Preparation method of agaro-oligosaccharide

Abstract

The preparation method comprises the following steps: (1) dissolving agar in a citric acid monohydrate solution, and carrying out acidolysis to obtain an acidolysis solution; centrifuging, filtering, adding beta-agarase AgWH50B, and carrying out enzymolysis for 20-28 hours at the temperature of 30-35 DEG C; boiling, centrifuging and filtering to obtain supernate, namely crude oligosaccharide liquid containing agarotriose; (2) dissolving agar in a phosphoric acid solution, and degrading for 55-65 minutes at the temperature of 97-103 DEG C; centrifuging and filtering to obtain supernate, namely crude oligosaccharide liquid containing agarobiose; (3) dissolving agar in deionized water, adding alpha-agarase A33, and carrying out enzymolysis at 38-42 DEG C for 20-28 hours; and boiling, centrifuging and filtering to obtain supernate, namely crude oligosaccharide liquid containing agarotetraose. The preparation method of the agaro-oligosaccharide is used for preparing agarotriose, agarobiose and agarotetraose, and the product purity is high. The method can provide guidance for subsequent large-scale industrial production of agaro-oligosaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of agar oligosaccharides, and belongs to the technical field of agar degradation. BACKGROUND

[0002] Agar oligosaccharides are oligosaccharides with a degree of polymerization of 2-20 obtained by hydrolysis of agarose, mainly composed of repeating units of agarobiose, including agar oligosaccharides and neoagar oligosaccharides, with 3,6-endothelial alpha-L-galactose residues as the reducing end of agar oligosaccharides and beta-D-galactose residues as the reducing end of neoagar oligosaccharides. Agar oligosaccharides have antioxidant, anti-inflammatory, liver protection, and virus inhibition activities, and are widely used.

[0003] Currently, agar oligosaccharides are mostly prepared by enzymatic hydrolysis, which has the advantages of mild conditions and strong substrate specificity, but also has the disadvantages of low enzyme activity, low product purity, long reaction time, and many impurities. Therefore, it is of great significance to explore the preparation method of agar oligosaccharides in order to improve the enzymatic efficiency and product purity, which is of great significance for the large-scale production and application of agar oligosaccharides. SUMMARY

[0004] In view of the above prior art, the present application provides a preparation method of agar oligosaccharides.

[0005] The present application is achieved by the following technical solutions: The preparation method of agar oligosaccharides comprises the following steps: (1) 12-18 g of agar is dissolved in 100 mL of 0.02-0.03 g / mL citric acid monohydrate solution, and acid hydrolysis is carried out at 88-92℃ for 55-65 min to obtain an acid hydrolysis solution; then, the pH of the acid hydrolysis solution is adjusted to 6.5-7.5, centrifuged, filtered, and β-agarase AgWH50B is added to the supernatant, with an enzyme amount of 0.48-0.5 U / mL, and enzyme hydrolysis is carried out at 30-35℃ for 20-28 h; boiling, centrifugation, filtration, and the supernatant is the crude oligosaccharide solution containing agarotriose; The amino acid sequence of the β-agarase AgWH50B is shown in SEQ ID NO. 1; (2) 8-12 g of agar is dissolved in 100 mL of 0.08-0.12 mol / L phosphoric acid solution, and degradation is carried out at 97-103℃ for 55-65 min; the degradation solution is centrifuged, filtered, and the supernatant is the crude oligosaccharide solution containing agarobiose; (3) 1.2-1.8 g agarose is dissolved in 100 mL deionized water, and alpha-agarase A33 is added, the enzyme amount is 0.45-0.55 U / mL, and the enzyme is hydrolyzed at 38-42°C for 20-28 h; the enzyme hydrolysis solution is boiled, centrifuged, filtered, and the supernatant is the crude oligosaccharide solution containing agarotetraose; The amino acid sequence of the alpha-agarase A33 is shown in SEQ ID NO. 2.

