CN112941131B - Method for preparing microbial source chitosan oligosaccharide by fermentation method - Google Patents
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Abstract
The invention relates to the technical field of microbial preparation, in particular to a method for preparing microbial source chitosan oligosaccharide by a fermentation method. The method for preparing the chitosan oligosaccharide by three-bacteria mixed fermentation by using aspergillus, bacillus licheniformis and lactobacillus comprises the following steps: (1) Aspergillus fermentation; (2) bacillus licheniformis fermentation; (3) lactobacillus fermentation; (4) deslagging and degerming treatment; (5) adsorption treatment; and (6) alcohol precipitation and drying treatment. The invention has the advantages that: (1) The process for producing the chitosan oligosaccharide by the fermentation method is not influenced and limited by raw material sources and the external environment in seasons, the production process is more controllable, and the product quality is more stable. (2) The whole process is green in production, the discharge of strong acid, strong alkali and fermentation waste liquid is small, the pollution to the environment is small, the production condition is mild, the energy consumption is low, and the national energy conservation and emission reduction policies are met. The molecular weight distribution of the microbial source chitosan oligosaccharide prepared by the method is more concentrated, and the chitosan oligosaccharide accounts for 92.3% under the optimal condition.
Description
Technical Field
The invention relates to a method for preparing microbial source chitosan oligosaccharide by a fermentation method, and belongs to the technical field of microbial preparation.
Background
Chitosan (CTS), also known as chitosan, is the only basic polysaccharide in nature, but its use is greatly limited because it can only be dissolved in acidic solutions.
Chitosan Oligosaccharide (COS) is an oligomer obtained by enzymatic reaction or chemical degradation of CTS, has a polymerization degree of 2-20, and is the only basic amino oligosaccharide with positive charges in nature at present. COS is nontoxic, has low relative molecular mass, good water solubility, high biological activity and is easy to be absorbed and utilized by human bodies, has various biological activities of regulating immunity, inhibiting bacteria, resisting inflammation, reducing blood fat, resisting tumor and the like, and has important research significance and application value in the aspects of medicines, health care, cosmetics and functional foods.
The traditional chitosan oligosaccharide preparation process mainly comprises a two-step method, wherein chitosan is firstly extracted from shrimp and crab shells by an acid-base method, and then chitosan is used as a raw material to prepare the chitosan oligosaccharide by a chemical method, a physical method or an enzymatic method. However, a large amount of acid and alkali are consumed in the process of extracting chitosan from the shrimp and crab shells, a large amount of acid and alkali waste liquid is generated, and the problem of environmental pollution is extremely serious. The traditional production mode is contrary to the current environmental protection policy in China, and is unfavorable for the continuous benign development of related industries.
Therefore, how to provide a preparation method of chitosan oligosaccharide with mild production conditions, high molecular weight concentration of the chitosan oligosaccharide product and small waste liquid discharge in the production process is a technical problem which needs to be solved by the technicians in the field.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the existing preparation method of the microbial source chitosan oligosaccharide generates a large amount of waste liquid in the production process, pollutes the environment, and has low concentration of the molecular weight of the chitosan oligosaccharide product.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for preparing microbial source chitosan oligosaccharide by a fermentation method, which is characterized by comprising the following steps:
step 1): and (3) aspergillus fermentation: inoculating the aspergillus inclined spores into seed liquid for culture, inoculating the cultured seed liquid into a fermentation medium for culture, and carrying out aspergillus fermentation;
step 2): bacillus licheniformis fermentation: adding sterilized nutrient solution into the aspergillus fermentation liquid obtained in the step 1), uniformly stirring, adjusting the pH of the fermentation liquid to 5.5-7, and then inoculating 5-30g/L bacillus licheniformis powder under aseptic operation to continue fermentation;
step 3): lactic acid bacteria fermentation: inoculating 5-20g/L lactobacillus powder into the fermentation liquid obtained in the step 2) under aseptic operation, and continuing fermenting to obtain a liquid fermentation product containing chitosan oligosaccharide;
step 4): deslagging and degerming: adding solid calcium hydroxide into the liquid fermentation product containing the chitosan oligosaccharide obtained in the step 3) under the stirring state to adjust the pH value to 6.5-7.5, filtering, removing residues and sterilizing to obtain clear and transparent liquid containing the chitosan oligosaccharide;
step 5): adsorption treatment: adjusting the pH value of the liquid obtained in the step 4) to 8.0-10.5 by using a sodium hydroxide solution, adding a weak alkaline ion exchange resin according to the solid-liquid mass ratio of 1:10-1:30, adsorbing, filtering to remove the resin after the adsorption is finished, and adjusting the solution to be neutral to obtain a chitosan oligosaccharide solution;
step 6): alcohol precipitation and drying treatment: adding 3-4 times of absolute ethyl alcohol into the chitosan oligosaccharide solution obtained in the step 5), uniformly stirring, standing overnight, taking precipitate, washing the precipitate with the ethyl alcohol solution for 4-6 times, and drying in a freeze dryer to obtain white snowflake chitosan oligosaccharide solid powder.
Preferably, the aspergillus used for the fermentation in the step 1) is aspergillus ochraceus, its classification is named aspergillus ochraceus, latin name is Aspergillus ochraceus, and the aspergillus is preserved in China general microbiological culture collection center (CGMCC), and the strain preservation number is: CGMCC No.15668, the preservation date is: 25 th 2018, 04 th month, deposit unit address: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Preferably, the preparation method of the seed liquid in the step 1) comprises the following steps: sucking 0.1-0.12mL of strain under aseptic operation, uniformly coating in slant culture medium, culturing at 27-30deg.C for 4-6 days to obtain strain required for fermentation, scraping yellow spore under aseptic operation, transferring into sterile physiological saline, and preparing into spore with concentration of 4.0X10 8 -7.0×10 8 Spore suspension of individual spores/mL; transferring the spore suspension into a seed culture medium according to the inoculation amount of 10-15wt%, and culturing for 24-30h in a constant temperature shaking table at 27-30 ℃ at 170-200 rpm; transferring the seed solution into fermentation medium under aseptic operation at 27-30deg.C and rotation speed of 100-450rpm with aeration ratio of 0.4v/v/min-1.6v/v/mCulturing for 36-48h under in condition, heating to 45-65deg.C, adjusting pH to 6.0-8.0 with sodium hydroxide solution, maintaining temperature, and stirring for 12-24h.
More preferably, the raw materials of the slant culture medium comprise 200-220 parts of peeled potatoes, 20-25 parts of glucose, 20-23 parts of agar and 1000 parts of water in parts by weight, wherein the pH value is 6-6.8, and the slant culture medium is subjected to moist heat sterilization at 121 ℃ for 30min; the raw materials of the seed culture medium comprise 8-15 parts by weight of sucrose, 8-15 parts by weight of yeast powder, 2-8 parts by weight of molasses and 1000 parts by weight of water, wherein the pH value is 6-7, and the seed culture medium is subjected to damp-heat sterilization at 121 ℃ for 30min; the raw materials of the fermentation medium comprise, by weight, 20-30 parts of sucrose, 10-20 parts of soluble starch, 5-10 parts of yeast powder, 8-15 parts of soybean powder, 1.5-4 parts of molasses, 0.1-1 part of sodium acetate and 1000 parts of water, wherein the pH value is 5.5-7, and the fermentation medium is subjected to wet heat sterilization at 121 ℃ for 30min.
Preferably, the nutrient solution supplemented in the step 2) comprises 17.5-70 parts of glucose, 0.35-2.8 parts of NaCl and K in parts by weight 2 HPO 4 0.6-1.8 parts of MgSO 4 ·7H 2 0.4-0.9 part of O, feSO 4 ·7H 2 0.01-0.04 part of O and CaCl 2 0.1-1.0 part and 200 parts of water are added, and after the nutrient solution is prepared, the nutrient solution is sterilized by moist heat at 121 ℃ for 30min and is cooled for later use.
Preferably, the conditions for the fermentation of bacillus licheniformis in step 2) are as follows: the fermentation time is 12-24h, the fermentation temperature is 33-39 ℃, the rotation speed is 100-350rpm, and the ventilation ratio is 0.2v/v/min-1.0v/v/min.
Preferably, the conditions for the fermentation of the lactic acid bacteria in the step 3) are as follows: the fermentation time is 168-240h, the aseptic nitrogen is kept at positive pressure of 0.01-0.04MPa, the fermentation is kept still, the intermittent stirring is carried out for 1 time every 12h, the stirring time is 5.0min, and the stirring rotating speed is 30rpm-80rpm.
Preferably, the sterilization and deslagging treatment in the step 4) is specifically: removing slag and bacteria from the liquid fermentation product of the chitosan oligosaccharide with the pH value adjusted through a four-stage filtering device, wherein the pore diameters of the filtering media of the four-stage filtering device are as follows: 600-620 mesh, 1-1.2 μm, 0.45-0.47 μm and 0.22-0.25 μm.
Preferably, the adsorption treatment in step 5) specifically includes: maintaining the temperature of the system at 30-40 ℃, stirring at 200-220rpm, adsorbing for 1-8 h, sampling and detecting the protein concentration change in the solution at regular time, and finishing the adsorption step after the protein content in the solution cannot be detected.
Preferably, the volume fraction of the ethanol solution used for washing in the step 6) is 75%, and the time of freeze drying is 24-36h.
The invention utilizes aspergillus, bacillus and lactobacillus to perform three-bacteria fermentation, and provides an effective method for preparing microbial source chitosan oligosaccharide, which has relatively simple production process and small discharge of acid, alkali and fermentation waste liquid.
Compared with the prior art, the invention has the following beneficial effects:
(1) The process for producing the chitosan oligosaccharide by the fermentation method is not influenced and limited by raw material sources and the external environment in seasons, the production process is more controllable, and the product quality is more stable. (2) The whole process is green in production, the discharge of strong acid, strong alkali and fermentation waste liquid is small, the pollution to the environment is small, the production condition is mild, the molecular weight concentration of the chitosan oligosaccharide product is high, the energy consumption is low, and the national energy saving and emission policy is met.
Detailed Description
In order to make the present invention more comprehensible, preferred embodiments accompanied with the present invention are described in detail below.
In order to detect the effect of the method for preparing the microbial source chitosan oligosaccharide by the fermentation method, the invention adopts the following chitosan oligosaccharide concentration detection method and molecular weight detection method:
(1) Chitosan oligosaccharide concentration detection (DNS method)
Drawing a standard curve: and drawing a standard curve by using the glucosamine as a standard DNS method.
Sample treatment and measurement: and (3) centrifuging a proper amount of chitosan oligosaccharide solution, adding 3 times of ethanol into the supernatant to precipitate chitosan oligosaccharide, centrifuging to obtain a precipitate, dehydrating the precipitate with absolute ethanol for 4 times, and drying the precipitate at 60 ℃ to constant weight to obtain a chitosan oligosaccharide crude product. Precisely weighing 5mg of chitosan oligosaccharide crude product, adding 5mL of concentrated hydrochloric acid, hydrolyzing in a constant-temperature oscillating water bath kettle at (100+/-1) ℃ for 5h, regulating the pH to 7.0 by using a sodium hydroxide solution with the mass fraction of 30%, transferring the hydrolyzed solution into a 50mL volumetric flask, fixing the volume of purified water, shaking uniformly, and measuring the concentration of reducing sugar by a DNS method after proper dilution.
(2) Chitosan oligosaccharide molecular weight detection (GPC method)
Drawing a standard curve: the standard curve is drawn by taking glucosamine, chitosan pentasaccharide and chitosan heptasaccharide as standard substances, taking the peak-out time of the standard substances as an abscissa and taking the DP value of chitosan oligosaccharide as an ordinate.
Chromatographic conditions: a chromatographic column; mobile phase 0.12M acetic acid-1M sodium acetate; the flow rate is 0.8mL/min; the sample injection volume is 50uL; column temperature is 30 ℃; run time was 36min.
Sample measurement: and (3) centrifuging a proper amount of chitosan oligosaccharide solution, adding 3 times of ethanol into the supernatant to precipitate chitosan oligosaccharide, centrifuging to obtain a precipitate, dehydrating the precipitate with absolute ethanol for 4 times, and drying the precipitate at 60 ℃ to constant weight to obtain a chitosan oligosaccharide crude product. The crude chitosan oligosaccharide product was dissolved in a mobile phase, subjected to GPC detection after microfiltration through a 0.22 μm membrane, and the DP value of the chitosan oligosaccharide was calculated according to a standard curve.
Example 1
A method for preparing microbial source chitosan oligosaccharide by fermentation method comprises the following steps:
(1) And (3) aspergillus fermentation:
sucking 0.1mL of the preserved strain under aseptic operation, uniformly coating in slant culture medium, culturing at 27deg.C for 4 days to obtain strain required for fermentation, scraping yellow spore under aseptic operation, transferring into sterile physiological saline, and preparing into spore with concentration of 4.0X10 8 Spore suspension of individual spores/mL; transferring the spore suspension into a seed culture medium according to 10wt% of inoculation amount, and culturing for 24 hours in a shaking table at a constant temperature of 27 ℃ at 170rpm to obtain seed liquid;
fermenting in a 5L fermentation tank: 3.5L of liquid loading amount, inoculating 350mL of seed liquid into 3150mL of fermentation medium in a sterile operation mode, controlling the culture temperature to 27 ℃, controlling the rotation speed to be 100rpm, controlling the aeration ratio to be 0.4v/v/min, after fermenting for 36h, raising the temperature of the fermentation liquid to 45 ℃, adjusting the pH value to 6 by using sodium hydroxide solution, and carrying out heat preservation and stirring for 12h;
the slant culture medium comprises the following raw materials in parts by weight: 200 parts of peeled potatoes, 20 parts of glucose, 20 parts of agar, 1000 parts of water, and carrying out wet heat sterilization for 30min at the temperature of 121 ℃ at the pH of 6;
the seed culture medium comprises the following raw materials in parts by weight: 8 parts of sucrose, 8 parts of yeast powder, 2 parts of molasses, 1000 parts of water with pH of 6, and carrying out damp-heat sterilization at 121 ℃ for 30min; the fermentation medium comprises the following components in parts by weight: 20 parts of sucrose, 10 parts of soluble starch, 5 parts of yeast powder, 8 parts of soybean powder, 1.5 parts of molasses, 0.1 part of sodium acetate, 1000 parts of water, pH5.5 and sterilization at 121 ℃ under moist heat for 30min.
(2) Bacillus licheniformis fermentation:
adding 200mL of sterilized nutrient solution into the aspergillus fermentation liquid in the step (1), uniformly stirring, regulating the pH value of the fermentation liquid to 5.5, reducing the temperature of the fermentation liquid to 33 ℃, inoculating 17.5g of bacillus licheniformis powder into the fermentation liquid under aseptic operation, controlling the rotating speed to 100rpm and the ventilation ratio to 0.2v/v/min, and fermenting for 12 hours;
the nutrient solution comprises the following components in parts by weight: 17.5 parts of glucose, 0.35 part of NaCl and K 2 HPO 4 0.6-1.8 parts of MgSO 4 ·7H 2 0.4 part of O FeSO 4 ·7H 2 O0.01 part, caCl 2 0.1 part of water and 200 parts of water, and sterilizing the nutrient solution at 121 ℃ for 30min in a damp heat mode.
(3) Lactic acid bacteria fermentation:
and (3) inoculating 17.5g of lactobacillus powder into the fermentation broth in the step (2) under aseptic operation, maintaining positive pressure in a tank under 0.01MPa with aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time every 12 hours for 5.0min at a stirring speed of 30rpm, and stopping fermentation after fermentation for 168 hours to obtain the liquid fermentation product containing chitosan oligosaccharide.
Fermenting for 168-240h, maintaining positive pressure of sterile nitrogen at 0.01-0.04MPa, standing for fermentation, intermittently stirring for 1 time every 12h for 5.0min, and stirring at 30-80 rpm.
(4) Deslagging and degerming:
regulating the pH value of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and then removing slag and bacteria by using a four-stage filtering device; the pore diameters of the filter media of the four-stage filter device are as follows: 600 mesh, 1 μm, 0.45 μm and 0.22 μm, clear transparent chitosan oligosaccharide filtrate was obtained.
(5) Adsorption treatment:
adjusting the pH value of the filtrate in the step (4) to 8, adding the activated weak alkaline resin according to the solid-to-liquid ratio of 1:10, maintaining the temperature of the system at 30 ℃, and stirring for 1h under the condition of 200 rpm; and (3) sampling and detecting the protein concentration change in the solution at regular time, filtering to remove resin after the protein content in the solution cannot be detected, and regulating the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 7.8g/L.
(6) Alcohol precipitation and drying treatment:
adding 3 times of absolute ethyl alcohol into the chitosan oligosaccharide solution in the step (5), uniformly stirring, standing overnight, filtering, taking precipitate, washing with 75% ethyl alcohol solution for 4 times, and drying in a freeze dryer for 24 hours to obtain 22.8g of white snowflake chitosan oligosaccharide solid powder.
TABLE 1 molecular weight distribution of chitooligosaccharides
| Chitosan oligosaccharide DP value | 2 | 3 | 5 |
| Duty cycle (%) | 5.2 | 5.3 | 89.7 |
Example 2
A method for preparing microbial source chitosan oligosaccharide by fermentation method comprises the following steps:
(1) And (3) aspergillus fermentation:
sucking 0.11mL of the preserved strain under aseptic operation, uniformly coating in a slant culture medium, and culturing at constant temperature of 28deg.C for 5 days to obtain the desired fermentation productScraping yellow spore under aseptic operation, transferring into sterile physiological saline to obtain spore with concentration of 5.5X10 8 Spore suspension of individual spores/mL; transferring the spore suspension into a seed culture medium according to 13wt% of inoculation amount, and culturing for 27h in a constant-temperature shaking table at 28 ℃ at 185rpm to obtain seed liquid;
fermenting in a 5L fermentation tank: 3.5L of liquid loading amount, 445mL of seed liquid is aseptically inoculated into 3055mL of fermentation medium, the culture temperature is 28 ℃, the rotating speed range is controlled to be 100 rpm-250 rpm, the aeration ratio is controlled to be 1v/v/min, after fermentation is carried out for 42h, the temperature of the fermentation liquid is raised to 55 ℃, the pH value is regulated to 7 by sodium hydroxide solution, and the heat preservation and stirring are carried out for 18h;
the slant culture medium comprises the following raw materials in parts by weight: 210 parts of peeled potatoes, 22 parts of glucose, 21 parts of agar, 1000 parts of water, pH 6.4 and sterilization at 121 ℃ for 30min under moist heat;
the seed culture medium comprises the following raw materials in parts by weight: 11 parts of sucrose, 12 parts of yeast powder, 5 parts of molasses, pH6.5, 1000 parts of water and carrying out damp-heat sterilization at 121 ℃ for 30min; the fermentation medium comprises the following components in parts by weight: 25 parts of sucrose, 15 parts of soluble starch, 8 parts of yeast powder, 7 parts of soybean powder, 3 parts of molasses, 0.5 part of sodium acetate, 1000 parts of water, and carrying out wet heat sterilization at the pH of 6 and 121 ℃ for 30min;
(2) Bacillus licheniformis fermentation:
adding 200mL of sterilized nutrient solution into the aspergillus fermentation liquid in the step (1), uniformly stirring, regulating the pH value of the fermentation liquid to 6.3, reducing the temperature of the fermentation liquid to 36 ℃, inoculating 40g of bacillus licheniformis powder into the fermentation liquid under aseptic operation, controlling the rotating speed to 150rpm and the ventilation ratio to 0.4v/v/min, and fermenting for 18 hours.
The nutrient solution comprises the following components in parts by weight: 45 parts of glucose, 1.5 parts of NaCl and K 2 HPO 4 1.2 parts of MgSO 4 ·7H 2 0.7 part of O FeSO 4 ·7H 2 O0.02 part, caCl 2 0.6 part of water and 200 parts of water, and sterilizing the nutrient solution at 121 ℃ for 30min in a damp heat mode.
(3) Lactic acid bacteria fermentation:
and (3) inoculating 55g of lactobacillus powder into the fermentation broth in the step (2) under aseptic operation, maintaining positive pressure in a tank under 0.02MPa with aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time every 12 hours for 5.0min at a stirring speed of 50rpm, and stopping fermentation after 188 hours of fermentation to obtain the liquid fermentation product containing chitosan oligosaccharide.
(4) Deslagging and degerming:
regulating the pH value of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and then removing slag and bacteria by using a four-stage filtering device; the pore diameters of the filter media of the four-stage filter device are as follows: 610 mesh, 1.1 μm, 0.46 μm and 0.23 μm, clear transparent chitosan oligosaccharide filtrate was obtained.
(5) Adsorption treatment:
adjusting the pH value of the filtrate in the step (4) to 9, adding the activated weak alkaline resin according to the solid-to-liquid ratio of 1:20, maintaining the system temperature at 35 ℃, and stirring for 4.5 hours under the condition of 210 rpm; and (3) sampling and detecting the protein concentration change in the solution at regular time, filtering to remove resin after the protein content in the solution cannot be detected, and regulating the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 10.2g/L.
(6) Alcohol precipitation and drying treatment:
adding 3 times of absolute ethyl alcohol into the chitosan oligosaccharide solution in the step (5), uniformly stirring, standing overnight, filtering, taking precipitate, washing with 75% ethyl alcohol solution for 5 times, and drying in a freeze dryer for 30h to obtain 29.6g of white snowflake chitosan oligosaccharide solid powder.
TABLE 2 molecular weight distribution of chitooligosaccharides
| Chitosan oligosaccharide DP value | 2 | 3 | 5 |
| Duty cycle (%) | 3.1 | 4.6 | 92.3 |
Example 3
A method for preparing microbial source chitosan oligosaccharide by fermentation method comprises the following steps:
(1) And (3) aspergillus fermentation:
sucking 0.1-0.12mL of the preserved strain under aseptic operation, uniformly coating in slant culture medium, culturing at 30deg.C for 6 days to obtain strain required for fermentation, scraping yellow spore under aseptic operation, transferring into sterile physiological saline, and preparing into spore with concentration of 7.0X10 8 Spore suspension of individual spores/mL; transferring the spore suspension into a seed culture medium according to 15wt% of inoculation amount, and culturing for 30 hours at 200rpm in a constant temperature shaking table at 30 ℃ to prepare seed liquid;
fermenting in a 5L fermentation tank: 3.5L of liquid loading amount, inoculating 525mL of seed liquid into 2975mL of fermentation medium in a sterile mode, controlling the culture temperature to be 30 ℃, controlling the rotation speed to be 250rpm, controlling the aeration ratio to be 1.6v/v/min, after fermenting for 36h, raising the temperature of the fermentation liquid to be 60 ℃, adjusting the pH value to be 8 by using sodium hydroxide solution, and carrying out heat preservation and stirring for 24h;
the slant culture medium comprises the following raw materials in parts by weight: 220 parts of peeled potatoes, 25 parts of glucose, 23 parts of agar, 1000 parts of water, pH6.8 and sterilizing for 30min at 121 ℃ in a wet heat mode;
the seed culture medium comprises the following raw materials in parts by weight: 15 parts of sucrose, 15 parts of yeast powder, 8 parts of molasses, pH7, 1000 parts of water and carrying out damp-heat sterilization at 121 ℃ for 30min; the fermentation medium comprises the following components in parts by weight: 30 parts of sucrose, 20 parts of soluble starch, 10 parts of yeast powder, 15 parts of soybean powder, 4 parts of molasses, 1 part of sodium acetate, 1000 parts of water, and carrying out wet heat sterilization for 30min at the pH of 7 and 121 ℃;
(2) Bacillus licheniformis fermentation:
adding 200mL of sterilized nutrient solution into the aspergillus fermentation liquid in the step (1), uniformly stirring, regulating the pH value of the fermentation liquid to 7, reducing the temperature of the fermentation liquid to 39 ℃, inoculating 105g of bacillus licheniformis powder into the fermentation liquid under aseptic operation, controlling the rotating speed to 350rpm, controlling the aeration ratio to 1v/v/min, and fermenting for 24 hours.
The nutrient solution comprises the following components in parts by weight: glucose 70 parts, naCl2.8 parts, K 2 HPO 4 1.8 parts of MgSO 4 ·7H 2 0.9 part of O FeSO 4 ·7H 2 O0.04 part, caCl 2 1.0 part and 200 parts of water, and sterilizing the nutrient solution at 121 ℃ for 30min under damp heat.
(3) Lactic acid bacteria fermentation:
and (3) inoculating 70g of lactobacillus powder into the fermentation broth in the step (2) under aseptic operation, maintaining positive pressure in a tank under 0.04MPa with aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time every 12 hours for 5.0min at a stirring speed of 80rpm, and stopping fermentation after fermentation for 240 hours to obtain the liquid fermentation product containing chitosan oligosaccharide.
(4) Deslagging and degerming:
regulating the pH value of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and then removing slag and bacteria by using a four-stage filtering device; the pore diameters of the filter media of the four-stage filter device are as follows: 620 mesh, 1.2 μm, 0.47 μm and 0.25 μm to obtain clear transparent chitosan oligosaccharide filtrate.
(5) Adsorption treatment:
adjusting the pH value of the filtrate in the step (4) to 10.5, adding the activated weak alkaline resin according to the solid-to-liquid ratio of 1:30, maintaining the system temperature at 40 ℃, and stirring for 8 hours under the condition of 220 rpm; and (3) sampling and detecting the protein concentration change in the solution at regular time, filtering to remove resin after the protein content in the solution cannot be detected, and regulating the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 8.3g/L.
(6) Alcohol precipitation and drying treatment:
adding 4 times of absolute ethyl alcohol into the chitosan oligosaccharide solution in the step (5), uniformly stirring, standing overnight, filtering, taking precipitate, washing with 75% ethyl alcohol solution for 6 times, and drying in a freeze dryer for 36h to obtain 24.5g of white snowflake chitosan oligosaccharide solid powder.
TABLE 3 molecular weight distribution of Chitosan oligosaccharides
| Chitosan oligosaccharide DP value | 2 | 3 | 5 |
| Duty cycle (%) | 8.6 | 9.2 | 82.2 |
From the above examples, the present invention provides a method for preparing microbial source chitosan oligosaccharide by fermentation. The molecular weight distribution of the microbial source chitosan oligosaccharide prepared by the method is more concentrated, and the chitosan oligosaccharide accounts for 92.3% under the optimal condition.
Claims (9)
1. A method for preparing microbial source chitosan oligosaccharide by a fermentation method is characterized by comprising the following steps:
step 1): and (3) aspergillus fermentation: inoculating the aspergillus inclined spores into seed liquid for culture, inoculating the cultured seed liquid into a fermentation medium for culture, and carrying out aspergillus fermentation; the aspergillus used for fermentation is aspergillus ochraceus, the classification of which is named aspergillus ochraceus, the Latin name is Aspergillus ochraceus, and the aspergillus is preserved in China general microbiological culture collection center (CGMCC), and the strain preservation number is: CGMCC No.15668, the preservation date is: 25 th 2018, 04 th month, deposit unit address: beijing, chaoyang area, north Chenxi Lu No.1, 3;
step 2): bacillus licheniformis fermentation: adding sterilized nutrient solution into the aspergillus fermentation liquid obtained in the step 1), uniformly stirring, adjusting the pH of the fermentation liquid to 5.5-7, and then inoculating 5-30g/L bacillus licheniformis powder under aseptic operation to continue fermentation;
step 3): lactic acid bacteria fermentation: inoculating 5-20g/L lactobacillus powder into the fermentation liquid obtained in the step 2) under aseptic operation, and continuing fermenting to obtain a liquid fermentation product containing chitosan oligosaccharide;
step 4): deslagging and degerming: adding solid calcium hydroxide into the liquid fermentation product containing the chitosan oligosaccharide obtained in the step 3) under the stirring state to adjust the pH value to 6.5-7.5, filtering, removing residues and sterilizing to obtain clear and transparent liquid containing the chitosan oligosaccharide;
step 5): adsorption treatment: adjusting the pH value of the liquid obtained in the step 4) to 8.0-10.5 by using a sodium hydroxide solution, adding a weak alkaline ion exchange resin according to the solid-liquid mass ratio of 1:10-1:30, adsorbing, filtering to remove the resin after the adsorption is finished, and adjusting the solution to be neutral to obtain a chitosan oligosaccharide solution;
step 6): alcohol precipitation and drying treatment: adding 3-4 times of absolute ethyl alcohol into the chitosan oligosaccharide solution obtained in the step 5), uniformly stirring, standing overnight, taking precipitate, washing the precipitate with the ethyl alcohol solution for 4-6 times, and drying in a freeze dryer to obtain white snowflake chitosan oligosaccharide solid powder.
2. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the seed solution in step 1) is prepared by the following steps: sucking 0.1-0.12mL of strain under aseptic operation, uniformly coating in slant culture medium, culturing at 27-30deg.C for 4-6 days to obtain strain required for fermentation, scraping yellow spore under aseptic operation, transferring into sterile physiological saline, and preparing into spore with concentration of 4.0X10 8 -7.0×10 8 Spore suspension of individual spores/mL; transferring the spore suspension into a seed culture medium according to the inoculation amount of 10-15wt%, and culturing for 24-30h in a constant temperature shaking table at 27-30 ℃ at 170-200 rpm; transferring the seed solution into fermentation medium under aseptic operation at 27-30deg.C and rotation speed of 100-450rpm with aeration ratio of 0.4v/v/min-1.6 v/v%Culturing for 36-48h under min, heating to 45-65deg.C, adjusting pH to 6.0-8.0 with sodium hydroxide solution, maintaining the temperature, and stirring for 12-24h.
3. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 2, wherein the raw materials of the slant culture medium comprise, by weight, 200-220 parts of peeled potatoes, 20-25 parts of glucose, 20-23 parts of agar and 1000 parts of water, wherein the pH value is 6-6.8, and the slant culture medium is subjected to wet heat sterilization at 121 ℃ for 30min; the raw materials of the seed culture medium comprise 8-15 parts by weight of sucrose, 8-15 parts by weight of yeast powder, 2-8 parts by weight of molasses and 1000 parts by weight of water, wherein the pH value is 6-7, and the seed culture medium is subjected to damp-heat sterilization at 121 ℃ for 30min; the raw materials of the fermentation medium comprise, by weight, 20-30 parts of sucrose, 10-20 parts of soluble starch, 5-10 parts of yeast powder, 8-15 parts of soybean powder, 1.5-4 parts of molasses, 0.1-1 part of sodium acetate and 1000 parts of water, wherein the pH value is 5.5-7, and the fermentation medium is subjected to wet heat sterilization at 121 ℃ for 30min.
4. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the nutrient solution supplemented in the step 2) comprises 17.5-70 parts by weight of glucose, 0.35-2.8 parts by weight of NaCl and K 2 HPO 4 0.6-1.8 parts of MgSO 4 ·7H 2 0.4-0.9 part of O, feSO 4 ·7H 2 0.01-0.04 part of O and CaCl 2 0.1-1.0 part and 200 parts of water are added, and after the nutrient solution is prepared, the nutrient solution is sterilized by moist heat at 121 ℃ for 30min and is cooled for later use.
5. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the conditions of the bacillus licheniformis fermentation in the step 2) are as follows: the fermentation time is 12-24h, the fermentation temperature is 33-39 ℃, the rotation speed is 100-350rpm, and the ventilation ratio is 0.2v/v/min-1.0v/v/min.
6. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the conditions of the lactobacillus fermentation in the step 3) are as follows: the fermentation time is 168-240h, the aseptic nitrogen is kept at positive pressure of 0.01-0.04MPa, the fermentation is kept still, the intermittent stirring is carried out for 1 time every 12h, the stirring time is 5.0min, and the stirring rotating speed is 30rpm-80rpm.
7. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the sterilization and deslagging treatment in the step 4) is specifically as follows: removing slag and bacteria from the liquid fermentation product of the chitosan oligosaccharide with the pH value adjusted through a four-stage filtering device, wherein the pore diameters of the filtering media of the four-stage filtering device are as follows: 600-620 meshes,
1-1.2 μm, 0.45-0.47 μm and 0.22-0.25 μm.
8. The method for preparing microbial source chitosan oligosaccharide by fermentation according to claim 1, wherein the adsorption treatment in step 5) specifically comprises: maintaining the temperature of the system at 30-40 ℃, stirring at 200-220rpm, adsorbing for 1-8 h, sampling and detecting the protein concentration change in the solution at regular time, and finishing the adsorption step after the protein content in the solution cannot be detected.
9. The method for preparing microbial source chitosan oligosaccharide according to claim 1, wherein the volume fraction of the ethanol solution used for washing in the step 6) is 75%, and the time of freeze-drying is 24-36 hours.
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