Red algae oligosaccharide with bowel relaxing function, composition, preparation method and application

Abstract

The invention belongs to the technical field of biology, and discloses red algae oligosaccharide with a bowel relaxing function, a composition, a preparation method and application. The compound enzyme agent (obtained by compounding red algae polysaccharide enzyme, galactokinase and galactosyltransferase) in the composition for preparing red algae oligosaccharide provided by the invention can directly take a red algae coarse material subjected to simple treatment as a fermentation substrate, and meanwhile, Zn < 2 + > with a specific concentration in a fermentation solution is coordinated with the compound enzyme agent; the yield of the red algae oligosaccharide obtained through fermentation is increased, and the content of the red algae oligosaccharide with the polymerization degree of 4-6 is 95% or above; when the composition is applied to preparation of red algae oligosaccharide, the composition has the advantages of low substrate purity requirement, high product polymerization degree uniformity, high yield and the like, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a red algal oligosaccharide with laxative function, a composition, a preparation method and an application thereof. Background Art

[0002] Red algal oligosaccharide is an important marine active substance, which has the effects of antioxidant, anti-aging, moisturizing, regulating intestinal microbiota, antibacterial and anti-inflammatory. Red algal polysaccharide has great application prospects in the fields of functional foods, medicine, etc.

[0003] Currently, red algal oligosaccharides are generally prepared from red algae such as Gracilaria and Gelidium as raw materials by chemical methods, enzymatic methods and other methods. Compared with the chemical method with high energy consumption, large pollution and complex product components, the preparation of red algal oligosaccharides by the enzymatic method has the advantages of mild reaction conditions, easy control, and easy separation and purification of products, so it is more widely used. However, since most of the commonly used α-agarase and β-agarase in the prior art can only use high-purity agarose as a substrate, it is difficult to directly use crude agar as a substrate, and the obtained product has a non-uniform degree of polymerization, and there is also a problem of low yield, which has great limitations. Summary of the Invention

[0004] The first object of the present invention is to provide a composition for preparing red algal oligosaccharides. The complex enzyme agent obtained by compounding red algal polysaccharide enzyme, galactokinase and galactosyltransferase in the composition can directly use the simply treated red algal crude material as a fermentation substrate; meanwhile, the Zn 2+ concentration in the fermentation solution will affect the three-dimensional spatial structure and catalytic activity of each enzyme molecule in the complex enzyme agent, and further affect the reaction and transformation of substances during the fermentation process. When the Zn 2+ concentration in the fermentation solution is controlled at 0.01 - 0.5 mmol / L, the degree of polymerization of the fermentation product is 4 - 6, and the yield of the product is also increased to a certain extent. When this composition is applied to the preparation of red algal oligosaccharides, it has the advantages of low substrate purity requirements, high degree of polymerization uniformity of the product and high yield, and has good application prospects.

[0005] The second object of the present invention is to provide a method for preparing agarotetraose.

[0006] The third object of the present invention is to provide agarotetraose prepared by the above method for preparing agarotetraose.

[0007] The fourth object of the present invention is to provide a method for preparing agaropentaose.

[0008] The fifth object of the present invention is to provide agaropentaose prepared by the above method for preparing agaropentaose.

[0009] The sixth object of the present invention is to provide a red algal oligosaccharide composition.

[0010] The seventh object of the present invention is to provide the use of the above-mentioned agarotetraose, agarose hexasaccharide and / or red algal oligosaccharide composition in the preparation of defecation function foods and / or drugs.

[0011] Specifically, the composition for preparing red algal oligosaccharides provided by the present invention includes: a complex enzyme agent, which includes a red algal polysaccharide enzyme, a galactokinase and a galactosyltransferase with an added enzyme activity ratio of 1:(0.0001-0.01):(0.0001-0.01), the amino acid sequence of the red algal polysaccharide enzyme is as shown in SEQ ID NO:2, the amino acid sequence of the galactokinase is as shown in SEQ ID NO:4, and the amino acid sequence of the galactosyltransferase is as shown in SEQ ID NO:6; a fermentation solution, which includes Zn 2+ and ATP, and the concentration of the Zn 2+ is 0.01-0.5 mmol / L.

