CN112941131A - 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 chitosan oligosaccharide by a fermentation method. The method for preparing the chitosan oligosaccharide by performing three-bacterium mixed fermentation by using aspergillus, bacillus licheniformis and lactic acid bacteria comprises the following steps: (1) fermenting aspergillus; (2) b, fermenting the bacillus licheniformis; (3) fermenting by using lactic acid bacteria; (4) deslagging and degerming; (5) adsorption treatment; (6) and (5) carrying out 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 the source of raw materials and the external environment of seasons, the production process is more controllable, and the product quality is more stable. (2) The whole process is green, the discharge of strong acid, strong alkali and fermentation waste liquid is small, the environmental pollution is small, the production condition is mild, the energy consumption is low, and the national energy-saving and emission-reduction policy is met. The microbial chitosan oligosaccharide prepared by the method has concentrated molecular weight distribution, and the ratio of the chitosan pentasaccharide can reach 92.3% under the optimal condition.
Description
Technical Field
The invention relates to a method for preparing microbial chitosan oligosaccharide by a fermentation method, belonging to the technical field of microbial preparation.
Background
Chitosan (CTS), also called chitosan, is the only basic polysaccharide in nature, but its application is greatly limited because it can only be dissolved in acidic solution.
Chitosan Oligosaccharides (COS) are oligomers obtained by enzymatic reaction or chemical degradation of CTS, have the polymerization degree of 2-20, and are the only basic amino oligosaccharides with positive charges in the nature at present. COS is non-toxic, has low relative molecular mass, good water solubility and high biological activity, 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 tumors 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, firstly, chitosan is extracted from shrimp and crab shells by an acid-base method, and then the 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 is consumed in the process of extracting chitosan from the starting materials of shrimp and crab shells, a large amount of acid and alkali waste liquid is generated, and the problem of environmental pollution is very serious. The traditional production mode is contrary to the current environmental protection policy in China, and is not beneficial to the continuous and benign development of related industries.
Therefore, the technical problem to be solved by those skilled in the art is how to provide a method for preparing chitosan oligosaccharide, which has mild production conditions, high concentration of molecular weight of the product chitosan oligosaccharide and less waste liquid discharge in the production process.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the prior preparation method of the chitosan oligosaccharide of microbial source generates a large amount of waste liquid in the production process, pollutes the environment, and has the problems of low concentration of the molecular weight of the chitosan oligosaccharide product and the like.
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): fermenting by using aspergillus: transferring aspergillus slant spores into a seed solution for culture, transferring the cultured seed solution into a fermentation culture medium for culture, and performing aspergillus fermentation;
step 2): b, fermentation of the bacillus licheniformis: supplementing sterilized nutrient solution into the aspergillus fermentation liquor obtained in the step 1), uniformly stirring, adjusting the pH of the fermentation liquor to 5.5-7, adding 5-30g/L of bacillus licheniformis powder under aseptic operation, and continuing fermentation;
step 3): and (3) fermenting lactic acid bacteria: inoculating 5-20g/L of lactobacillus powder into the fermentation liquor obtained in the step 2) under aseptic operation, and continuing fermentation to obtain a liquid fermentation product containing chitosan oligosaccharide;
step 4): deslagging and degerming treatment: 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 of the liquid obtained in the step 4) to 8.0-10.5 by using a sodium hydroxide solution, adding a weakly basic ion exchange resin according to the solid-liquid mass ratio of 1:10-1:30, adsorbing, filtering to remove the resin after adsorption is finished, and adjusting the solution to be neutral to obtain a chitosan oligosaccharide solution;
step 6): carrying out alcohol precipitation drying treatment: adding 3-4 times of anhydrous ethanol into the chitosan oligosaccharide solution obtained in the step 5), uniformly stirring, standing overnight, taking a precipitate, washing the precipitate for 4-6 times by using the ethanol solution, and drying in a freeze dryer to obtain white snowflake-shaped chitosan oligosaccharide solid powder.
Preferably, the Aspergillus used for fermentation in step 1) is Aspergillus ochraceus which is classified and named as Aspergillus ochraceus, the name of latin is Aspergillus ochracea, the Aspergillus ochraceus is deposited in the common microorganism center of China committee for culture collection of microorganisms, namely CGMCC, and the culture collection number is as follows: CGMCC No.15668, the preservation date is: 25/04/2018, depository address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Preferably, the preparation method of the seed liquid in the step 1) comprises the following steps: sucking the strain under aseptic condition0.1-0.12mL, uniformly coating in slant culture medium, culturing at 27-30 deg.C for 4-6 days to obtain strain required for fermentation, scraping yellow spore under aseptic operation, transferring into aseptic normal saline to obtain spore with concentration of 4.0 × 108-7.0×108spores/mL of spore suspension; transferring the spore suspension into a seed culture medium according to the inoculum size of 10-15 wt%, and culturing for 24-30h in a constant temperature shaking table at 27-30 ℃ and 200 rpm; inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 10-15 wt% under aseptic operation, culturing for 36-48h under the conditions of 27-30 ℃, 100rpm and 450rpm of rotation speed and 0.4-1.6 v/v/min of aeration ratio, then heating to 45-65 ℃, adjusting the pH to 6.0-8.0 by using a sodium hydroxide solution, and preserving heat and stirring for 12-24 h.
More preferably, the raw materials of the slant culture medium comprise 220 parts by weight of peeled potato 200-; the raw materials of the seed culture medium comprise 8-15 parts of sucrose, 8-15 parts of yeast powder, 2-8 parts of molasses and 1000 parts of water by weight, the pH value is 6-7, and the seed culture medium is subjected to moist heat sterilization at 121 ℃ for 30 min; 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 meal, 1.5-4 parts of molasses, 0.1-1 part of sodium acetate and 1000 parts of water, the pH value of the fermentation medium is 5.5-7, and the fermentation medium is subjected to moist heat sterilization at 121 ℃ for 30 min.
Preferably, the nutrient solution supplemented in the step 2) comprises 17.5 to 70 parts by weight of glucose, 0.35 to 2.8 parts by weight of NaCl and K2HPO40.6 to 1.8 portions of MgSO4·7H20.4-0.9 part of O, FeSO4·7H20.01 to 0.04 portion of O and CaCl20.1 to 1.0 portion and 200 portions of water are added, and after the nutrient solution is prepared, the wet heat sterilization is carried out for 30min at the temperature of 121 ℃, and the cooling is carried out for standby.
Preferably, the conditions for fermentation of the bacillus licheniformis in the step 2) are as follows: the fermentation time is 12-24h, the fermentation temperature is 33-39 ℃, the rotating speed is 100-.
Preferably, the conditions for fermenting the lactic acid bacteria in the step 3) are as follows: fermenting for 168-240 hr, maintaining the positive pressure of 0.01-0.04MPa with sterile nitrogen, standing for fermenting, and intermittently stirring for 1 time every 12 hr at stirring speed of 30-80 rpm for 5.0 min.
Preferably, the degerming and deslagging treatment in the step 4) is specifically as follows: the liquid fermentation product of the chitosan oligosaccharide with the adjusted pH value is subjected to deslagging and degerming by a four-stage filtering device, and the aperture of a filtering medium of the four-stage filtering device is as follows in sequence: 600-620 mesh, 1-1.2 μm, 0.45-0.47 μm and 0.22-0.25 μm.
Preferably, the adsorption treatment of step 5) is specifically: maintaining the temperature of the system at 30-40 ℃, stirring at the speed of 200-220rpm, adsorbing for 1-8 h, sampling at regular time during the period to detect the concentration change of the protein in the solution, and finishing the adsorption step after the content of the protein 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 freeze-drying time is 24-36 h.
The invention utilizes aspergillus, bacillus and lactic acid bacteria to carry out three-bacteria fermentation, and provides an effective method for preparing the microbial chitosan oligosaccharide, which has relatively simple production process and small discharge amount 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 the source of raw materials and the external environment of seasons, the production process is more controllable, and the product quality is more stable. (2) The whole process is green, the discharge of strong acid, strong alkali and fermentation waste liquid is small, the environmental pollution is small, the production condition is mild, the concentration of the molecular weight of the product chitosan oligosaccharide is high, the energy consumption is low, and the method conforms to the national energy-saving and emission-reducing policy.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments 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 (5) drawing a standard curve by using a DNS method with glucosamine as a standard substance.
Sample treatment and determination: centrifuging appropriate amount of chitosan oligosaccharide solution, collecting supernatant, adding 3 times of ethanol to precipitate chitosan oligosaccharide, centrifuging to obtain precipitate, dehydrating the precipitate with anhydrous ethanol for 4 times, and oven drying the precipitate at 60 deg.C to constant weight to obtain crude product of chitosan oligosaccharide. Precisely weighing 5mg of the crude product of the chitosan oligosaccharide, adding 5mL of concentrated hydrochloric acid, hydrolyzing in a constant-temperature oscillation water bath kettle at (100 +/-1) DEG C for 5h, then adjusting the pH to 7.0 by using a sodium hydroxide solution with the mass fraction of 30%, transferring the hydrolysate to a 50mL volumetric flask, fixing the volume of purified water, shaking up, and then properly diluting to determine the concentration of reducing sugar by using a DNS method.
(2) Molecular weight measurement of Chitosan oligosaccharide (GPC method)
Drawing a standard curve: and (3) drawing a standard curve by using glucosamine, chitotriose, chitopentasaccharide and chitoheptasaccharide as standard substances, taking the peak-out time of the standard substances as an abscissa and the DP value of the chitosan oligosaccharide as an ordinate.
Chromatographic conditions are as follows: a chromatographic column; mobile phase 0.12M acetic acid-1M sodium acetate; the flow rate is 0.8 mL/min; sample introduction volume is 50 uL; the column temperature is 30 ℃; run time 36 min.
And (3) sample determination: centrifuging appropriate amount of chitosan oligosaccharide solution, collecting supernatant, adding 3 times of ethanol to precipitate chitosan oligosaccharide, centrifuging to obtain precipitate, dehydrating the precipitate with anhydrous ethanol for 4 times, and oven drying the precipitate at 60 deg.C to constant weight to obtain crude product of chitosan oligosaccharide. Dissolving the crude product of the chitosan oligosaccharide by using a mobile phase, performing GPC detection after microfiltration by using a 0.22 mu m membrane, and calculating the DP value of the chitosan oligosaccharide according to a standard curve.
Example 1
A method for preparing a microbial source chitosan oligosaccharide by a fermentation method comprises the following steps:
(1) fermenting by using aspergillus:
sucking 0.1mL of the preserved strain under aseptic condition, uniformly coating the strain on a slant culture medium, culturing at a constant temperature of 27 ℃ for 4 days to obtain the strain required by fermentation, scraping yellow spores under aseptic condition, transferring the yellow spores into aseptic normal saline, and preparing to obtain the spore with the concentration of 4.0 × 108spores/mL of spore suspension; transferring the spore suspension into a seed culture medium according to the inoculation amount of 10 wt%, and culturing for 24h in a constant temperature shaking table at 27 ℃ and at 170rpm to obtain a seed solution；
5L fermentation tank fermentation: the liquid loading amount is 3.5L, 350mL of the seed liquid is inoculated into 3150mL of fermentation medium in a sterile operation mode, the culture temperature is 27 ℃, the rotation speed range is controlled to be 100rpm, the aeration ratio is 0.4v/v/min, after fermentation is carried out for 36h, the temperature of the fermentation liquid is raised to 45 ℃, the pH value is adjusted to 6 by using a sodium hydroxide solution, and the heat preservation and the stirring are carried out for 12 h;
the slant culture medium comprises the following raw materials in parts by weight: peeling potato 200 parts, glucose 20 parts, agar 20 parts, water 1000 parts, pH6, and performing moist heat sterilization at 121 ℃ for 30 min;
the seed culture medium comprises the following raw materials in parts by weight: 8 parts of cane sugar, 8 parts of yeast powder, 2 parts of molasses, 6 parts of pH, 1000 parts of water and 30min of moist heat sterilization at 121 ℃; the fermentation medium comprises the following components in parts by weight: 20 parts of cane sugar, 10 parts of soluble starch, 5 parts of yeast powder, 8 parts of soybean meal, 1.5 parts of molasses, 0.1 part of sodium acetate, 1000 parts of water, pH5.5 and carrying out moist heat sterilization at 121 ℃ for 30 min.
(2) B, fermentation of the bacillus licheniformis:
adding 200mL of sterilized nutrient solution into the Aspergillus fermentation liquor obtained in the step (1), uniformly stirring, adjusting the pH value of the fermentation liquor to 5.5, cooling the temperature of the fermentation liquor to 33 ℃, inoculating 17.5g of Bacillus licheniformis powder into the fermentation liquor under aseptic operation, controlling the rotating speed to be 100rpm, controlling the aeration ratio to be 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 NaCl0, and K2HPO40.6 to 1.8 portions of MgSO4·7H20.4 part of O in FeSO4·7H20.01 portion of O and CaCl20.1 part of water and 200 parts of water, and sterilizing for 30min at 121 ℃ by moist heat after the nutrient solution is prepared.
(3) And (3) fermenting lactic acid bacteria:
and (3) inoculating 17.5g of lactic acid bacteria powder into the fermentation liquor in the step (2) under the aseptic operation, maintaining the positive pressure in the tank to be 0.01MPa by using aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time every 12 hours for 5.0min at the stirring speed of 30rpm, and stopping fermentation after 168 hours of fermentation to obtain the liquid fermentation product containing the chitosan oligosaccharide.
Fermenting for 168-168 times, maintaining the positive pressure of 0.01-0.04MPa with sterile nitrogen, standing for fermentation, intermittently stirring for 1 time every 12h, stirring for 5.0min, and stirring at 30-80 rpm.
(4) Deslagging and degerming treatment:
adjusting the pH of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and removing slag and bacteria by a four-stage filter device; the aperture of the filter medium of the four-stage filter device is as follows: 600 mesh, 1 μm, 0.45 μm and 0.22 μm, a clear and transparent chitosan oligosaccharide filtrate was obtained.
(5) Adsorption treatment:
adjusting the pH value of the filtrate obtained in the step (4) to 8, adding activated weak alkaline resin according to the solid-to-liquid ratio of 1:10, maintaining the system temperature at 30 ℃, and stirring at 200rpm for 1 h; and sampling at regular time during the period to detect the change of the protein concentration in the solution, filtering to remove the resin after the protein content in the solution cannot be detected, and adjusting the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 7.8 g/L.
(6) Carrying out alcohol precipitation drying treatment:
and (3) adding 3 times of absolute ethyl alcohol into the chitosan oligosaccharide solution obtained in the step (5), uniformly stirring, standing overnight, filtering, taking a precipitate, washing with a 75% ethanol solution for 4 times, and drying in a freeze dryer for 24 hours to obtain 22.8g of white snowflake-shaped chitosan oligosaccharide solid powder.
TABLE 1 Chitosan oligosaccharide molecular weight distribution
| DP value of Chitosan oligosaccharide | 2 | 3 | 5 |
| Ratio (%) | 5.2 | 5.3 | 89.7 |
Example 2
A method for preparing a microbial source chitosan oligosaccharide by a fermentation method comprises the following steps:
(1) fermenting by using aspergillus:
sucking 0.11mL of the preserved strain under aseptic condition, uniformly coating the strain on a slant culture medium, culturing at 28 deg.C for 5 days to obtain strain required for fermentation, scraping yellow spore under aseptic condition, transferring into aseptic normal saline to obtain the final product with spore concentration of 5.5 × 108spores/mL of spore suspension; transferring the spore suspension into a seed culture medium according to the inoculation amount of 13 wt%, and culturing for 27h in a constant temperature shaking table at 28 ℃ and 185rpm to prepare a seed solution;
5L fermentation tank fermentation: liquid loading amount is 3.5L, 445mL of seed liquid is aseptically inoculated into 3055mL of fermentation medium, the culture temperature is 28 ℃, the rotation speed range is controlled to be 100 rpm-250 rpm, the aeration ratio is 1v/v/min, after fermentation is carried out for 42h, the temperature of the fermentation liquid is raised to 55 ℃, the pH value is adjusted to 7 by using sodium hydroxide solution, and the fermentation liquid is kept warm and stirred for 18 h;
the slant culture medium comprises the following raw materials in parts by weight: peeling potato 210 parts, glucose 22 parts, agar 21 parts, water 1000 parts, pH 6.4, and performing moist heat sterilization at 121 ℃ for 30 min;
the seed culture medium comprises the following raw materials in parts by weight: 11 parts of cane sugar, 12 parts of yeast powder, 5 parts of molasses, pH6.5, 1000 parts of water, and carrying out damp-heat sterilization at 121 ℃ for 30 min; the fermentation medium comprises the following components in parts by weight: 25 parts of cane sugar, 15 parts of soluble starch, 8 parts of yeast powder, 7 parts of soybean meal, 3 parts of molasses, 0.5 part of sodium acetate, 1000 parts of water, 6 parts of pH, and carrying out moist heat sterilization at 121 ℃ for 30 min;
(2) b, fermentation of the bacillus licheniformis:
adding 200mL of sterilized nutrient solution into the Aspergillus fermentation liquor obtained in the step (1), uniformly stirring, adjusting the pH value of the fermentation liquor to 6.3, cooling the temperature of the fermentation liquor to 36 ℃, adding 40g of Bacillus licheniformis powder into the fermentation liquor under aseptic operation, controlling the rotation speed to be 150rpm and the aeration ratio to be 0.4v/v/min, and fermenting for 18 h.
The nutrient solution comprises the following components in parts by weight: 45 parts of glucose, 1.5 parts of NaCl1, and K2HPO41.2 parts of MgSO 24·7H20.7 part of O in FeSO4·7H20.02 portion of O and CaCl20.6 part of water and 200 parts of water, and sterilizing for 30min at 121 ℃ by moist heat after the nutrient solution is prepared.
(3) And (3) fermenting lactic acid bacteria:
adding 55g of lactobacillus powder into the fermentation liquid of the step (2) under aseptic operation, maintaining the positive pressure in the tank at 0.02MPa by aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time at intervals of 12h, stirring for 5.0min at the stirring speed of 50rpm, and stopping fermentation after 188h of fermentation to obtain the liquid fermentation product containing the chitosan oligosaccharide.
(4) Deslagging and degerming treatment:
adjusting the pH of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and removing slag and bacteria by a four-stage filter device; the aperture of the filter medium of the four-stage filter device is as follows: 610 mesh, 1.1 μm, 0.46 μm and 0.23 μm, a clear transparent chitosan oligosaccharide filtrate was obtained.
(5) Adsorption treatment:
adjusting the pH value of the filtrate obtained in the step (4) to 9, adding activated weak alkaline resin according to the solid-to-liquid ratio of 1:20, maintaining the system temperature at 35 ℃, and stirring at 210rpm for 4.5 hours; and sampling at regular time during the period to detect the change of the protein concentration in the solution, filtering to remove the resin after the protein content in the solution cannot be detected, and adjusting the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 10.2 g/L.
(6) Carrying out alcohol precipitation drying treatment:
and (3) adding 3 times of absolute ethyl alcohol into the chitosan oligosaccharide solution obtained in the step (5), uniformly stirring, standing overnight, filtering, taking a precipitate, washing with a 75% ethanol solution for 5 times, and drying in a freeze dryer for 30 hours to obtain 29.6g of white snowflake-shaped chitosan oligosaccharide solid powder.
TABLE 2 Chitosan oligosaccharide molecular weight distribution
| DP value of Chitosan oligosaccharide | 2 | 3 | 5 |
| