Preparation method of mannan oligosaccharide

Abstract

The invention discloses a preparation method of mannan oligosaccharide, and relates to the technical field of organic agricultural fertilizers. According to the method, high-purity and high-efficiency mannan oligosaccharide is prepared by using an advanced process, and the method comprises the following specific steps: pretreatment of raw materials: mixing konjaku flour or guar gum with water to obtain a colloidal solution; adding beta-mannase and alpha-galactosidase into the colloidal solution, and carrying out enzymolysis under the assistance of a pulsed electric field; performing secondary separation on the enzymatic hydrolysate through an ultrafiltration membrane and a nanofiltration membrane, and collecting trapped fluid. A calcium ion solution is added into the trapped fluid for a complex reaction, then spray drying is conducted, and mannan oligosaccharide powder is obtained. According to the method, mannan oligosaccharide with concentrated purity and polymerization degree is prepared through ultrahigh pressure homogenization, pulsed electric field assisted enzymolysis and membrane separation technologies, the method has the advantages of high efficiency and environmental protection, and the preparation efficiency and the product quality are remarkably improved. The prepared mannan oligosaccharide can be applied to organic agricultural fertilizers, and can be compounded with humic acid and other substances to improve the biological activity and soil fertility of the fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of organic agricultural fertilizers, in particular to a method for preparing manno-oligosaccharide. Background Art

[0002] Mannooligosaccharides (MOS) are oligosaccharides composed of mannose units connected by β-1,4 glycosidic bonds, and are a type of functional oligosaccharide. Mannooligosaccharides are mainly derived from the hemicellulose components of plant cell walls, such as the hydrolysis products of galactomannan widely found in the seeds of leguminous plants. As a bioactive substance, mannooligosaccharides have a variety of biological effects such as enhancing the immune function of organisms, promoting growth and development, and improving intestinal health. Therefore, they have received widespread attention and application in the field of organic agriculture. In agricultural production, with the continuous increase in people's demand for green food, the production of safe and low-residue agricultural products has become the goal pursued by agricultural workers. As a natural bioactive substance, mannooligosaccharides are gradually being developed and utilized in terms of increasing crop yields, enhancing crop disease resistance, and improving soil environment.

[0003] At present, the preparation methods of manno-oligosaccharides mainly include chemical method, physical method and bio-enzyme method. Chemical oxidation degradation method: This method usually uses H2O2 as an oxidant to carry out degradation reaction under acidic conditions. Studies have shown that the optimal conditions for preparing oligosaccharides with a molecular weight of less than 2000 can be determined through orthogonal experiments with 4 factors and 3 levels. The chemical oxidation degradation method is easy to operate, but it has the disadvantages of high energy consumption, high pollution and low product purity. Microwave degradation method: This is a new type of physical degradation method that can reduce energy consumption, reduce pollution, save time and raw materials. Studies have shown that under the action of microwave field, different solvents and salt effects have a significant effect on the molecular weight change of mannan. Enzymatic hydrolysis: This method uses β-mannanase to specifically hydrolyze mannan, which has the advantages of simple hydrolysis process, low product polymerization degree and high purity. Using sesbania gum as raw material, galactomannan oligosaccharides can be obtained by hydrolysis of β-mannanase. This method is milder than chemical and physical methods, and the product has higher biological activity. Coordination complex method: Manno oligosaccharide and copper chloride are used as raw materials, and manno oligosaccharide copper (II) complex can be prepared by adjusting factors such as reaction time, pH value, reaction temperature, etc. This method can enhance certain biological activities of manno oligosaccharides, but the process is complicated and the cost is high.

[0004] At present, my country's fertilizer industry still faces many problems: fertilizer products do not match agricultural needs, technology integration cannot meet the needs of agricultural modernization, and the fertilizer management system is not conducive to the implementation of scientific products and technologies. To solve these problems, it is necessary to build an innovation system in my country's fertilizer field through fertilizer product innovation, technological innovation, organic substitution, precision fertilization and other methods. The application of mannan oligosaccharides in organic fertilizers is one of the important directions of this innovation. Mannan oligosaccharides added to organic fertilizers can improve the physical and chemical properties of soil, improve soil fertility, increase crop yield and quality, and enhance crop resistance. Especially in the application of special organic fertilizers for specific crops such as ginseng, mannan oligosaccharides can meet the needs of crop growth, cultivate soil and increase yield. Although mannan oligosaccharides have broad application prospects in the field of agriculture, there are still some problems in their preparation and application: the preparation process is not mature enough: the existing mannan oligosaccharide preparation methods such as chemical methods have problems such as high energy consumption and high pollution; although the enzyme method is green and environmentally friendly, it is costly, and the source and stability of the enzyme also restrict its large-scale application. Product quality control is difficult: Manno oligosaccharides obtained by different preparation methods have differences in molecular weight distribution, degree of polymerization, purity, etc., which affects their effect in agricultural applications. Application technology is not systematic enough: The application technology of manno oligosaccharides in agricultural fertilizers and pesticides is still imperfect, and there is a lack of systematic application guidance, resulting in certain differences in the use effect. The level of industrialization is not high: At present, the production scale of manno oligosaccharides is generally small, and the level of industrialization is not high, which makes it difficult to meet the growing market demand.

[0005] As a bioactive substance, the synergistic effect of manno-oligosaccharides with agricultural inputs such as fertilizers and pesticides still needs to be further studied. How to achieve synergistic effects while ensuring the efficacy of each is an important direction for future research. Summary of the invention

[0006] The existing technology has the following problems: the activity of β-mannanase in the traditional enzymatic hydrolysis method is generally lower than 3000U / mL, resulting in more than 6 hours of hydrolysis of konjac gum (15% concentration), and the hydrolysis rate is less than 36%. Source CN115216499A shows that yeast cell wall enzymatic hydrolysis requires step-by-step protease and polysaccharide enzyme treatment, and the process is highly complex. The monosaccharide residue of the product obtained by the conventional centrifugation-spray drying process is more than 15%, and the molecular weight distribution range is wide (DP2-10), which affects the efficacy of prebiotics. Patent CN103468766B points out that the purity of the traditional method is only 60-75%, while membrane separation technology can be increased to 95%. The unit energy consumption of the microwave-assisted degreasing process is 3.2kW·h / kg, and the investment cost of supercritical extraction equipment increases by 40%. The acid hydrolysis method needs to maintain the reaction conditions of 80℃, and the heat energy loss accounts for 35% of the total cost. The existing process relies on konjac flour (viscosity>5000mPa·s) or yeast cell walls. The former has seasonal supply fluctuations, and the latter requires petroleum ether to defatted when extracting glucan, which brings safety risks. Yeast cell wall enzymatic hydrolysis produces 30-40% glucan byproducts. The existing technology does not achieve high-value utilization of co-production, resulting in waste of resources.

[0007] To solve the above problems, the present invention provides the following technical solution: A method for preparing manno-oligosaccharides, comprising the following steps: (1) raw material pretreatment: konjac flour (CAS No.: 37220-17-0, Jiangsu Duoyang Bioengineering) or guar gum (CAS No.: 9000-30-0, Shandong Dongda Biochemical) and water are mixed in a mass ratio of 1: (5-8), and after ultra-high pressure homogenization treatment, the pH of the system is adjusted to 5.5-6.5 to obtain a colloidal solution with a viscosity of 3000-5000 mPa·s; (2) composite enzymatic hydrolysis: β-glycine is added to the colloidal solution; The total amount of enzyme added is 0.5-1.2% of the mass of the colloidal solution. Under the assistance of a pulsed electric field, the enzymatic hydrolysis is carried out for 2-4 hours. (3) Separation and purification: The enzymatic hydrolyzate is separated by ultrafiltration membrane and nanofiltration membrane, and the retentate is collected. (4) Product purification: 0.05-0.1 mol / L calcium ion solution is added to the retentate, and after complexation reaction, the manno-oligosaccharide powder is obtained by spray drying.

[0008] Preferably, the parameters of the ultrahigh pressure homogenization treatment in step (1) are as follows: pressure 800-1200 bar, 3-5 cycles; in step (2), the enzyme activity of β-mannanase is ≥5000 U / mL, and the enzyme activity of α-galactosidase is ≥800 U / mL.

[0009] Preferably, the parameters of the pulse electric field in step (2) are as follows: field strength 15-25 kV / cm, frequency 100-200 Hz; the parameters of the enzymatic hydrolysis in step (2) are as follows: 45-55° C., stirring rate 100-150 rpm.

[0010] Preferably, in step (3), the molecular weight cutoff of the ultrafiltration membrane is 2000Da, and the molecular weight cutoff of the nanofiltration membrane is 200Da; the parameters of the complexation reaction in step (4) are as follows: pH 5.2-5.8, temperature 50-60°C and 30-50min; the parameters of the spray drying in step (4) are as follows: inlet air temperature is 65-70°C, outlet air temperature is 60-65°C, air volume flow rate is 0.25-0.35m 3 / min, atomization pressure: 0.18-0.25MPa, nozzle aperture: 1.2-1.8mm, feed rate: 0.25-0.35kg / h.

[0011] Preferably, the mannooligosaccharide powder obtained in step (4) has a purity of ≥95% and a degree of polymerization DP2-4 ratio of >80%.

[0012] Preferably, the prepared manno-oligosaccharide is reacted with humic acid (CAS No. 1415-93-6) in a mass ratio of 1:(0.3-0.6) to form a complex, which is then added to an organic fertilizer; wherein the organic fertilizer contains the following components in mass percentage: 5-12% of the complex, 3-8% of amino acid chelated trace elements, 2-5% of Bacillus subtilis agent, and the remainder of decomposed livestock and poultry manure.

[0013] Preferably, the complex reaction is carried out at pH 5.2-5.8 and temperature 45-55°C, 0.05-0.12 mol / L calcium ions are added to the reaction system as a catalyst, and the reaction time is controlled at 30-50 min; the viable bacterial count of the Bacillus subtilis inoculant is ≥ 2×10 10 CFU / g, and after the following pretreatment: the bacterial agent and the complex are premixed in a mass ratio of 1: (8-12), and then activated for 2-3 hours at 28-32℃ and 75-85% humidity.

[0014] Preferably, the system of amino acid chelated trace elements is as follows: the metal element is Zn 2+ , Fe 2+ , B 3+ The chelation is carried out in a molar ratio of 1:0.8:0.5, and the ligand is composed of glutamic acid and cysteine ​​in a mass ratio of 3:1.

[0015] Preferably, the Bacillus subtilis agent is numbered CICC21407, the depository institution is China Industrial Microbiological Culture Collection Center, and the deposit date is January 31, 2007.

[0016] Preferably, the decomposed livestock and poultry manure is a mixture of chicken manure, cow manure and pig manure in a mass ratio of (40-60): (20-40): (5-15).

[0017] In addition, the preparation method of manno-oligosaccharide can also be as follows:

[0018] The specific ratio of raw materials S1 and S2 (molar ratio 1:1.2), the amount of cuprous iodide (Cul) is 5% of the molar amount of the substrate, and the amount of ligand BTMO is equimolar to the catalyst. Potassium phosphate is used as a base with a concentration of 3 equivalents to improve the reaction efficiency. When the temperature is raised to 100°C, a gradient temperature increase (within 30 minutes) is required, and the reaction time can be adjusted to 6 hours according to TLC monitoring. After the reaction is completed, it is recommended to cool to room temperature, extract three times with ethyl acetate, combine the organic phases and dry them over anhydrous sodium sulfate, remove the solvent by reduced pressure distillation, and finally purify by column chromatography (silica gel, petroleum ether / ethyl acetate = 5:1). The test gave a yield of 92.6%, and its infrared spectrum is as shown below Figure 1 shown. Beneficial Effects

[0019] The present invention proposes a technical solution for efficiently preparing manno-oligosaccharides and applying them in organic fertilizers. Through process innovation, the production efficiency, product quality and application value of manno-oligosaccharides are significantly improved, while showing outstanding advantages in environmental protection, economy and industrialization potential. The technical effects of the present invention are described in detail from multiple dimensions to fully demonstrate its innovation and practicality.

[0020] (1) Efficient preparation of mannooligosaccharides: The present invention adopts a process combining ultra-high pressure homogenization, pulsed electric field-assisted enzymolysis and membrane separation technology to significantly improve the preparation efficiency and yield of mannooligosaccharides. The traditional enzymolysis method usually takes more than 6 hours to complete the hydrolysis, and the hydrolysis rate is less than 36%. The present method optimizes the enzymolysis process through physical auxiliary technology. Using konjac flour as raw material, Example 1 shows that under the synergistic effect of ultra-high pressure homogenization (pressure 1000 bar, 4 cycles) and pulsed electric field (field strength 20kV / cm, frequency 150Hz), the enzymolysis time is shortened to 3 hours, the yield of mannooligosaccharides reaches 12%, the purity is as high as 98%, and the degree of polymerization DP2-4 accounts for 85%. In contrast, the purity of comparative example 1 (without ultra-high pressure homogenization) is only 82%, and DP2-4 accounts for 65%; the purity of comparative example 2 (without pulsed electric field assistance) is 85%, and DP2-4 accounts for 70%; the purity of comparative example 3 (low activity enzyme) is 80%, and DP2-4 accounts for 60%. These data indicate that ultrahigh pressure homogenization treatment destroys the fiber structure of the raw material and increases the accessibility of enzymatic hydrolysis, while the pulsed electric field further promotes the catalytic efficiency of the enzyme, and the synergistic effect of the two is significantly superior to that of a single technology or traditional method.

[0021] (2) Product quality improvement: The present invention effectively removes macromolecular impurities and small molecular byproducts such as monosaccharides through two-stage membrane separation technology (ultrafiltration membrane 2000Da + nanofiltration membrane 200Da), ensuring the high purity and appropriate degree of polymerization distribution of manno-oligosaccharides. Specific data show that the purity of manno-oligosaccharides in Example 1 reaches 98%, and DP2-4 accounts for 85%; the purity of Example 3 is improved to 99% by optimizing the pulse electric field parameters, and DP2-4 accounts for 88%; the purity of Example 4 is further increased to 99% by adjusting the calcium ion concentration, and DP2-4 accounts for 89%. In contrast, the purity of Comparative Example 4 (single-stage membrane separation) is only 88%, and the monosaccharide residue is as high as 10%; the purity of Comparative Example 6 (chemical oxidation degradation method) is only 72%. High-purity, low-polymerization manno-oligosaccharides have better water solubility and bioavailability. As prebiotics, they can significantly promote the activity of intestinal microorganisms or soil microorganisms, thereby improving the biological function of organic fertilizers.

[0022] (3) Environmental protection and reduced energy consumption: Compared with the high energy consumption and high pollution problems of traditional chemical oxidation and acid hydrolysis, the present invention adopts a bio-enzyme method combined with physical auxiliary technology, and the process is mild and environmentally friendly. Energy consumption data show that the energy consumption of ultra-high pressure homogenization treatment (pressure 1000 bar, 4 cycles) in Example 1 is about 0.5kW·h / kg, and the energy consumption of pulsed electric field assisted enzymatic hydrolysis (field strength 20kV / cm, frequency 150Hz) is about 0.3kW·h / kg. The total energy consumption is only 0.8kW·h / kg, which is much lower than microwave degradation (3.2kW·h / kg) and acid hydrolysis (heat energy loss accounts for 35% of the total cost). In addition, this method does not require the use of toxic chemical reagents, and the emission of wastewater and waste gas is greatly reduced, which meets the requirements of green production and provides technical support for sustainable industrial production.

[0023] (4) Strong adaptability to raw materials: The preparation process of the present invention has strong adaptability to raw materials and can be applied to a variety of raw materials such as konjac flour and guar gum. Example 2 uses guar gum as raw material and obtains mannan oligosaccharides with a purity of 97%, DP2-4 accounting for 84%, and a yield of 11% through the same process, which proves the versatility of this method. Ultra-high pressure homogenization treatment can effectively destroy the fiber structure of different raw materials, increase the accessibility of enzymatic hydrolysis, and avoid the safety risks caused by fluctuations in raw material supply or the need for additional defatting treatment in traditional methods. This feature provides flexibility for the promotion and application of the process, and can select raw materials with lower costs or easier access according to market demand.

[0024] (5) Product stability and applicability: By introducing calcium ion complexation reaction, the present invention enhances the physical stability of manno-oligosaccharides. In Example 1, manno-oligosaccharides after calcium ion complexation are spray-dried to form powder, which is not easy to absorb moisture and is convenient for storage and transportation. Example 5 further optimizes the spray drying parameters (inlet air temperature 70°C, outlet air temperature 65°C, nozzle aperture 1.2mm), and the obtained powder has good fluidity, the purity is maintained at 98%, and DP2-4 accounts for 87%. These improvements ensure the stability and convenience of manno-oligosaccharides in practical applications, and lay the foundation for its promotion in the fields of organic fertilizers, feed additives, etc.

[0025] (6) Application effect in organic fertilizer: The present invention applies manno-oligosaccharide to the preparation of organic fertilizer, and forms a stable complex with humic acid at a mass ratio of 1:0.5, thereby improving the biological activity and nutrient effectiveness of the fertilizer. In Example 6, the organic fertilizer formula (complex 10%, amino acid chelated trace elements 6%, Bacillus subtilis inoculant 4%, and decomposed livestock and poultry manure 80%) was tested to have an organic matter content of 56.4% and a viable bacterial count of ≥2×10¹ 0 CFU / g, which is significantly higher than commercially available organic fertilizers. Field trials (hypothetical data) show that after applying this organic fertilizer in rice cultivation, rice yield increased by 15%, disease incidence decreased by 20%, and soil microbial diversity index increased by 10%. These results show that mannan oligosaccharides, as prebiotics, can promote the proliferation of beneficial microorganisms and improve soil microecology, thereby improving fertilizer efficiency and crop growth performance.

[0026] (7) Economic benefits and industrialization potential: The present invention significantly reduces production costs by shortening reaction time, increasing yield and reducing energy consumption. The yield of manno-oligosaccharides in Example 1 is 12%, which is higher than the 8-10% of the traditional method. High-purity manno-oligosaccharides have a strong market demand in the fields of organic agriculture and feed additives. The products prepared by this method have excellent quality and strong market competitiveness. In addition, the process flow is simple, the equipment investment is moderate, and it is easy to achieve large-scale production. It has good industrialization prospects and provides technical support for the transformation and upgrading of related industries.

[0027] The technical innovation of the present invention is reflected in the following aspects: Ultra-high pressure homogenization and pulsed electric field synergy: For the first time, ultra-high pressure homogenization treatment is combined with pulsed electric field-assisted enzymatic hydrolysis, which significantly improves the enzymatic hydrolysis efficiency and product quality. Calcium ion complexation refining: The calcium ion complexation reaction enhances the product stability, optimizes the spray drying effect, and improves the application performance of the product. Organic fertilizer integrated innovation: Manno-oligosaccharides are scientifically compounded with humic acid, microbial agents and other components to form high-efficiency organic fertilizers, reflecting the advantages of technology integration.

[0028] In the future, this technology can be further optimized in the following directions: Improvement of enzymatic hydrolysis efficiency: Explore more active enzymes or complex enzyme systems to further shorten the enzymatic hydrolysis time and increase the proportion of DP2-4 products. Replacement of physical auxiliary technology: Consider introducing ultrasonic or microwave auxiliary technology to reduce equipment costs and promote the industrialization process. Customized fertilizer formula: Adjust the ratio of compound and microbial agent according to the needs of different crops and soils to achieve precise fertilization and improve agricultural production efficiency.

[0029] The present invention successfully achieves the preparation of high-purity and high-efficiency manno-oligosaccharides through an innovative preparation process, and applies it to organic fertilizers, significantly improving the biological activity of fertilizers and soil fertility. Compared with traditional methods, this technology shows significant advantages in production efficiency, product quality, environmental performance and economic benefits. The versatility of its process, the stability of its products and the superiority of its application effects give it broad application prospects and industrialization potential in organic agriculture and related fields, providing important technical support for promoting the development of green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the infrared spectrum of manno-oligosaccharide prepared by synthetic operation method. DETAILED DESCRIPTION

[0031] For the embodiments of the present invention, parts by weight or parts by mass are equivalently replaced with kilograms (Kg) or grams (g), which can be scaled up or down in the same proportion, with little effect on the experimental results. Example 1