[0012] Further, the fermentation solution uses Tris-HCl buffer as a solvent, and the concentration of the Tris-HCl buffer is 5-10 mM and the pH is 7-7.5.

[0013] Further, the concentration of the ATP is 0.5-2 mmol / L.

[0014] The method for preparing agarotetraose provided by the present invention using the above-mentioned composition for preparing red algal oligosaccharides includes: taking the complex enzyme agent, a red algal raw material and the fermentation solution for mixing and fermentation to obtain agarotetraose; the added enzyme activity ratio of the red algal polysaccharide enzyme, the galactokinase and the galactosyltransferase is 1:(0.0005-0.001):(0.0005-0.001), and the concentration of the Zn 2+ is 0.2-0.3 mmol / L.

[0015] Further, based on the total mass of the fermentation solution, the addition amount of the complex enzyme agent is 10-100 U / g, and the addition amount of the red algal raw material is 10-30%.

[0016] Further, the temperature of the fermentation is 20-35 °C and the time is 12-48 h.

[0017] The agarotetraose provided by the present invention is prepared by the above method for preparing agarotetraose.

[0018] The method for preparing agarose hexasaccharide using the above-mentioned composition for preparing red algal oligosaccharide provided by the present invention includes: mixing a complex enzyme agent, a red algal raw material and a fermentation solution, and fermenting to obtain agarose hexasaccharide; the addition enzyme activity ratio of the red algal polysaccharase, galactokinase and galactosyltransferase is 1:(0.005-0.01):(0.005-0.01), and the concentration of Zn 2+ is 0.015-0.02 mmol / L.

[0019] Further, based on the total mass of the fermentation solution, the addition amount of the complex enzyme agent is 10-100 U / g, and the addition amount of the red algal raw material is 10-30%.

[0020] Further, the temperature of the fermentation is 20-35 °C, and the time is 12-48 h.

[0021] The agarose hexasaccharide provided by the present invention is prepared by the above method for preparing agarose hexasaccharide.

[0022] The red algal oligosaccharide composition provided by the present invention includes agarose tetrasaccharide and agarose hexasaccharide, and the mass ratio of the agarose tetrasaccharide to the agarose hexasaccharide is 1:(0.8-1.2).

[0023] The present invention also provides the application of the above agarose tetrasaccharide, agarose hexasaccharide and / or red algal oligosaccharide composition in the preparation of laxative functional foods and / or drugs. Description of the Drawings

[0024] Figure 1 is the electrophoresis diagram of the red algal polysaccharase provided in Preparation Example 1 of the present invention;

[0025] Figure 2 is the electrophoresis diagram of the galactokinase provided in Preparation Example 1 of the present invention;

[0026] Figure 3 is the electrophoresis diagram of the galactosyltransferase provided in Preparation Example 1 of the present invention. Detailed Description of the Invention

[0027] The composition for preparing red algal oligosaccharides provided by the present invention specifically includes a complex enzyme agent and a fermentation solution. In the present invention, the complex enzyme agent includes red algal polysaccharase, galactokinase and galactosyltransferase, and the added enzyme activity ratio of the red algal polysaccharase, galactokinase and galactosyltransferase is 1:(0.0001-0.01):(0.0001-0.01), such as 1:0.009:0.01, 1:0.005:0.001, 1:0.0001:0.0001 or any value therebetween; the amino acid sequence of the red algal polysaccharase is as shown in SEQ ID NO:2, the amino acid sequence of the galactokinase is as shown in SEQ ID NO:4, and the amino acid sequence of the galactosyltransferase is as shown in SEQ ID NO:6.

[0028] In the present invention, the fermentation solution includes Zn 2+ and ATP, and the concentration of the Zn 2+ is 0.01-0.5 mmol / L, such as 0.01 mmol / L, 0.03 mmol / L, 0.1 mmol / L, 0.25 mmol / L, 0.4 mmol / L, 0.5 mmol / L or any value therebetween.