[0006] Further, in the step (1), the beta-agarase AgWH50B is prepared by the following method: the activated beta-agarase AgWH50B producing engineering bacteria (a conventional method of constructing an Escherichia coli recombinant strain) is inoculated into a fermentation medium at an inoculation amount of 1% (volume ratio), and is cultured at 37°C, 200 r / min on a shaking table for 4 h, then 1.5‰ isopropyl thiogalactoside (IPTG) is added, and the culture is cultured at 20°C, 200 r / min on a shaking table for 4 h; the culture is centrifuged and broken, and the crude enzyme solution is obtained, and the pure enzyme powder is obtained by nickel column purification and freeze-drying; the components of the fermentation medium are 2% yeast powder, 1.5% sucrose, 1% sodium chloride, and the rest is water, and the initial pH is 7.0.

[0007] Further, in the steps (1), (2), and (3), after the crude oligosaccharide solution is obtained, it is freeze-dried into powder, then the freeze-dried powder is dissolved in deionized water, and purified by Bio-gel P2 column, and the purified solution is collected and freeze-dried to obtain agarotriose, agarobiose or agarotetraose.

[0008] Further, the specific operation of the step (1) is as follows: 15 g agarose is dissolved in 100 mL 2.5% citric acid monohydrate solution, and acid hydrolysis is carried out at 90°C for 60 min to obtain an acid hydrolysis solution; then, the pH of the acid hydrolysis solution is adjusted to 7.0, centrifuged, filtered, and the supernatant is added with beta-agarase AgWH50B pure enzyme powder, the enzyme amount is 0.485 U / mL, and the enzyme is hydrolyzed at 30°C for 24 h; boiled, centrifuged, filtered, and the supernatant is the crude oligosaccharide solution; freeze-dried into powder, then the freeze-dried powder is dissolved in deionized water, and purified by Bio-gel P2 column, and the purified solution is collected and freeze-dried to obtain agarotriose.

[0009] Further, the specific operation of the step (2) is as follows: 10 g agarose is dissolved in 100 mL 0.1 mol / L phosphoric acid solution, and degradation is carried out at 100°C for 60 min; the degradation solution is centrifuged, filtered, and the supernatant is the crude oligosaccharide solution; freeze-dried into powder, then the freeze-dried powder is dissolved in deionized water, and purified by Bio-gel P2 column, and the purified solution is collected and freeze-dried to obtain agarobiose.

[0010] Further, the specific operation of the step (3) is: 1.5 g agarose is dissolved in 100 mL deionized water, and alpha-agarase A33 is added, the enzyme amount is 0.5 U / mL, and the enzyme is hydrolyzed at 40 DEG C, 200 r / min water bath shaker for 24 h; boiling, centrifugation, filtration, and the supernatant is the crude oligosaccharide solution; freeze-drying into powder, then dissolving the freeze-dried powder in deionized water, purifying by using Bio-gel P2 column, collecting the purified liquid, and freeze-drying, to obtain agarotetraose.

[0011] The preparation method of the agarose oligosaccharide of the present application is used for preparing agarotriose, agarobiose and agarotetraose, the enzyme activity of the enzyme used is high, the product purity is high, and the reaction time is short. The fermentation culture conditions of the beta-agarase AgWH50B for preparing agarotriose are optimized, and the beta-agarase AgWH50B can be prepared with high efficiency. The present application constructs a preparation system for efficiently preparing agarose oligosaccharide, which can provide guidance for subsequent large-scale industrial production of agarose oligosaccharide.

[0012] Various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Effect of nitrogen source on relative enzyme activity, wherein the letter annotation method is used to represent the difference significance, and the same below.

[0014] Figure 2 Effect of carbon source on relative enzyme activity.

[0015] Figure 3 Optimization results of sucrose addition amount.

[0016] Figure 4 Effect of metal ions on relative enzyme activity.

[0017] Figure 5 Effect of initial pH on relative enzyme activity.

[0018] Figure 6 Effect of fermentation temperature on relative enzyme activity.

[0019] Figure 7 Effect of IPTG addition amount on relative enzyme activity.

[0020] Figure 8 Effect of induction time on relative enzyme activity.

[0021] Figure 9 High-phase liquid chromatography detection results of agarose oligosaccharide.

[0022] Figure 10 Mass spectrometry detection results of agarose oligosaccharide.

[0023] Figure 11 Results of high phase liquid chromatography detection of agarobile.

[0024] Figure 12 Mass spectrometry results of agarobionic acid.

[0025] Figure 13 Results of high phase liquid chromatography detection of agartetraose.

[0026] Figure 14 Mass spectrometry results of agarose. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0028] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0029] Experiment 1: Optimization of preparation conditions for β-agarase AgWH50B 1.1 Basic Information β-Agarase AgWH50B is an agarase reported in the prior art that can be used to prepare new agarotetrasaccharides, and its amino acid sequence is shown in SEQ ID NO.1. However, β-Agarase AgWH50B prepared by conventional fermentation culture has low enzyme activity. Therefore, this experiment aims to explore and optimize the fermentation culture conditions of β-Agarase AgWH50B to achieve efficient preparation of β-Agarase AgWH50B.

[0030] The amino acid sequence of β-agarase AgWH50B is shown in SEQ ID NO.1, as follows: MTFTKSKIATVLSLSLLGIYGCASTTPQNEQAAAGEQVVEDMGGALPDFESDKFFSKLKAEHAKASAVTDTGVTAGSQALKIDFDSVNEANKFKFWPNVKLHPDTGNWNWNAKGSLTLDVTNPTDSTANIILKIADNVGVMGAGDNQLNYALSVPAGETVPVEMIFNGSKRKLDGYWGGEKINLRKLVEFQIFVQGPIDQQSVIVDNFALVDATGDFVEASGAEEVVTGPVPTVLAITDFEKGQDSFISAERSVATTISPVKTDDGAAIDVLFSASNSYPNITFRPDVPWDWSGQGDFNVAFDMVNKSDEPLQLFVRVDDDEHEAFGGTANGVQNSWSGYVTIAPNDEGTYYLSLMPAGDQMVSGMRGEPPKKSYKAQAISYGWGDNNLDLSNIYSMQLYLQNPTADQKLQISSVRLIPNLESDTSRYEGLLDEFGQYTGQDWAQKVKSLEDLQAAGAAELDSLEHPTQLPDRSKFGGWADGPKLEATGFFRAEKVDGKWALVDPEGYLFFVTGLDNIRMDDTVTITGVDFSNKETREGREVASELRNSMFTWLPEYDDVLAESYDYADWIHTGALKKGEVFSFYSANLQRKYQTSREEALKIWKDVTLNRMQDWGFTTLGNWADPKFYDNQQIAYAANGWIFGDHARISTGNDYWGPIHDPFDPEFAVSTRKMAEKVASEVSKDDPWLMGIFVDNEISWGNTKNEANHYGLVVNALSYDIKESPAKAAFTKHLQDKYSSIDALNQSWGTKVTSWADFEVSFDHRSRLSSSMKKDYSEMLQMLSEKYFSTVQAELKKVLPNHMYLGARFADWGVTPEIARGAAPYVDVMSYNLYAEDLNSKGDWSLLPELDKPSIIGEFHFGATDTGLFHGGIVSASNQADRAKKYTHYMQSIVDNPYFVGAHWFQYLDSPTTGRAWDGENYNVGFVSITDTPYQELIDAAKQFNRDLYNLRYKK.

[0031] 1.2 Experimental Methods 1.2.1 Activation of Agarase AgWH50B The engineered bacteria producing Agarase AgWH50B were preserved in the laboratory at -80℃.

[0032] 1.2.1.1 Plate screening 1 μL of the bacteria was taken from the bacteria preservation tube containing the engineered bacteria producing Agarase AgWH50B by using a pipette gun and streaked on a plate containing solid culture medium with 0.5‰ kanamycin (Kana) resistance, and incubated in a 37℃ incubator for 12 h. Well-grown single colonies were selected for test tube culture activation.

[0033] 1.2.1.2 Test tube activation The single colonies obtained by plate streaking were transferred to LB medium and incubated at 37℃, 200 r / min on a shaking table for 8-10 h to obtain a seed solution.

[0034] 1.2.1.3 Fermentation and disruption In a clean bench, 0.5‰ Kana resistance was added to the sterilized 50 mL LB medium, and then 50 μL of the seed solution was added thereto, and incubated at 37℃, 200 r / min on a shaking table for about 4 h until the OD 600 was 0.6-0.8, 1‰ IPTG was added and the expression was induced at 20℃, 200 r / min on a shaking table for 16-18 h.

[0035] After the fermentation was completed, the fermentation liquid was centrifuged at 8000 r / min for 15 min to obtain a bacterial precipitate, which was resuspended. The resuspended bacterial solution was ultrasonically disrupted (400 W, 30 min), followed by centrifugation at 8000 r / min for 15 min to obtain a supernatant, i.e., a crude enzyme solution, and the enzyme activity was determined.

[0036] The crude enzyme solution was filtered through a 0.22 μm filter membrane, and subjected to nickel column purification. For each 10 mL sample, a certain volume of 5 mM imidazole solution was used for equilibration. After the sample was added, the target protein was eluted, and the elution was performed in order from low concentration to high concentration imidazole solution (40 mM, 80 mM, 120 mM, 200 mM). The 120 mM eluate was collected, desalted, and freeze-dried to obtain a pure enzyme powder.

[0037] 1.2.2 Determination of enzyme activity The activity of crude enzyme solution of agarase AgWH50B was determined by DNS method. 200 μL of crude enzyme solution was added with 200 μL of 0.3% agarose solution, and reacted at 40°C for 60 min. After the reaction, the enzyme was inactivated by boiling water for 10 min. After inactivation, 100 μL of the reaction solution was taken out and centrifuged at 12000 r / min for 5 min. 150 μL of DNS was added to the reaction solution, and boiled for 10 min. After cooling, 200 μL of the supernatant was added to a 96-well plate, and the absorbance value at 540 nm was determined by an enzyme marker.

[0038] The agarase unit enzyme activity (U) was defined as the amount of enzyme required for hydrolyzing 1 μmol of agarose to produce reducing sugar per minute under the optimal reaction conditions.

[0039] 1.2.3 Optimization of medium components 1.2.3.1 Selection of nitrogen source LB medium (yeast powder 5 g / L, i.e. 0.5%; peptone 10 g / L, i.e. 1%; NaCl 10 g / L, i.e. 1%) was used as a control. Seven components (2% yeast powder, 2% peptone, 2% urea, 2% ammonium sulfate, 2% ammonium chloride, 1% yeast powder + 1% ammonium sulfate, and 1% peptone + 1% ammonium sulfate) were selected as nitrogen sources of the medium (i.e. one of the seven components was used to replace yeast powder and peptone in the LB medium), sterilized at 121°C for 20 min, cooled, and 0.5‰ Kana resistance was added in a clean bench. 1% activated bacterial solution was inoculated, and cultured at 37°C and 200 r / min for 4 h. The OD 600 was 0.6-0.8. 1‰ IPTG was added, and the culture was incubated at 20°C and 200 r / min for 16 h. The relative enzyme activity under each nitrogen source condition was calculated based on the highest enzyme activity of the experimental group as 100%.

[0040] 1.2.3.2 Selection of carbon source Based on the optimal nitrogen source determined above, 2% yeast powder and 1% NaCl were used as a control. Six components (1% glucose, 1% lactose, 1% sucrose, 1% corn dextrin, 1% soluble starch, and 1% potato starch) were selected as carbon sources of the medium (i.e. one of the six components was added to the control). The medium was detected as described above in 1.2.3.1.

[0041] 1.2.3.3 Selection of metal ions Based on the optimal nitrogen source and the optimal carbon source determined above, the medium (2% yeast powder, 1.5% sucrose, and 1% NaCl) was used as a control. 1% NaCl in the control medium was replaced with Mg 2+ , Mn 2+, Cu 2+ , K + , Fe 3+ (all are chloride salts). The medium detection is the same as 1.2.3.1 above.

[0042] 1.2.4 Optimization of culture conditions 1.2.4.1 Optimization of initial pH On the basis of the above-mentioned determination of the optimal fermentation medium, the initial pH of the medium was adjusted to 5.0, 6.0, 7.0, 8.0, respectively, using low concentration hydrochloric acid or sodium hydroxide solution. The medium detection is the same as 1.2.3.1 above.

