriose

Abstract

A preparation method for agarotriose, belonging to the field of agar oligosaccharide preparation. A method of, in optimal enzymolysis temperature conditions, pre-hydrolyzing agar with an acid for a short time, then neutralizing and performing enzymolysis, is used to achieve specific and efficient preparation of agarotriose. Compared with methods of preparing odd-numbered agar oligosaccharides by acid hydrolysis, the reaction conditions are mild and easy to control, and the influence of the viscosity of the system rising after the agar is cooled down after high temperature treatment on subsequent enzymolysis is reduced. Compared with methods of preparing odd-numbered agar oligosaccharides by enzymolysis, substrate concentration is high, and α-agarase, the main enzymolysis product of which contains agarotriose, is used, so that the yield of agarotriose is significantly increased, which can provide a certain theoretical basis for the industrial preparation of functional agar oligosaccharides, and is applicable to the fields of food, cosmetics and pharmaceuticals.

Description

A preparation method of agarotriose Technical Field

[0001] The invention relates to a method for preparing agarotriose, and belongs to the field of agar oligosaccharide preparation. Background Art

[0002] Agar is a colloidal polysaccharide found in the cell walls of red algae, composed of ionic thioagar and neutral agarose. The former is primarily composed of a series of complex polysaccharide chains, while the latter is a chain-like linear sugar with alternating β-1,4- and α-1,3-glycosidic bonds, and is the primary component of agar in its gel-like state. Agar oligosaccharides, derived from agarose, generally refer to oligosaccharides with a degree of polymerization of 2 to 10 formed by the hydrolysis of agar polysaccharides. They can be divided into agar oligosaccharides and neo-agarose oligosaccharides based on the difference in their non-reducing ends. Numerous studies have shown that agar oligosaccharides possess broad application prospects in the food, cosmetics, pharmaceutical, and other industries due to their various physiological activities, including anti-inflammatory, hepatoprotective, prebiotic, and antioxidant properties. However, agar oligosaccharides have not yet been industrialized.

[0003] Agar oligosaccharide preparation methods primarily include acid degradation and biodegradation. While the acid degradation method offers advantages such as high substrate concentration and high oligosaccharide yield, it also presents challenges such as product inhomogeneity, loss of oligosaccharide activity, and high energy consumption during high-temperature acid hydrolysis. Furthermore, high-temperature acid hydrolysis of agar can lead to a rebound in viscosity after a short cooling period. Biodegradation, also known as enzymatic hydrolysis, utilizes agarase to specifically cleave the glycosidic bonds of agar polysaccharides. This method offers advantages such as easily controlled reaction conditions, high product specificity, and a green process. However, this method also results in relatively low substrate concentrations and low product yields.

[0004] Summary of the Invention

[0005] To address the issues of uneven product quality, easy loss of activity, and high energy consumption associated with high-temperature acid hydrolysis during the acid hydrolysis process, as well as the low substrate concentrations associated with enzymatic hydrolysis, which hinder the industrial production of functional agar oligosaccharides on a large scale, the present invention utilizes a method that efficiently produces agarotriose by controlling the acid hydrolysis conditions under optimal enzymatic hydrolysis temperature. This method effectively combines the advantages of both acid and enzymatic methods while minimizing the impact of the viscosity increase caused by cooling the agar after high-temperature treatment on subsequent enzymatic hydrolysis. This method is of great significance for the industrial production of agar oligosaccharides and the expansion of technologies for the specific preparation of agar oligosaccharides.

[0006] To address at least one of the above problems, the present invention uses acid hydrolysis followed by enzymatic hydrolysis to hydrolyze agar to prepare agar oligosaccharides, significantly improving the product yield. At the same time, the present invention uses agar as a substrate, hydrochloric acid as a solvent, and sodium hydroxide as a neutralizing agent, effectively reducing industrial costs. Furthermore, the present invention utilizes α-agarase, whose end product of enzymatic hydrolysis contains agarose, and uses a mild optimal enzymatic hydrolysis temperature of 40°C as the reaction temperature. By optimizing the substrate concentration, the hydrochloric acid concentration of the acid pretreatment, the acid hydrolysis time, and the amount of enzyme added, the yield of agarose is effectively increased, offering advantages in terms of process simplification, energy reduction, and environmental protection. Furthermore, the present invention uses high-performance anion exchange chromatography and ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry to analyze the product.

[0007] The first object of the present invention is to provide a method for efficiently preparing agarotriose using α-agarase, comprising the following steps:

[0008] (1) adding agar to a dilute hydrochloric acid solution, mixing uniformly, and obtaining a mixed solution for reaction;

[0009] (2) taking an appropriate amount of the mixed solution of step (1) and adjusting the pH with NaOH;

[0010] (3) adding crude α-agarase enzyme solution to the acid hydrolysis neutralization solution in step (2) for further reaction;

[0011] (4) The product obtained in step (3) is concentrated and purified, and then analyzed using a high performance anion exchange chromatography and an ultra-high performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometer.

[0012] In one embodiment of the present invention, the mass percentages of the substances in the agar acid hydrolyzate in step (1) are 10-20% agar and 90-80% dilute hydrochloric acid solution, and the total is 100%.

[0013] In one embodiment of the present invention, the method comprises the following steps:

[0014] (1) Weigh agar, add it to a hydrochloric acid solution, stir it at 30-50°C and 300-400 rpm for 10-280 minutes, and mix it evenly to obtain an agar acid solution;

[0015] (2) adjusting the pH of the agar acid hydrolysis solution prepared in step (1) to 7.0 to 9.0 to obtain an agar acid hydrolysis neutralization solution;

[0016] (3) The agar acid hydrolyzed neutralized solution obtained in step (2) is kept warm at 30-50° C. and 150-250 rpm for 10-50 min, and crude α-agarase enzyme solution is added thereto for enzymatic hydrolysis for 8-24 h. The acid hydrolyzed solution containing agarose is obtained by boiling for 10-30 min to inactivate the enzyme.

[0017] In one embodiment of the present invention, the concentration of hydrochloric acid in the agar acid hydrolyzate in step (1) is 0.25-3.00M.

[0018] In one embodiment of the present invention, the concentration of the agar acid hydrolysis solution is 10-20% (w / w).

[0019] In one embodiment of the present invention, the mixing in step (1) is carried out by stirring at 40° C. and 300-400 rpm for 10-280 min.

[0020] In one embodiment of the present invention, the concentration of NaOH in step (2) is 6M.

[0021] In one embodiment of the present invention, the α-agarase in step (3) is α-AGA (Cm-AGA) derived from Catenovulum maritimum, and the end products of the degradation of agar by the enzyme include agarotriose and agaropentaose.

[0022] In one embodiment of the present invention, the nucleotide sequence encoding the α-agarase is shown as SEQ ID NO.1.

[0023] In one embodiment of the present invention, the amount of α-agarase Cm-AGA added in step (3) is 10 to 60 U / g, and the enzymatic hydrolysis time is 8 to 24 hours.

[0024] In one embodiment of the present invention, step (2) is to adjust the pH of the agar acid hydrolysis solution to 8.0 using NaOH to obtain an agar acid hydrolysis neutralization solution.

[0025] In one embodiment of the present invention, the added amount of the α-agarase is 40 to 60 U / g.

[0026] In one embodiment of the present invention, the concentration and purification in step (4) is performed by freeze-drying first and then purification using a Superdex 30 Increase 10 / 300GL gel chromatography column.

[0027] In one embodiment of the present invention, the product analysis in step (4) is performed using a high performance anion exchange chromatograph or an ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometer.

[0028] In one embodiment of the present invention, the method comprises the following steps:

[0029] (1) Weigh agar, add it to a 2 M hydrochloric acid solution, and stir at 40°C and 380 rpm for 40 min to mix evenly to obtain a 15% (w / w) agar acid solution;

[0030] (2) adjusting the pH of the agar acid hydrolysis solution prepared in step (1) to 8.0 to obtain an agar acid hydrolysis neutralization solution;

[0031] (3) The agar acid hydrolyzed neutralized solution obtained in step (2) was kept warm at 40°C and 180 rpm for 30 min, and 40 U / g α-agarase crude enzyme solution was added thereto for enzymatic hydrolysis for 12 h. The acid hydrolyzed solution containing agarose was obtained after boiling for 15 min to inactivate the enzyme.

[0032] The second object of the present invention is agarose prepared by the method of the present invention.

[0033] The third object of the present invention is the application of agarose in the fields of food, cosmetics and pharmaceuticals. Beneficial effects

[0034] The present invention utilizes a method that pre-hydrolyzes agar with acid for a short period of time, then neutralizes it, and then further enzymatically hydrolyzes it under optimal enzymatic hydrolysis temperature conditions, achieving the specific and efficient production of agarotriose. Compared to methods for preparing odd-numbered agar oligosaccharides by acid hydrolysis, the present invention offers milder and more controllable reaction conditions, while also minimizing the impact of the subsequent enzymatic hydrolysis on the viscosity of the system caused by cooling the agar after high-temperature treatment. Compared to methods for preparing odd-numbered agar oligosaccharides by enzymatic hydrolysis, the present invention operates at a higher substrate concentration and utilizes α-agarase, whose enzymatic hydrolysis end product contains agarotriose, significantly increasing agarotriose yield. This method provides a theoretical basis for the industrial production of functional agar oligosaccharides and is suitable for use in the food, cosmetics, and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 is a graph showing the HPAEC-PAD results of A3 prepared by hydrochloric acid pretreatment combined with enzymatic hydrolysis in Example 2.

[0036] FIG2 is a graph showing the yield of A3 prepared by acid hydrolysis combined with enzymatic hydrolysis under different substrate concentrations in Example 3.

[0037] FIG3 is a graph showing the yield of A3 prepared by acid hydrolysis combined with enzymatic hydrolysis under different hydrochloric acid concentrations in Example 4.

[0038] FIG4 is a graph showing the yield of A3 prepared by acid hydrolysis combined with enzymatic hydrolysis under different hydrochloric acid hydrolysis time conditions in Example 5.

[0039] FIG5 is a graph showing the yield of A3 prepared by acid hydrolysis combined with enzymatic hydrolysis under different enzyme addition conditions after acid pretreatment in Example 6.

[0040] FIG6 is a graph showing the yield of A3 prepared by acid hydrolysis combined with enzymatic hydrolysis under different enzymatic hydrolysis time conditions after acid pretreatment in Example 7.

[0041] FIG7 is a graph showing the HPAEC-PAD results of purified A3 obtained by combining short-term hydrochloric acid hydrolysis with Cm-AGA enzymatic hydrolysis of agar in Example 8.

[0042] FIG8 is a graph showing the UPLC-Q-TOF-MS results of purified A3 obtained by combining short-term hydrochloric acid hydrolysis with Cm-AGA enzymatic hydrolysis of agar in Example 8. DETAILED DESCRIPTION

[0043] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0044] The culture medium involved in the following examples is as follows:

[0045] LB medium: 0.5% (w / v) yeast extract, 1% (w / v) tryptone, 1% (w / v) NaCl, pH 7.0.

[0046] TB medium: 2.4% (w / v) yeast extract, 1.2% (w / v) tryptone, 17 mM KH2PO4, 72 mM K2HPO4, 0.4% (w / v) glycerol.

[0047] Solid plate medium: LB medium, 1.5% (w / v) agar.

[0048] The detection methods involved in the following embodiments are as follows:

[0049] Determination of recombinant α-agarase Cm-AGA enzyme activity

[0050] Prepare a 0.25% (w / w) agarose substrate solution in Tris-HCl buffer (20 mM, pH 8.0). Pipette 0.9 mL of substrate and add 0.1 mL of enzyme solution. Incubate at 40°C for 30 min. Terminate the reaction by adding 1 mL of 3,5-dinitrosalicylic acid (DNS) solution. Boil in a water bath for 5 min, then rapidly cool. Dilute with 2 mL of deionized water, and measure absorbance at 540 nm. A standard curve was constructed using the boiled enzyme solution as a control and galactose as a standard.

[0051] Enzyme activity definition: Each unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (calculated as galactose) per minute.

[0052] Detection of agarotriose or agaropentaose

[0053] Agar samples hydrochloric acid hydrolysis and Cm-AGA enzymatic hydrolysis were boiled for 20 minutes to inactivate the enzyme. After centrifugation, dilution, and filtration through a 0.22 μm aqueous filter, the products were analyzed using high-performance anion exchange chromatography (HPAEC-PAD) coupled with ultra-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). HPAEC-PAD conditions included a Dionex CarboPac PA-200 anion exchange column, a column temperature of 35°C, and a mobile phase of 100 mM NaOH, 100 mM NaOH, and 500 mM NaAc at a flow rate of 0.5 mL / min. The UPLC-Q-TOF-MS conditions were as follows: an ACQUITY UPLC BEH Amide column, a column temperature of 40°C, mobile phases A consisting of 0.1% ammonia-acetonitrile solution and B consisting of water-methanol (2:1, v / v) solution, at a flow rate of 0.3 mL / min, and mass spectrometry analysis was performed in negative ionization mode using an electrospray ionization (ESI) source.

[0054] Example 1: Expression of recombinant α-agarase

[0055] The specific steps are as follows:

[0056] (1) Strain construction:

[0057] The construction of the recombinant strain E. coli BL21(DE3) / pET-28a(+)-Cm-aga refers to the literature (You Y, Xie W, Li C, et al. Characterization and efficient production of anα-agarase from marine bacterium Catenovulum maritimum STB14[J]. Food Bioengineering, 2022, 2).

[0058] The recombinant Cm-aga gene (nucleotide sequence shown in SEQ ID NO. 1) was cloned into the plasmid pET-28a(+) using restriction endonucleases Nco I and Xho I. The resulting expression vector pET-28a(+) / Cm-aga was transformed into E. coli JM109 and plated on a plate containing kanamycin. Transformants were selected for sequencing and double enzyme digestion verification. The correctly sequenced expression vector was transformed into competent E. coli BL21(DE3) cells, and the recombinant strain E. coli BL21(DE3) / pET-28a(+)-Cm-aga was cultured.

[0059] The Cm-aga gene sequence is shown below (SEQ ID NO.1):

[0060] (2) Scratching the plate:

[0061] Use an inoculating loop to streak the recombinant E. coli BL21(DE3) / pET-28a(+)-Cm-aga bacterial solution onto LB solid medium containing 100 μg / mL kanamycin, and incubate in a 37°C constant temperature incubator for 12 h.

[0062] (3) Activation:

[0063] Pick a single colony and transfer it to LB medium containing 100 μg / mL kanamycin. Incubate in a shake flask at 37°C and 200 rpm for 8-12 h to prepare a seed solution.

[0064] (4) Fermentation:

[0065] The seed solution obtained in step (3) was transferred to 50 mL of TB medium containing 100 μg / mL kanamycin at an inoculum volume of 4% (v / v). 600 When the value reached 0.6, isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.01 mM) was added, and the flask was shaken at 25°C and 200 rpm for 20 h. The fermentation broth was collected and centrifuged at 4°C and 10,000 rpm for 20 min to obtain the bacteria. An equal volume of lysis buffer (20 mM Tris-HCl, pH 8.0) was added to resuspend the bacteria, and the cells were treated with an ultrasonic cell disruptor for 15 min.

[0066] The supernatant was collected by low-temperature high-speed centrifugation to obtain the intracellular crude enzyme solution. The Cm-AGA enzyme activity was measured by DNS method and was 30.79 U / mL.

[0067] Example 2: Method for preparing agarose

[0068] The specific steps are as follows:

[0069] (1) Weigh 68 g of agar and add it to a 0.25 M hydrochloric acid solution. Stir at 40°C and 380 rpm for 130 min to obtain a 20% (w / w) agar acid solution.

[0070] (2) Weigh 18 g of the agar acid hydrolysis solution obtained in step (1), add NaOH to adjust the pH to 8.0 to obtain an agar acid hydrolysis neutralization solution;

[0071] (3) The agar acid hydrolyzed neutralized solution obtained in step (2) was placed in a constant temperature shaking water bath at 40°C and 180 rpm for 30 min, and then the 30 U / g Cm-AGA crude enzyme solution prepared in Example 1 was added thereto. After enzymatic hydrolysis for 12 h, the solution was boiled for 15 min to inactivate the enzyme.

[0072] (4) 1 g of the acid enzymatic hydrolysate obtained in step (3) was sampled and fixed to 10 mL. After dilution 2000 times, the sample was filtered through a 0.22 μm aqueous filter membrane for HPAEC-PAD detection. The results are shown in FIG1 , and the main product was agarose.

[0073] (5) Calculation of agarose production and yield.

[0074] The mass fraction of the agar acid hydrolysis solution on a dry basis is Q, the total mass is Mg, the amount of NaOH added is Kg, the amount of enzyme added is Ng, the sample weight after acid hydrolysis is mg, and the total volume after dilution and constant volume is V mL. The sample is further diluted n-fold, passed through a membrane, and then analyzed by ion chromatography. The concentration of the agarotriose standard is 10 μg / mL, the corresponding peak area of ​​the standard is S1, and the total peak area corresponding to the acid hydrolysis sample is S2.

[0075] The mass of agarose contained in mg of sample is Y g.

[0076] The total agarose content in agarate hydrolysate is Z g

[0077] Agarose yield in product (g / g agar):

[0078] Agarose yield in the product (%):

[0079] In summary, we can get:

[0080] The results showed that the yield of agarose was 0.0577 g / g agar and the yield of agarose was 5.77%.

[0081] Example 3: Method for preparing agarose

[0082] The specific steps are as follows:

[0083] 34.0, 42.5, 51.0, 59.5, and 68.0 g of agar were weighed respectively, added to 0.25 M hydrochloric acid solution, stirred at 40° C. and 380 rpm for 130 min, and mixed evenly to prepare agar acid hydrolysis solutions with concentrations of 10.0, 12.5, 15.0, 17.5, and 20.0% (w / w), respectively.

[0084] Other methods were followed as in Example 2, and the final yield and yield of agarose were as shown in Table 1.

[0085] The analysis and calculation were performed against agar oligosaccharide standards. The A3 yields after acid hydrolysis combined with enzymatic hydrolysis under different substrate concentrations are shown in Figure 2.

[0086] Table 1 Effects of different agar substrate concentrations on the yield and yield of A3 prepared by acid enzymatic hydrolysis

[0087] The results showed that: as can be seen from the chart, the yield of A3 obtained by acid hydrolysis combined with enzymatic hydrolysis first increased and then decreased with the increase of substrate concentration; when the substrate concentration was 15% (w / w), the final A3 yield was the highest at 12.87%.

[0088] Presumed cause: The main component of agar that forms a gel is agarose. When heated and dissolved, agarose molecules are distributed randomly like threads. Hydrogen bonds form between the hydroxyl groups on the sugar molecules between the chains, causing them to coil around each other to form rope-like agarose bundles. The higher the agarose concentration, the more gelled it becomes. Therefore, under certain hydrochloric acid concentrations, the higher the agar concentration, the easier it is to form a gel, resulting in a decrease in the yield of agarose triose.

[0089] Subsequent experiments were performed with an agar substrate concentration of 15% (w / w).

[0090] Example 4: Method for preparing agarose

[0091] The specific steps are as follows:

[0092] The specific implementation is the same as that of Example 2, except that the adjustment step (1) is:

[0093] Weigh 51 g of agar and add it to 0.25 M, 0.50 M, 1.00 M, 2.00 M, and 3.00 M hydrochloric acid solutions, respectively. Stir at 40° C. and 380 rpm for 130 min to mix evenly to obtain an agar acid hydrolysis solution with a concentration of 15% (w / w).

[0094] Other methods were followed as in Example 2, and the final yield and yield of agarose were as shown in Table 2.

[0095] The analysis and calculation were performed against agar oligosaccharide standards. The A3 yields after acid hydrolysis combined with enzymatic hydrolysis under different hydrochloric acid concentrations are shown in Figure 3.

[0096] Table 2 Effect of different hydrochloric acid concentrations on the yield and yield of A3 prepared by acid enzymatic hydrolysis

[0097] As can be seen from the chart, the yield of A3 obtained by acid hydrolysis combined with enzymatic hydrolysis first increases and then decreases with the increase of hydrochloric acid concentration; when the hydrochloric acid concentration is 2M, the final A3 yield is the highest at 27.12%.

[0098] Speculated reasons: Under certain acid hydrolysis time conditions, as the concentration of hydrochloric acid increases, the large molecular fragments of agar are more easily cut into small molecules, the monosaccharide content will increase significantly, and the yield of agarose will gradually decrease.

[0099] Subsequent experiments were conducted with an agar substrate concentration of 15% (w / w) and a hydrochloric acid concentration of 2M.

[0100] Example 5: Method for preparing agarose

[0101] The specific steps are as follows:

[0102] The specific implementation is the same as that of Example 2, except that the adjustment step (1) is:

[0103] Weigh 51 g of agar, add it to 2 M hydrochloric acid solution, stir at 40°C and 380 rpm for 10, 20, 30, 40, 55, 70, 100, 130, and 280 min, mix well, and obtain agar acid hydrolysis solution with a concentration of 15% (w / w).

[0104] Other methods were followed as in Example 2, and the final yield and yield of agarose were as shown in Table 3.

[0105] The analysis and calculation were performed against agar oligosaccharide standards. The A3 yields after acid hydrolysis combined with enzymatic hydrolysis under different hydrochloric acid hydrolysis time conditions are shown in Figure 4.

[0106] Table 3 Effect of different hydrochloric acid hydrolysis time on the yield and yield of A3 prepared by acid enzymatic hydrolysis

[0107] The results showed that: as can be seen from the chart, the yield of A3 obtained by acid hydrolysis combined with enzymatic hydrolysis first increased and then decreased with the increase of hydrochloric acid hydrolysis time; when the acid hydrolysis time was 40 minutes, the final A3 yield was the highest at 42.07%.

[0108] Speculated reason: Under certain hydrochloric acid concentration conditions, as the acid hydrolysis time increases, the large molecular fragments of agar are further cut into small molecules, the monosaccharide content will increase significantly, and the agarotriose yield will gradually decrease.

[0109] The subsequent experiments were conducted with an agar substrate concentration of 15% (w / w), a hydrochloric acid concentration of 2 M, and an enzymatic hydrolysis time of 40 min.

[0110] Example 6: Method for preparing agarose

[0111] The specific steps are as follows:

[0112] The specific implementation is the same as that of Example 2, except that the adjustment step (1) is:

[0113] 51 g of agar was weighed and added to a 2 M hydrochloric acid solution. The mixture was stirred at 40° C. and 380 rpm for 40 min and mixed evenly to obtain an agar acid solution with a concentration of 15% (w / w).

[0114] Adjustment step (3) is:

[0115] The agar acid hydrolyzed neutralized solution obtained in step (2) was placed in a constant temperature shaking water bath at 40°C and 180 rpm for 30 min, and then the 10, 20, 30, 40, and 60 U / g Cm-AGA crude enzyme solutions prepared in Example 1 were added thereto respectively for enzymatic hydrolysis for 12 h, and then boiled for 15 min to inactivate the enzyme.

[0116] Other methods were followed as in Example 2, and the final yield and yield of agarose were as shown in Table 4.

[0117] The analysis and calculation were performed against agar oligosaccharide standards. The A3 yields after acid pretreatment and enzymatic hydrolysis at different enzyme addition conditions were shown in Figure 5.

[0118] Table 4 Effect of different enzyme addition amounts on the yield and yield of A3 prepared by acid enzymatic hydrolysis

[0119] As shown in the chart, the yield of A3 from acid-enzyme hydrolysis combined with enzymatic hydrolysis gradually increases with increasing enzyme dosage. When the enzyme dosage is 40-60 U / g, the final A3 yield reaches a maximum of 52.84-55.27%, with a slightly slower increase. Considering the high industrial costs associated with enzymatic hydrolysis, the cost-effective enzyme dosage of 40 U / g was selected.

[0120] Subsequent experiments were conducted with an agar substrate concentration of 15% (w / w), an enzymatic hydrolysis time of 40 min, a hydrochloric acid concentration of 2 M, and an enzyme dosage of 40 U / g.

[0121] Example 7: Method for preparing agarose

[0122] The specific steps are as follows:

[0123] The specific implementation is the same as that of Example 2, except that the adjustment step (1) is:

[0124] 51 g of agar was weighed and added to a 2 M hydrochloric acid solution. The mixture was stirred at 40° C. and 380 rpm for 40 min and mixed evenly to obtain an agar acid solution with a concentration of 15% (w / w).

[0125] Adjustment step (3) is:

[0126] The agar acid hydrolyzed neutralized solution obtained in step (2) was placed in a constant temperature shaking water bath at 40°C and 180 rpm for 30 min, and 40 U / g Cm-AGA crude enzyme solution was added thereto for enzymolysis. After enzymolysis for 8, 12, 16, 20, and 24 h, the solution was boiled for 15 min to inactivate the enzyme.

[0127] Other methods were followed as in Example 2, and the final yield and yield of agarose were as shown in Table 5.

[0128] The analysis and calculation were performed against agar oligosaccharide standards. The A3 yields after acid pretreatment and enzymatic hydrolysis at different enzymatic hydrolysis times were shown in Figure 6.

[0129] Table 5 Effect of different enzymatic hydrolysis times on the yield and yield of A3 prepared by acid enzymatic hydrolysis

[0130] As shown in the chart, the yield of A3 from acid-enzyme hydrolysis combined with enzymatic hydrolysis gradually increases with enzymatic hydrolysis time before leveling off. Longer enzymatic hydrolysis times increase the likelihood of bacterial growth and conversion of reducing sugars into other substances. Therefore, the highest A3 yield, 53.21%, was achieved when the enzymatic hydrolysis time was 16 hours.

[0131] As shown in FIG6 , by comparing the peak area of ​​the agarose standard, it can be seen that the conversion rate of agarose prepared by using 51 g agar as the substrate can reach up to 53.21%, that is, 27.1371 g agarose is obtained.

[0132] The literature (Jiang, Liu, Sun, et al. A Novel Route for Agarooligosaccharide Production with the Neoagarooligosaccharide-Producing β-Agarase as Catalyst [J]. Catalysts, 2020, 10 (2): 214.) reported that after 15% (w / v) agarose was initially liquefied with 2.5% (w / v) citric acid at 90 ° C and 0.5 MPa for 50 minutes, the yield of agarose obtained by catalysis of β-agarase AgWH50B (neoagarooligosaccharide-producing β-Agarase) was 48%, while the yield of agarose obtained by catalysis of β-agarase DagA (neoagarooligosaccharide-producing β-Agarase) was 14%. It can be seen that the method for preparing agarose in Example 2 is superior to the existing preparation technology.

[0133] Example 8: Preparation and purification of agarose

[0134] The specific steps are as follows:

[0135] (1) Weigh 51 g of agar and add it to a 2 M hydrochloric acid solution. Stir at 40°C and 380 rpm for 40 min to mix evenly to obtain an agar acid solution with a concentration of 15% (w / w);

[0136] (2) Weigh 18 g of the agar acid hydrolysis solution obtained in step (1), add NaOH to adjust the pH to 8.0 to obtain an agar acid hydrolysis neutralization solution;

[0137] (3) The agar acid hydrolyzed neutralized solution obtained in step (2) was placed in a constant temperature shaking water bath at 40°C and 180 rpm for 30 min, 40 U / g Cm-AGA crude enzyme solution was added thereto, and enzymatic hydrolysis was carried out for 16 h, followed by boiling for 15 min to inactivate the enzyme.

[0138] (4) The reaction system obtained in step (3) was centrifuged to obtain the supernatant, which was lyophilized, concentrated, and weighed. After reconstitution, the sample was separated by size exclusion chromatography (SEC) using a Superdex 30 Increase 10 / 300GL column. Milli-Q water was used for elution at a flow rate of 0.1 mL / min, and the sample was collected in separate tubes. The purity of the agarose was verified by HPAEC-PAD and UPLC-Q-TOF-MS analysis.

[0139] Example 9: Characterization of agarose products

[0140] (1) The ion chromatography results of the product obtained in Example 8 are shown in FIG7 . The purified agarose is free of other miscellaneous sugars and has a high purity of 98.3%. Patent Publication No. CN108715841B provides an immobilized enzyme that produces 3,6-endo-L-galactose for use in degrading agarose to produce agarose. The purified agarose has a purity exceeding 96.0%. This indicates that the method for preparing agarose in Example 8 is superior to existing preparation technologies.

[0141] (2) The liquid chromatography mass spectrometry results of the product obtained in Example 8 are shown in FIG8 , and the component analysis is as follows: agarose [MH] - The doubly charged ion (m / z 242.0021) in the mass spectrum appeared at half the mass of the singly charged ion (m / z 485.0080) of the same mass unit, indicating that the two peaks were identical. The mass spectrometry results further demonstrated that agarose had been isolated and purified to high purity.

[0142] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing agarotriose, characterized in that: The method comprises the following steps: (1) Weigh agar, add it to a hydrochloric acid solution, stir it at 30 to 50° C. and 300 to 400 rpm for 10 to 280 min, mix it evenly, and obtain an agar acid solution; (2) adjusting the pH of the agar acid hydrolysis solution prepared in step (1) to 7.0 to 9.0 to obtain an agar acid hydrolysis neutralization solution; (3) The agar acid hydrolyzed neutralized solution obtained in step (2) is kept warm at 30-50° C. and 150-250 rpm for 10-50 min, and then crude α-agarase solution is added thereto for enzymolysis for 8-24 h. The acid hydrolyzed solution containing agarose is obtained after boiling for 10-30 min to inactivate the enzyme.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the hydrochloric acid solution is 0.25M to 3.00M.

3. The preparation method according to claim 2, characterized in that: The concentration of the agar acid hydrolysis solution is 10-20% (w / w).

4. The preparation method according to claim 3, characterized in that: The α-agarase is α-AGA derived from Catenovulum maritimum, and the nucleotide sequence encoding the α-agarase is shown in SEQ ID NO.

1.

5. The preparation method according to claim 4, characterized in that: Step (2) is to adjust the pH of the agar acid hydrolysis solution to 8.0 using NaOH to obtain an agar acid hydrolysis neutralization solution.

6. The method according to claim 5, characterized in that The added amount of the α-agarase is 10-60 U / g.

7. The method according to claim 6, characterized in that The added amount of the α-agarase is 40-60 U / g.

8. The method according to claim 7, characterized in that The method comprises the following steps: (1) Weigh agar, add it to a 2 M hydrochloric acid solution, stir it at 40° C. and 380 rpm for 40 min, mix it evenly, and obtain a 15% (w / w) agar acid solution; (2) adjusting the pH of the agar acid hydrolysis solution prepared in step (1) to 8.0 to obtain an agar acid hydrolysis neutralization solution; (3) The agar acid hydrolyzed neutralized solution obtained in step (2) was kept warm at 40° C. and 180 rpm for 30 min, and 40 U / g α-agarase crude enzyme solution was added thereto for enzymolysis for 12 h. The acid hydrolyzed solution containing agarose was obtained after boiling for 15 min to inactivate the enzyme.

9. The preparation method according to claim 1, characterized in that: In step (1), the mixing is carried out at 40° C., 300 to 400 rpm, and stirring is performed for 10 to 280 minutes.

10. The preparation method according to claim 1, characterized in that: In step (2), the NaOH concentration is 6M.

11. The preparation method according to claim 1, characterized in that: The method further comprises (4) concentrating and purifying the product obtained in step (3), and then analyzing the product using a high performance anion exchange chromatograph or an ultra high performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometer.

12. The preparation method according to claim 11, characterized in that: The concentration and purification in step (4) is performed by first freeze-drying and then purification using a Superdex 30 Increase 10 / 300 GL gel chromatography column.

13. Agarotriose prepared by the method according to any one of claims 1 to 12.

14. Use of agarotriose according to claim 13 in the fields of food, cosmetics and pharmaceuticals.

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