[0029] In some specific embodiments, the fermentation solution preferably uses Tris-HCl buffer as the solvent, and the concentration of the Tris-HCl buffer is preferably 5-10 mM, such as 5 mM, 5.1 mM, 5.6 mM, 6 mM, 8 mM, 10 mM or any value therebetween; the pH is preferably 7-7.5, such as 7, 7.1, 7.2, 7.3, 7.5 or any value therebetween.

[0030] In some specific embodiments, the concentration of ATP in the fermentation solution is preferably 0.5-2 mmol / L, such as 0.5 mmol / L, 0.8 mmol / L, 1 mmol / L, 1.3 mmol / L, 1.5 mmol / L, 1.8 mmol / L, 2 mmol / L or any value therebetween.

[0031] Based on the above composition for preparing red algal oligosaccharides, the inventors of the present invention, through further in-depth research and a large number of experiments, obtained a method for preparing high-purity agarotetraose. The method specifically includes: mixing a composite enzyme agent, a red algal raw material, and a fermentation solution, and fermenting to obtain agarotetraose; and the added enzyme activity ratio of the red algal polysaccharidase, galactokinase, and galactosyltransferase is 1:(0.0005 - 0.001):(0.0005 - 0.001), such as 1:0.001:0.001, 1:0.0005:0.0005, 1:0.0009:0.0005, 1:0.001:0.0005, or any value therebetween; the concentration of Zn 2+ is 0.2 - 0.3 mmol / L, such as 0.2 mmol / L, 0.21 mmol / L, 0.23 mmol / L, 0.28 mmol / L, 0.3 mmol / L, or any value therebetween.

[0032] In some specific embodiments, based on the total mass of the fermentation solution, the addition amount of the composite enzyme agent is preferably 10 - 100 U / g, such as 10 U / g, 15 U / g, 18 U / g, 25 U / g, 30 U / g, 38 U / g, 50 U / g, 75 U / g, 100 U / g, or any value therebetween; the addition amount of the red algal raw material is preferably 10 - 30%, such as 10%, 12%, 15%, 17.5%, 20%, 25%, 30%, or any value therebetween.

[0033] In some specific embodiments, the fermentation temperature is preferably 20 - 35 °C, such as 20 °C, 21 °C, 23 °C, 25 °C, 30 °C, 35 °C, or any value therebetween; the time is preferably 12 - 48 h, such as 12 h, 13 h, 15 h, 18 h, 24 h, 48 h, or any value therebetween.

[0034] The present invention also provides agarotetraose prepared by the above method for preparing agarotetraose, and its purity is greater than 95%.

[0035] Based on the above composition for preparing red algal oligosaccharides, the inventors of the present invention, through further in-depth research and a large number of experiments, obtained a method for preparing high-purity agaropentaose. The method specifically includes: mixing a composite enzyme agent, a red algal raw material, and a fermentation solution, and fermenting to obtain agaropentaose; and the added enzyme activity ratio of the red algal polysaccharidase, galactokinase, and galactosyltransferase is 1:(0.005 - 0.01):(0.005 - 0.01), such as 1:0.005:0.005, 1:0.009:0.01, 1:0.01:0.01, or any value therebetween; the concentration of Zn 2+The concentration is 0.015 - 0.02 mmol / L, such as 0.015 mmol / L, 0.016 mmol / L, 0.018 mmol / L, 0.02 mmol / L or any value therebetween.

[0036] In some specific embodiments, based on the total mass of the fermentation solution, the addition amount of the complex enzyme agent is preferably 10 - 100 U / g, such as 10 U / g, 13 U / g, 17 U / g, 20 U / g, 30 U / g, 40 U / g, 50 U / g, 75 U / g, 100 U / g or any value therebetween; the addition amount of the red alga raw material is preferably 10 - 30%, such as 10%, 10.5%, 18%, 20%, 25%, 30% or any value therebetween.

[0037] In some specific embodiments, the fermentation temperature is preferably 20 - 35 °C, such as 20 °C, 21 °C, 23 °C, 27.3 °C, 35 °C or any value therebetween; the time is preferably 12 - 48 h, such as 12 h, 18 h, 24 h, 48 h or any value therebetween.

[0038] The present invention also provides agarose hexasaccharide prepared by the above method for preparing agarose hexasaccharide, and its purity is greater than 96%.

[0039] The red algal oligosaccharide composition provided by the present invention comprises agarotetraose and agarose hexasaccharide, and the mass ratio of the agarotetraose to the agarose hexasaccharide is 1:(0.8 - 1.2), such as 1:0.8, 1:0.85, 1:0.9, 1:0.98, 1:1, 1:1.1, 1:1.2 or any value therebetween. The red algal oligosaccharide composition containing a specific ratio of agarotetraose and agarose hexasaccharide has excellent laxative effect on mice and has great potential for application as a laxative functional food and / or medicine.

[0040] The present invention also provides the application of the above agarotetraose, agarose hexasaccharide and / or red algal oligosaccharide composition in the preparation of a laxative functional food and / or medicine.

[0041] The embodiments of the present invention are described in detail below. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those not specified in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. The reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0042] Preparation Example 1

[0043] This preparation example is used to illustrate the preparation of red algal polysaccharide enzyme, specifically including:

[0044] 1. Preparation of recombinant plasmid: The gene fragment encoding porphyranase was chemically synthesized (the nucleotide sequence is shown in SEQ ID NO: 1); NdeⅠ and XhoⅠ restriction enzyme sites were added to both ends of the obtained gene fragment and then synthesized. After digestion with NdeⅠ and XhoⅠ enzymes, it was recombined onto the vector pET24a(+) to obtain the recombinant plasmid.

[0045] The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3), placed in LB liquid medium, and cultured overnight by shaking at 37 °C and 160 rpm to extract a large amount of recombinant plasmid.

[0046] 2. Expression of porphyranase: The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain I; recombinant strain I was inoculated into LB medium containing 50 mg / L kanamycin sulfate at an inoculation amount of 1%, and cultured by shaking in a shaker at 37 °C, 200 rpm and 5% CO2 until OD 600 = 0.8, and isopropyl-β-D-thiogalactoside with a final concentration of 0.1 mmol was added for induced overnight expression.

[0047] 3. Extraction of porphyranase: The above cell culture solution was centrifuged to remove the supernatant; the precipitate was resuspended in 15 mL of Tris-HCl buffer (pH = 7.5), and the cells were broken by ice bath and ultrasonic treatment for 10 min. After centrifugation, the supernatant was taken to obtain an enzyme solution containing porphyranase with the amino acid sequence shown in SEQ ID NO: 2.

[0048] The SDS-PAGE gel electrophoresis pattern is as Figure 1 shown. It can be seen from Figure 1 that the molecular mass of this porphyranase is 33.2 kDa.

[0049] 4. Determination of porphyranase activity: The DNS method was used to determine the enzyme activity of porphyranase, specifically including: taking 20 μL of the enzyme solution and adding it to 500 μL of 0.2% agarose solution (pH = 7.0, prepared with 2 mM Tris-HCl buffer), incubating at 37 °C for 5 min and then stopping the reaction at high temperature; using the inactivated enzyme solution as a control, adding 500 μL of DNS reagent, reacting at 100 °C for 7 min, and detecting OD 540 , and defining 1 enzyme activity unit as: the porphyranase required to generate 1 μmol / L galactose in 1 min. The enzyme activity of porphyranase in this enzyme solution is 38.17 U / mL.

[0050] Preparation Example 2

[0051] This preparation example is used to illustrate the preparation of galactokinase, specifically including:

[0052] 1. Preparation of recombinant plasmid: The galactokinase-encoding gene with the sequence shown in SEQ ID NO:3 was obtained by codon optimization and chemical synthesis; NdeⅠ and XhoⅠ restriction sites were added to both ends of the obtained gene fragment, followed by synthesis. After digestion with NdeⅠ and XhoⅠ enzymes, it was recombined onto the vector pET24a(+), resulting in a recombinant plasmid.

[0053] The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) and placed in LB liquid medium. It was cultured overnight with shaking at 37°C and 160 rpm, and a large amount of recombinant plasmid was extracted.

[0054] 2. Expression of galactokinase: The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain I; Recombinant strain I was inoculated into LB medium containing 50 mg / L kanamycin sulfate at an inoculation amount of 1%, and cultured with shaking in a shaker at 37°C, 200 rpm and 5% CO2 until OD 600 = 0.8, and isopropyl-β-D-thiogalactoside with a final concentration of 0.1 mmol was added for overnight induction expression.

[0055] 3. Extraction of galactokinase: The above cell culture solution was centrifuged to remove the supernatant; The precipitate was resuspended in 15 mL of Tris-HCl buffer (pH = 7.5), and the cells were broken by ice bath and ultrasonic treatment for 10 min. The supernatant was collected by centrifugation to obtain an enzyme solution containing galactokinase with the amino acid sequence shown in SEQ ID NO:4.

[0056] The SDS-PAGE gel electrophoresis pattern is as Figure 2 shown. It can be seen from Figure 2 that the molecular mass of this galactokinase is 37.55 kDa.

[0057] 4. Determination of galactokinase enzyme activity: Using D-galactose as the substrate (5 mM), and adopting the Kinase-Glo TM kinase activity detection kit (Promega) and referring to the instruction manual to determine the enzyme activity of galactokinase. One enzyme activity unit was defined as: galactokinase required to consume 1 μmol / L ATP in 1 min. The enzyme activity of galactokinase in this enzyme solution was 47.04 U / mL.

[0058] Preparation Example 3

[0059] This preparation example is used to illustrate the preparation of galactosyltransferase, specifically including:

[0060] 1. Preparation of recombinant plasmid: The gene fragment encoding galactosyltransferase (nucleotide sequence as shown in SEQ ID NO:5) was obtained by chemical synthesis; NdeⅠ and XhoⅠ restriction enzyme sites were added to both ends of the obtained gene fragment respectively, and then synthesized. After digestion with NdeⅠ and XhoⅠ enzymes, it was recombined onto the vector pET24a(+) to obtain the recombinant plasmid.

[0061] The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3), and placed in LB liquid medium, and cultured overnight by shaking at 37℃ and 160rpm to extract a large amount of recombinant plasmid.

[0062] 2. Expression of galactosyltransferase: The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain recombinant strain I; The recombinant strain I was inoculated into LB medium containing 50mg / L kanamycin sulfate at an inoculation amount of 1%, and cultured by shaking in a shaker at 37℃, 200rpm and 5% CO2 until OD 600 =0.8, and isopropyl-β-D-thiogalactoside with a final concentration of 0.1mmol was added for induced overnight expression.

[0063] 3. Extraction of galactosyltransferase: The above cell culture solution was centrifuged at 4℃ and 10000rpm for 10min to remove the supernatant; The precipitate was resuspended in 15mL of Tris-HCl buffer (pH = 7.5), and the cells were broken by ice bath and ultrasonic treatment for 10min, and then centrifuged at 4℃ and 8000rpm for 10min to take the supernatant to obtain an enzyme solution containing galactosyltransferase with the amino acid sequence as shown in SEQ ID NO:6.

[0064] Figure 3 is the electrophoresis pattern of the above enzyme solution. As Figure 3 can be seen, the molecular mass of this galactosyltransferase is 40.83kDa.

[0065] 4. Determination of galactosyltransferase enzyme activity: Using UDP-gal as the donor substrate and Glc-NAc as the acceptor substrate, the enzyme activity of galactosyltransferase was determined, specifically including: taking 20μL of the enzyme solution and adding it to 500μL of 0.2% Tris-HCl buffer (pH = 7.0, 2mM, containing 0.0mM phenol red, 0.1mM MnCl2, 2mM UDP-gal and 10mM GlcNAc), incubating at 30℃ for 3h, and measuring OD 557 . Defining 1 enzyme activity unit as: the galactosyltransferase required to consume 1μmol / L donor substrate / acceptor substrate per minute. The enzyme activity of galactosyltransferase in this enzyme solution is 28.57U / mL.

[0066] Example 1

[0067] This example is used to illustrate the preparation of red algal oligosaccharides, specifically including:

[0068] 1. Preparation of Gracilaria lemaneiformis crude material: Take 5 g of Gracilaria lemaneiformis, cut it into pieces, add 500 mL of water, heat it in a water bath at 80 °C for 2 h, filter it with eight layers of gauze, collect the aqueous solution, and obtain the red algal crude material.

[0069] 2. Preparation of red algal oligosaccharides: Take the complex enzyme preparation at an addition amount of 50 U / g, mix it evenly with 25 g of Gracilaria lemaneiformis crude material and 100 g of fermentation solution, place it in a shaker at 30 °C and 200 rpm for fermentation for 12 h, boil it for 15 min to inactivate the enzyme, and filter it with a 0.22 μm filter membrane to obtain an aqueous solution containing red algal oligosaccharides;

[0070] Take the aqueous solution containing red algal oligosaccharides, carry out vacuum distillation at 60 °C, filter to obtain the concentrated solution, take the concentrated solution for freeze-drying and pulverization to obtain red algal oligosaccharide I.

[0071] Among them, the complex enzyme preparation used in this example includes the red algal polysaccharide enzyme provided in Preparation Example 1, the galactokinase provided in Preparation Example 2, and the galactosyltransferase provided in Preparation Example 3, and the addition enzyme activity ratio of the red algal polysaccharide enzyme, galactokinase, and galactosyltransferase is 1:0.009:0.01; the fermentation solution is Tris-HCl buffer (7.25 mM, pH = 7.5), and includes 0.018 mmol of Zn 2+ and 1 mmol of ATP.

[0072] Examples 2 to 4

[0073] Examples 2 to 4 use the method provided in Example 1 to prepare red algal oligosaccharides. The difference is that in "Step 2. Preparation of red algal oligosaccharides", the reagents and conditions used are different, as shown in Table 1 specifically. Other conditions are the same, and red algal oligosaccharides II to IV are obtained.

[0074] Table 1. Reagents and conditions for the preparation of red algal oligosaccharides

[0075]

[0076] Note: The enzyme activity ratio refers to the addition enzyme activity ratio of the red algal polysaccharide enzyme, galactokinase, and galactosyltransferase.

[0077] Comparative Example 1

[0078] This comparative example uses the method provided in Example 1 to prepare red algal oligosaccharides. The difference is that in "Step 2. Preparation of red algal oligosaccharides", Zn is not added to the fermentation solution 2+ , other conditions are the same, and comparative red algal oligosaccharide I is obtained.

[0079] Comparative Example 2

[0080] This comparative example uses the method provided in Example 1 to prepare porphyran oligosaccharides. The difference is that in "Step 2, Preparation of porphyran oligosaccharides", Ba with an equimolar concentration is used in the fermentation solution 2+ instead of Zn 2+ , and other conditions are the same, obtaining comparative porphyran oligosaccharide II.

[0081] Comparative Example 3

[0082] This comparative example uses the method provided in Example 1 to prepare porphyran oligosaccharides. The difference is that in "Step 2, Preparation of porphyran oligosaccharides", porphyran polysaccharide enzyme with an equal added enzyme activity is used instead of galactosyltransferase in the complex enzyme agent, and other conditions are the same, obtaining comparative porphyran oligosaccharide III.

[0083] Comparative Example 4

[0084] This comparative example uses the method provided in Example 1 to prepare porphyran oligosaccharides. The difference is that in "Step 2, Preparation of porphyran oligosaccharides", the added enzyme activity ratio of porphyran polysaccharide enzyme, galactokinase and galactosyltransferase in the complex enzyme agent is 1:1:1, and other conditions are the same, obtaining comparative porphyran oligosaccharide IV.

[0085] Test Example

[0086] This test example is used to illustrate the specific components of the porphyran oligosaccharides provided in the above examples and the comparative porphyran oligosaccharides provided in the comparative examples. The test methods include:

[0087] 1. Drawing of the standard curve: Take the agar oligosaccharide standard (NA2 - 10) and mix it with deionized water to prepare a series of standard solutions with different concentrations. After sterilization through a 0.22 μm membrane, perform ion chromatography analysis to obtain the peak areas of each standard solution, and draw the standard curve;

[0088] 2. Determination of agar oligosaccharides: Take 20 mg of agar oligosaccharides, comparative porphyran oligosaccharides and deionized water respectively, and make up the volume to 1000 mL to prepare a test solution with a concentration of 20 mg / L. After sterilization through a 0.22 μm membrane, perform ion chromatography analysis to obtain the peak areas of each peak, and calculate the concentration of each agar oligosaccharide (C x1 , g / L) in the test solution according to the standard curve, and calculate the content C x2 of each agar oligosaccharide (disaccharide - hexasaccharide) in the porphyran oligosaccharides or comparative porphyran oligosaccharides according to the following formula.

[0089] C x2 (%) = C x1 / 20 × 100%

[0090] Among them, the detection conditions for the above ion chromatography analysis include: the chromatographic column is a Dionex CarboPac PA-100 anion exchange column (including an analytical column [4×250 mm] and a guard column [4×50 mm]); the mobile phase is 100 mmol / L NaOH and 150 mmol / L NaAc, and the flow rate is 0.25 mL / min; four-potential pulsed amperometry is used for detection; the column temperature is 25 °C; the injection volume is 25 μL. The test results are shown in Table 2.

[0091] Table 2. Specific components of red algal oligosaccharides

[0092]

[0093] Note: "-" indicates that no peak appears in the ion chromatogram or the peak area cannot be calculated for the peaks that appear.

[0094] It can be seen from the test results shown in Table 2 that when the method provided in Examples 1 to 4 of the present invention is used to treat the crude Gracilaria lemaneiformis, the yield of red algal oligosaccharides is above 25%. Moreover, when the method provided in Comparative Examples 1 to 4 is used to treat the crude Gracilaria lemaneiformis, the degree of polymerization of the obtained red algal oligosaccharides is heterogeneous (a large amount of agar oligosaccharides or agar polysaccharides with a degree of polymerization of 10 or higher are contained in Comparative Examples 1, 2, and 4), while the content of agarobiose to agarotetraose in the red algal oligosaccharides prepared by the method provided in Examples 1 to 4 of the present invention is above 95%, and the degree of polymerization of the red algal oligosaccharides is uniform.

[0095] It should be noted that when the conditions provided in Example 1 are adopted (that is, the addition enzyme activity ratio of red algal polysaccharase, galactokinase, and galactosyltransferase is 1:0.009:0.01, and the concentration of Zn 2+ is 0.018 mmol), the content of agarose hexasaccharide in the obtained red algal oligosaccharides is as high as 96.01%, which can be used to achieve the high-purity preparation of agarose hexasaccharide.

[0096] When the conditions provided in Example 3 are adopted (that is, the addition enzyme activity ratio of red algal polysaccharase, galactokinase, and galactosyltransferase is 1:0.005:0.001, and the concentration of Zn 2+ is 0.25 mmol), the content of agarotetraose in the obtained red algal oligosaccharides is as high as 95.72%, which can be used to achieve the high-purity preparation of agarotetraose.

[0097] Example 6

[0098] This example is used to illustrate the laxative function of the red algal oligosaccharides provided in the above examples. The test method includes:

[0099] 1. Construction of constipation mouse model: Adult healthy male mice weighing 18 - 22 g were selected as experimental animals, and each group was administered and raised according to the following protocol (3 mice per group, N = 18):

[0100] (1) Model group: Orally gavaged with loperamide hydrochloride (dosage 5 mg / KgBW) every day. After 14 days of continuous gavage, orally gavaged with normal saline (dosage 5 mg / KgBW) every day and continued continuous gavage for another 14 days;

[0101] (2) Experimental group: Orally gavaged with loperamide hydrochloride (dosage 5 mg / KgBW) every day. After 14 days of continuous gavage, orally gavaged with the red algal oligosaccharides provided in Examples 1 - 4 (dosage 10 mg / KgBW) every day and continued continuous gavage for another 14 days;

[0102] (3) Blank control group: Orally gavaged with normal saline (dosage 5 mg / KgBW) every day for 28 days of continuous gavage;

[0103] The total gavage time was 28 days. During the gavage period, the mice were kept in a quiet, clean, ventilated and warm environment with the environmental temperature of (22 ± 1) °C and the humidity of 45% - 65%, ensuring that the mice had free access to water and standard feed.

[0104] 2. After 28 days of continuous gavage, the mice in each group were gavaged with the same dose of ink, and the time for each mouse to defecate black stools for the first time and the number of black stools excreted within 6 h were recorded. The results are shown in Table 3.