[0043] 1.2.4.2 Optimization of fermentation temperature On the basis of the above-mentioned determination of the optimal initial pH, the fermentation temperature was set to 18℃, 20℃, 22℃, 24℃, respectively. The fermentation medium was sterilized at 121℃ for 20 min, cooled, 0.5‰ Kana resistance was added in the clean bench, 1% activated bacteria liquid was inoculated, and cultured in a 37℃, 200 r / min shaking incubator for 4 h until OD 600 was 0.6-0.8, 1‰ IPTG was added, and cultured in a 200 r / min shaking incubator at the set fermentation temperature for 16 h. The relative enzyme activity under each nitrogen source condition was calculated with the highest enzyme activity group as 100%.

[0044] 1.2.4.3 Optimization of inducer addition amount On the basis of the above-mentioned determination of the optimal initial pH and fermentation temperature, the addition amount of inducer was set to 0.5‰, 1‰, 1.5‰, 2‰, respectively. The fermentation medium was sterilized at 121℃ for 20 min, cooled, 0.5‰ Kana resistance was added in the clean bench, 1% activated bacteria liquid was inoculated, and cultured in a 37℃, 200 r / min shaking incubator for 4 h until OD 600 was 0.6-0.8, different addition amounts of IPTG were added, and cultured in a 200 r / min shaking incubator at 20℃ for 16 h. The relative enzyme activity under each nitrogen source condition was calculated with the highest enzyme activity group as 100%.

[0045] 1.2.4.4 Optimization of induction time On the basis of the above-mentioned determination of the optimal initial pH, fermentation temperature, and inducer addition amount, the induction time was set to 2 h, 4 h, 6 h, 8 h, 16 h, respectively. The fermentation medium was sterilized at 121℃ for 20 min, cooled, 0.5‰ Kana resistance was added in the clean bench, 1% activated bacteria liquid was inoculated, and cultured in a 37℃, 200 r / min shaking incubator for 4 h until OD 600The relative enzyme activity of each nitrogen source was calculated based on the highest enzyme activity of the experimental group. The relative enzyme activity of each nitrogen source was calculated based on the highest enzyme activity of the experimental group.

[0046] 1.2.5 Data analysis All experiments were tested in triplicate. IBM SPSS Statistics 27 statistical software was used for statistical analysis, with a significance level of 0.05. Data were expressed as mean ± standard deviation (SD). One-way ANOVA and Tukey's multiple comparison with 95% confidence intervals were used to determine significance. p <0.05).

[0047] 1.3 Results and discussion 1.3.1 Analysis of nitrogen source optimization results The effect of nitrogen source on relative enzyme activity is shown in Figure 1 It can be seen that different nitrogen sources and addition amounts have a greater impact on the enzyme activity of agarase AgWH50B. When the nitrogen source is 2% yeast powder, the relative enzyme activity of agarase AgWH50B in the prepared crude enzyme solution is the highest, which is nearly 300% higher than that of the control group (the enzyme activity of the crude enzyme solution of the control group is 27 U / mL). Therefore, 2% yeast powder was selected as the nitrogen source for subsequent experiments.

[0048] 1.3.2 Analysis of carbon source optimization results The effect of carbon source on relative enzyme activity is shown in Figure 2 It can be seen that when the carbon source is sucrose, the relative enzyme activity of agarase AgWH50B in the prepared crude enzyme solution is the highest, while when the carbon source is corn dextrin, soluble starch, and potato starch, the relative enzyme activity is reduced.

[0049] Subsequently, the addition amount of sucrose (0.5%, 1%, 1.5%, 2%, 2.5%, 3%) was optimized, and the optimization results of the addition amount of sucrose are shown in Figure 3 Within the range of 0.5% to 3%, the relative enzyme activity showed a trend of first increasing and then decreasing. When the addition amount of sucrose was 2%, the relative enzyme activity was the highest, followed by 1.5%, but there was no significant difference between the two. Considering factors such as production cost, 1.5% sucrose was selected as the carbon source for subsequent experiments.

[0050] 1.3.3 Analysis of metal ion optimization results The effect of metal ion on relative enzyme activity is shown in Figure 4 It can be seen that Mg 2+ , Mn 2+ , Cu 2+ , K + , and Fe 3+The relative enzyme activity of agarase was reduced to different degrees. Therefore, the selection of 1% NaCl as the medium component is the best strategy.

[0051] 1.3.4 Optimization analysis of culture conditions 1.3.4.1 Optimization analysis of initial pH The initial pH of the fermentation medium is crucial for the normal growth of Escherichia coli and the induced expression of enzymes. The effect of the initial pH on the relative enzyme activity is shown in Figure 5 It can be seen that when the initial pH of the fermentation medium is 5.0, the ability of Escherichia coli to induce and express enzymes is poor, which may be related to the fact that low pH is not conducive to the growth of bacteria. When the initial pH is 6.0-8.0, there is no significant difference in the relative enzyme activity of agarase secreted by Escherichia coli. Therefore, considering various factors such as operation, cost, and enzyme expression, the initial pH of the fermentation medium is finally determined to be 7.0.

[0052] 1.3.4.2 Optimization analysis of fermentation temperature In addition to the initial pH, the induction temperature of fermentation is also an important factor affecting the expression of agarase. The effect of the fermentation temperature on the relative enzyme activity is shown in Figure 6 It can be seen that the effect of the fermentation temperature on the relative enzyme activity of agarase shows a trend of first increasing and then decreasing, and the relative enzyme activity of agarase is the highest when the fermentation temperature is 20°C. Therefore, 20°C is selected as the fermentation temperature for subsequent experiments.

[0053] 1.3.4.3 Optimization analysis of IPTG addition amount The effect of the IPTG addition amount on the relative enzyme activity is shown in Figure 7 It can be seen that with the increase of the IPTG addition amount, the relative enzyme activity of agarase shows a trend of first increasing and then decreasing. When the addition amount is 1.5‰, the relative enzyme activity of agarase is the highest, and the difference is significant. Therefore, an addition amount of 1.5‰ is selected for subsequent experiments.

[0054] 1.3.4.4 Optimization analysis of induction time The effect of the induction time on the relative enzyme activity is shown in Figure 8 It can be seen that when the induction time is 2 h, the expression amount of agarase is low, and the relative enzyme activity is poor. When the induction time is 4 h, the relative enzyme activity of agarase is the highest, and the enzyme activity of the crude enzyme solution is 279 U / mL. When the induction time is greater than 4 h, the relative enzyme activity of agarase decreases, which may be due to the inhibitory effect of the expressed protein. Therefore, the induction time of 4 h is selected as the best induction time.

[0055] Based on the above optimization analysis results of the culture conditions, the optimal fermentation medium for the agarase AgWH50B is: 2% yeast powder, 1.5% sucrose, 1% sodium chloride, and the rest is water. The optimal culture conditions are: initial pH of 7.0, fermentation temperature of 20°C, IPTG addition amount of 1.5‰, and induction time of 4 h. Under these optimal conditions, the enzyme activity of the extracted crude enzyme solution is 279 U / mL, which is about 10 times that of the initial culture conditions (LB medium, initial pH of 7.0, fermentation temperature of 20°C, IPTG addition amount of 1‰, and induction time of 16 h), and the fermentation time is shortened to one fourth, greatly improving the fermentation efficiency of the agarase.

[0056] Experiment 2 High-efficiency preparation of agar oligosaccharide 2.1 Preparation of agarotriose 15 g of agar (purchased from Beijing Solabio Technology Co., Ltd.) was dissolved in 100 mL of 0.025 g / mL citric acid monohydrate solution, placed in a high-pressure steam sterilization pot, and acid hydrolyzed at 90°C for 60 min to reduce the degradation difficulty of agar, obtaining an acid hydrolysate. Then, the pH of the acid hydrolysate was adjusted to neutral (7.0), centrifuged at 10,000 r / min for 10 min, filtered, and β-agarase AgWH50B pure enzyme powder was added to the supernatant, with an enzyme addition amount of 0.485 U / mL, and the enzyme hydrolysis was carried out at 30°C for 24 h; boiling, centrifuging at 10,000 r / min for 10 min, filtering, and the supernatant was the crude oligosaccharide solution, which was freeze-dried into a powder.

[0057] Then, 1 g of the freeze-dried powder was dissolved in 15 mL of deionized water, purified by Bio-gel P2 column, and the purified liquid within the appropriate range was collected and freeze-dried to obtain agar oligosaccharide. The agar oligosaccharide was dissolved in deionized water to prepare a 1 mg / mL agar oligosaccharide solution, which was used for the following detection.