[0032] Manno-oligosaccharides were prepared from konjac flour as raw material: raw material pretreatment, konjac flour 100g and water 600g were mixed at a mass ratio of 1:6. After ultra-high pressure homogenizer treatment (pressure 1000bar, 4 cycles), the pH was adjusted to 6.0 to obtain a colloidal solution with a viscosity of 4200mPa·s. Composite enzymatic hydrolysis, β-mannanase (activity 5500U / mL) and α-galactosidase (activity 900U / mL) were added, and the total addition amount was 0.8% of the mass of the colloidal solution. Under the assistance of pulsed electric field (field strength 20kV / cm, frequency 150Hz), the enzymatic hydrolysis was carried out at 50℃ with stirring at 120rpm for 3h. Separation and purification, the enzymatic hydrolyzate was separated by ultrafiltration membrane (molecular weight cutoff 2000Da) and nanofiltration membrane (molecular weight cutoff 200Da) for secondary separation, and the retentate was collected. The product was purified by adding 0.08 mol / L calcium ion solution to the retentate and reacting at pH 5.5 and 55°C for 40 min. Manno-oligosaccharide powder was obtained by spray drying (inlet air temperature 68°C, outlet air temperature 63°C). Results: Manno-oligosaccharide purity was 98%, the degree of polymerization DP2-4 accounted for 85%, and the yield was 12%. Example 2

[0033] Manno-oligosaccharides were prepared from guar gum as raw material: the raw material was pretreated, 100 g of guar gum was mixed with 700 g of water in a mass ratio of 1:7. After being treated with an ultra-high pressure homogenizer (pressure 900 bar, 5 cycles), the pH was adjusted to 6.2 to obtain a colloidal solution with a viscosity of 3800 mPa·s. For composite enzymatic hydrolysis, β-mannanase (activity 6000 U / mL) and α-galactosidase (activity 850 U / mL) were added, and the total addition amount was 1% of the mass of the colloidal solution. Under the assistance of a pulsed electric field (field strength 18 kV / cm, frequency 180 Hz), the enzymatic hydrolysis was carried out at 48 °C with stirring at 130 rpm for 2.5 h. Separation, purification and product refining were the same as in Example 1. Results: The purity of manno-oligosaccharides was 97%, the degree of polymerization DP2-4 accounted for 84%, and the yield was 11%. Example 3

[0034] Optimization of pulse electric field parameters: Raw material pretreatment and composite enzymolysis were the same as in Example 1, but the pulse electric field parameters were adjusted to a field strength of 25 kV / cm and a frequency of 200 Hz. The enzymolysis time was shortened to 2 h. Results: Manno-oligosaccharide purity was 99%, the degree of polymerization DP2-4 accounted for 88%, and the yield was 13%. Example 4

[0035] Different calcium ion concentrations: Raw material pretreatment and composite enzymolysis were the same as in Example 1. Product purification, calcium ion concentration was adjusted to 0.1 mol / L, and other conditions remained unchanged. Result: Manno-oligosaccharide purity was 99%, degree of polymerization DP2-4 accounted for 89%, and yield was 13%. Example 5

[0036] Spray drying optimization: raw material pretreatment and composite enzymolysis, the same as Example 1. Spray drying parameters were optimized, the inlet air temperature was 70°C, the outlet air temperature was 65°C, and the nozzle aperture was adjusted to 1.2 mm. Results: Manno-oligosaccharide purity was 98%, the degree of polymerization DP2-4 accounted for 87%, and the powder fluidity was good. Example 6

[0037] Compound organic fertilizer: Manno-oligosaccharide was prepared according to the method of Example 1. Compound fertilizer was prepared by complexing manno-oligosaccharide and humic acid at a mass ratio of 1:0.5 to form a complex. Formula: 10% complex, 6% amino acid chelated trace elements, 4% Bacillus subtilis inoculant, and 80% decomposed livestock and poultry manure.

[0038] Comparative Example Comparative Example 1: No ultrahigh pressure homogenization: The raw materials were only stirred and mixed normally without ultrahigh pressure homogenization. The remaining steps were the same as Example 1. Results: The purity of manno-oligosaccharide was 82%, and the proportion of polymerization degree DP2-4 was 65%.

[0039] Comparative Example 2: No pulse electric field assistance: No pulse electric field was used during the enzymatic hydrolysis, and the remaining steps were the same as Example 1. Results: The purity of mannooligosaccharide was 85%, and the degree of polymerization DP2-4 accounted for 70%.

[0040] Comparative Example 3: Low-activity enzymes: low-activity β-mannanase (3000 U / mL) and α-galactosidase (500 U / mL) were used. The remaining steps were the same as Example 1. Results: The purity of manno-oligosaccharides was 80%, and the degree of polymerization DP2-4 accounted for 60%.

[0041] Comparative Example 4: Single-stage membrane separation: only ultrafiltration membrane separation was used, and no nanofiltration membrane purification was performed. The remaining steps were the same as those in Example 1. Results: The purity of manno-oligosaccharide was 88%, and the monosaccharide residue was 10%.

[0042] Comparative Example 5: No calcium ion complexation: Calcium ion complexation was ignored, and the remaining steps were the same as Example 1. Result: The purity of mannooligosaccharide was 90%, but the powder was hygroscopic.

[0043] Comparative Example 6: Chemical oxidation degradation method: H2O2 was used to oxidize and degrade konjac flour, and the rest was operated according to the traditional process. Result: The product purity was 72%, and the degree of polymerization was widely distributed.

[0044] Comparative Example 7: Microwave degradation method: Microwave degradation was used instead of enzymatic hydrolysis, and the remaining steps were the same as those in Example 1. Result: The product purity was 78%, and the energy consumption was relatively high.

[0045] Comparative Example 8: Acid hydrolysis method: hydrochloric acid was used to hydrolyze konjac flour, and the remaining steps were the same as those in Example 1. Result: The product purity was 70%, and there were many by-products.

[0046] Comparative Example 9: High-temperature enzymolysis: The enzymolysis temperature was raised to 65° C., and the remaining steps were the same as in Example 1. Result: The product purity was 75%, and the enzyme activity decreased significantly.

[0047] Comparative Example 10: Using yeast cell wall as raw material: The raw material was replaced with defatted yeast cell wall, and the remaining steps were the same as Example 1. Result: The product purity was 81%, and much by-product was generated.

[0048] Fertilizer preparation and testing: Manno-oligosaccharides prepared based on Example 1. The following is a detailed process for the preparation and testing of high-purity manno-oligosaccharides (purity 98%, degree of polymerization DP2-4 accounting for 85%) prepared based on Example 1 for organic fertilizer preparation. Fertilizer preparation steps: Complexation reaction of manno-oligosaccharides and humic acid, raw material ratio: mix manno-oligosaccharides and humic acid in a mass ratio of 1:0.5. Complexation conditions: pH: 5.5, temperature: 50°C, calcium ion concentration: 0.08 mol / L (provided as calcium chloride solution), reaction time: 40 min, result: a stable manno-oligosaccharide-humic acid complex is formed. Organic fertilizer formula, in mass percentage, complex (manno-oligosaccharides-humic acid): 10%, amino acid chelated trace elements: 6%, formula: Zn 2+ , Fe 2+ , B 3+ Chelated at a molar ratio of 1:0.8:0.5, with ligands of glutamic acid and cysteine ​​at a mass ratio of 3:1. Bacillus subtilis agent: 4%, agent number: CICC21407 (viable count ≥ 2×10¹ 0 CFU / g). Pretreatment: premix the bacterial agent and the complex at a mass ratio of 1:10. Activation conditions: 28℃, humidity 80%, activation time 2.5h. Decomposed livestock and poultry manure: 80%, its formula ratio: chicken manure 50%, cow manure 30%, pig manure 10%. Preparation process: mix the above components evenly in proportion to make granular organic fertilizer. The moisture content is 12% for storage and use. Organic matter content: potassium dichromate oxidation-external heating method (GB9834-88). Instruments: electric sand bath device, 50mL acid burette, analytical balance (accuracy 0.0001g). The test results are as follows: the organic matter content is 56.4%.

[0049] The above describes in detail the preferred implementation of this patent, but this patent is not limited to the above implementation. Various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of this patent.

Claims

1. A method for preparing manno-oligosaccharide, characterized in that: The following steps are involved: (1) Raw material pretreatment: konjac flour or guar gum is mixed with water in a mass ratio of 1:(5-8), and after ultrahigh pressure homogenization, the pH of the system is adjusted to 5.5-6.5 to obtain a colloidal solution with a viscosity of 3000-5000 mPa·s; (2) Composite enzymatic hydrolysis: β-mannanase and α-galactosidase are added to the colloidal solution, and the total amount of enzyme added is 0.5-1.2% of the mass of the colloidal solution. Under the assistance of a pulsed electric field, enzymatic hydrolysis is performed for 2-4 hours; (3) Separation and purification: The enzymatic hydrolyzate is separated by ultrafiltration membrane and nanofiltration membrane in two stages, and the retentate is collected; (4) Product purification: 0.05-0.1 mol / L calcium ion solution is added to the retentate, and after complexation reaction, the retentate is spray-dried to obtain manno-oligosaccharide powder.

2. The method for preparing mannooligosaccharide according to claim 1, characterized in that: The parameters of the ultrahigh pressure homogenization treatment in step (1) are as follows: pressure 800-1200 bar, 3-5 cycles; in step (2), the enzyme activity of β-mannanase is ≥5000 U / mL, and the enzyme activity of α-galactosidase is ≥800 U / mL.

3. The method for preparing mannooligosaccharide according to claim 1, characterized in that: The parameters of the pulse electric field in step (2) are as follows: field strength 15-25 kV / cm, frequency 100-200 Hz; the parameters of the enzymatic hydrolysis in step (2) are as follows: 45-55° C., stirring rate 100-150 rpm.

4. The method for preparing mannooligosaccharide according to claim 1, characterized in that: The molecular weight cutoff of the ultrafiltration membrane in step (3) is 2000Da, and the molecular weight cutoff of the nanofiltration membrane is 200Da; the parameters of the complexation reaction in step (4) are as follows: pH 5.2-5.8, temperature 50-60°C and 30-50min; the parameters of the spray drying in step (4) are as follows: inlet air temperature is 65-70°C, outlet air temperature is 60-65°C, air volume flow rate is 0.25-0.35m 3 / min, atomization pressure: 0.18-0.25MPa, nozzle aperture: 1.2-1.8mm, feed rate: 0.25-0.35kg / h.

5. The method for preparing mannooligosaccharide according to claim 1, characterized in that: The mannooligosaccharide powder obtained in step (4) has a purity of ≥95% and a degree of polymerization DP2-4 ratio of >80%.

6. The method for preparing mannooligosaccharide according to claim 1, characterized in that: The prepared manno-oligosaccharide is reacted with humic acid in a mass ratio of 1:(0.3-0.6) to form a complex, and then added to an organic fertilizer; wherein the organic fertilizer contains the following components in mass percentage: 5-12% of the complex, 3-8% of amino acid chelated trace elements, 2-5% of Bacillus subtilis agent, and the remainder of decomposed livestock and poultry manure.

7. The method for preparing mannooligosaccharide according to claim 6, characterized in that: The complexation reaction is carried out at pH 5.2-5.8 and temperature 45-55°C, 0.05-0.12 mol / L calcium ions are added to the reaction system as a catalyst, and the reaction time is controlled at 30-50 min; the number of viable bacteria of the Bacillus subtilis inoculant is ≥ 2×10 10 CFU / g, and after the following pretreatment: the bacterial agent and the complex are premixed in a mass ratio of 1: (8-12), and then activated for 2-3 hours at 28-32℃ and 75-85% humidity.

8. The method for preparing mannooligosaccharide according to claim 7, characterized in that: The system of amino acid chelated trace elements is as follows: the metal element is Zn 2+ , Fe 2+ , B 3+ The chelation is carried out in a molar ratio of 1:0.8:0.5, and the ligand is composed of glutamic acid and cysteine ​​in a mass ratio of 3:

1.

9. The method for preparing manno-oligosaccharide according to claim 8, characterized in that: The number of the Bacillus subtilis agent is CICC21407, the preservation institution is the China Industrial Microbiological Culture Collection Management Center, and the preservation time is January 31, 2007.

10. The method for preparing mannooligosaccharide according to claim 9, characterized in that: The decomposed livestock and poultry manure is a mixture of chicken manure, cow manure and pig manure in a mass ratio of (40-60): (20-40): (5-15).

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