[0105] Table 3. Laxative effect of red algal oligosaccharides

[0106]

[0107]

[0108] It can be seen from the test results shown in Table 3 that compared with the blank control group, the time for the model group to defecate black stools for the first time increased, and the number of stools excreted within 6 h decreased, indicating that the constipation mouse model was successfully established; compared with the model group, administering the red algal oligosaccharides provided in Examples 1 - 4 of the present invention to the mice shortened the time for the first defecation and increased the number of stools excreted within 6 h, having a certain laxative effect; among them, the red algal oligosaccharides provided in Example 2 had an excellent laxative effect.

[0109] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

[0110] The nucleotide and amino acid sequences involved in the present invention are specifically shown in Table 4 as follows.

[0111] Table 4. Nucleotide and Amino Acid Sequence Table

[0112]

[0113]

[0114]

[0115]

Claims

1. A composition for preparing red algal oligosaccharide, characterized in that, The composition comprises: A complex enzyme preparation, which comprises porphyranase, galactokinase and galactosyltransferase with an added enzyme activity ratio of 1:(0.0001-0.01):(0.0001-0.01), wherein the amino acid sequence of the porphyranase is as shown in SEQ ID NO:2, the amino acid sequence of the galactokinase is as shown in SEQ ID NO:4, and the amino acid sequence of the galactosyltransferase is as shown in SEQ ID NO:6; A fermentation solution, which comprises Zn 2+ and ATP, and the concentration of the Zn 2+ is 0.01 to 0.5 mmol / L.

2. The composition for preparing red algal oligosaccharide according to claim 1, characterized in that, The fermentation solution uses Tris-HCl buffer as a solvent, and the concentration of the Tris-HCl buffer is 5-10 mM, and the pH is 7-7.5; optionally, the concentration of ATP is 0.5-2 mmol / L.

3. A method for preparing agarotetraose using the composition for preparing red algal oligosaccharides according to claim 1 or 2, characterized in that, The method includes: taking a complex enzyme preparation, a red alga raw material and a fermentation solution, mixing and fermenting them to obtain agarotetraose; the added enzyme activity ratio of the red algal polysaccharase, galactokinase and galactosyltransferase is 1:(0.0005 - 0.001):(0.0005 - 0.001), and the concentration of Zn 2+ is 0.2 - 0.3 mmol / L.

4. The method for preparing agarotetraose according to claim 3, wherein Based on the total mass of the fermentation solution, the addition amount of the complex enzyme preparation is 10-100 U / g, and the addition amount of the red algae raw material is 10-30%; optionally, the fermentation temperature is 20-35 °C, and the time is 12-48 h.

5. An agarotetraose, characterized in that, Prepared by the method for preparing agarotetraose according to claim 3 or 4.

6. A method for preparing agarose hexasaccharide using the composition for preparing red algal oligosaccharide according to claim 1 or 2, characterized in that, The method includes: taking a composite enzyme agent, a red alga raw material and a fermentation solution, mixing and fermenting them to obtain agarose hexasaccharide; the added enzyme activity ratio of the red algal polysaccharase, galactokinase and galactosyltransferase is 1:(0.005-0.01):(0.005-0.01), and the concentration of Zn 2+ is 0.015-0.02 mmol / L.

7. The method for preparing agar hexasaccharide according to claim 6, characterized in that, Based on the total mass of the fermentation solution, the addition amount of the complex enzyme preparation is 10-100 U / g, and the addition amount of the red algae raw material is 10-30 wt%; optionally, the fermentation temperature is 20-35 °C, and the time is 12-48 h.

8. An agarose hexasaccharide, characterized in that, Prepared by the method for preparing agarotetraose according to claim 6 or 7.

9. A red algal oligosaccharide composition, characterized in that, Comprising agarotetraose and agaropentaose, and the mass ratio of the agarotetraose to the agaropentaose is 1:(0.8-1.2).

10. Use of the agarotetraose according to claim 5, the agaropentaose according to claim 8 and / or the red algal oligosaccharide composition according to claim 9 in the preparation of a laxative functional food and / or medicine.

Images