[0058] The high-performance liquid chromatography system equipped with a differential refractometer detector was used for detection, and the detection conditions were as follows: the Superdex 30 increese 10 / 300 gel filtration column was selected, the column temperature was set to 30°C, the mobile phase was 0.2 mol / L ammonium bicarbonate, and the flow rate was 0.4 mL / min. The sample was filtered by a 0.22 μm filter membrane, the injection amount was 100 μL, and the column was sealed with 20% anhydrous ethanol after use.

[0059] In addition, the ESI-MS method was used to determine the molecular weight of each sample on the microTOF-Q II device. The detection conditions were as follows: ion spray voltage: 4 kV, and ion source temperature: 350°C.

[0060] 2.2 Preparation of agarobiose Dissolve 10 g agar in 100 mL of 0.1 mol / L phosphoric acid solution, and degrade at 100°C for 60 min. Centrifuge, filter, and freeze-dry the supernatant to obtain a powder.

[0061] Then, dissolve 1 g of the powder in 15 mL of deionized water, purify using a Bio-gel P2 column, collect the purified solution in an appropriate range, and freeze-dry to obtain chitobiose. Dissolve the chitobiose in deionized water to prepare a 1 mg / mL chitobiose solution for the following detection.

[0062] Use a high-performance liquid chromatography system equipped with a differential refractometer detector for detection, and the detection conditions are as follows: use a Sugar Pak I chromatographic column (Waters, 6.5*300 mm), set the column temperature to 75°C, use 50 mg / L EDTA-Na2Ca as the mobile phase, and set the flow rate of the mobile phase to 0.5 mL / min. Filter the sample through a 0.22 μm filter, and inject 10 μL of the sample.

[0063] The mass spectrometry detection method is the same as that in 2.1 above.

[0064] 2.3 Preparation of chitotetraose Dissolve 1.5 g of agar in 100 mL of deionized water, add pure enzyme powder of α-agarase A33, and add 0.5 U / mL of enzyme, and then perform enzyme degradation at 40°C and 200 r / min on a water bath shaker for 24 h. Boil, centrifuge, filter, and freeze-dry the supernatant to obtain a powder.

[0065] Then, dissolve 1 g of the powder in 15 mL of deionized water, purify using a Bio-gel P2 column, collect the purified solution in an appropriate range, and freeze-dry to obtain chitotetraose. Dissolve the chitotetraose in deionized water to prepare a 1 mg / mL chitotetraose solution for the following detection.

[0066] The high-performance liquid chromatography detection and mass spectrometry detection methods are the same as those in 2.1 above.

[0067] The α-agarase A33 is an enzyme reported in the prior art, and its amino acid sequence is shown in SEQ ID NO. 2. Its preparation method is a conventional technical means.

[0068] The amino acid sequence of the α-agarase A33 is shown in SEQ ID NO. 2, and is as follows:

[0069] 2.4 Results and Discussion 2.4.1 Preparation of Yontriose The results of high phase liquid chromatography detection of oligoagarose are shown in Figure 9 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in Figure 10 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in

[0070] 2.4.2 Preparation of Yondiose The results of high phase liquid chromatography detection of oligoagarose are shown in Figure 11 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in Figure 12 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in

[0071] 2.4.3 Preparation of Yontriose The results of high phase liquid chromatography detection of oligoagarose are shown in Figure 13 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in Figure 14 It can be seen that the main product in the product is yontriose (A3), and the main impurity is yondiose (A2). The results of mass spectrometry detection of oligoagarose are shown in

[0072] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing oligoagarose, characterized in that, Comprise the following steps: (1) 12-18 g agar gel is dissolved in 100 mL concentration of 0.02-0.03 g / mL citric acid monohydrate solution, 88-92 ℃ acidolysis 55-65 min, the acidolysis liquid is obtained; then, the pH of the acidolysis liquid is adjusted to 6.5-7.5, centrifuged, filtered, and β-agarase AgWH50B is added to the supernatant, the enzyme amount is 0.48-0.5 U / mL, and the enzyme is hydrolyzed at 30-35 ℃ for 20-28 h; boiling, centrifugation, filtration, the supernatant is the crude oligosaccharide liquid containing agarose triose; The amino acid sequence of the β-agarase AgWH50B is shown in SEQ ID NO. 1; (2) 8-12 g agar gel is dissolved in 100 mL concentration of 0.08-0.12 mol / L phosphoric acid solution, and degraded at 97-103 ℃ for 55-65 min; the degradation liquid is centrifuged, filtered, and the supernatant is the crude oligosaccharide liquid containing agarose disaccharide; (3) 1.2-1.8 g agar gel is dissolved in 100 mL deionized water, and α-agarase A33 is added, the enzyme amount is 0.45-0.55 U / mL, and the enzyme is hydrolyzed at 38-42 ℃ for 20-28 h; the enzyme hydrolysis liquid is boiled, centrifuged, filtered, and the supernatant is the crude oligosaccharide liquid containing agarose tetrasaccharide; The amino acid sequence of the α-agarase A33 is shown in SEQ ID NO.

2.

2. The method for preparing oligoagar according to claim 1, characterized in that, In the step (1), the β-agarase AgWH50B is prepared by the following method: the activated β-agarase AgWH50B producing engineering bacteria are inoculated into the fermentation medium at an inoculation amount of 1%, and cultured at 37 ℃, 200 r / min on a shaking bed for 4 h, then 1.5 ‰ isopropylthio galactoside is added, and cultured at 20 ℃, 200 r / min on a shaking bed for 4 h; the culture liquid is centrifuged and broken, and the crude enzyme liquid is obtained; the composition of the fermentation medium is: 2% yeast powder, 1.5% sucrose, 1% sodium chloride, and the rest is water, and the initial pH is 7.

0.

3. The method for preparing agar oligosaccharides according to claim 1, characterized in that: In the steps (1), (2), (3), after the crude oligosaccharide liquid is obtained, it is freeze-dried into powder, then the freeze-dried powder is dissolved in deionized water, purified by Bio-gel P2 column, the purified liquid is collected, and freeze-dried, agarose triose, agarose disaccharide or agarose tetrasaccharide is obtained.

4. The method of preparing oligoagar according to claim 1, characterized in that: The specific operation of the step (1) is: 15 g agar gel is dissolved in 100 mL concentration of 2.5% citric acid monohydrate solution, 90 ℃ acidolysis 60 min, the acidolysis liquid is obtained; then, the pH of the acidolysis liquid is adjusted to 7.0, centrifuged, filtered, and β-agarase AgWH50B pure enzyme powder is added to the supernatant, the enzyme amount is 0.485 U / mL, and the enzyme is hydrolyzed at 30 ℃ for 24 h; boiling, centrifugation, filtration, the supernatant is the crude oligosaccharide liquid; freeze-dried into powder, then the freeze-dried powder is dissolved in deionized water, purified by Bio-gel P2 column, the purified liquid is collected, and freeze-dried, agarose triose is obtained.

5. The method of preparing oligoagar according to claim 1, characterized in that: The specific operation of the step (2) is as follows: 10 g agar is dissolved in 100 mL of a 0.1 mol / L phosphoric acid solution, and degraded at 100 ℃ for 60 min; the degradation solution is centrifuged, filtered, and the supernatant is the crude oligosaccharide solution; the solution is freeze-dried into a powder, then the freeze-dried powder is dissolved in deionized water, purified by using a Bio-gel P2 column, the purified solution is collected, and freeze-dried, to obtain agarobiose.

6. The method of preparing oligoagar according to claim 1, characterized in that: The specific operation of the step (3) is as follows: 1.5 g agar is dissolved in 100 mL of deionized water, and α-agarase A33 is added, with an enzyme amount of 0.5 U / mL, and the solution is enzymolyzed at 40 ℃ in a water bath shaker at 200 r / min for 24 h; the solution is boiled, centrifuged, filtered, and the supernatant is the crude oligosaccharide solution; the solution is freeze-dried into a powder, then the freeze-dried powder is dissolved in deionized water, purified by using a Bio-gel P2 column, the purified solution is collected, and freeze-dried, to obtain agarotetraose.

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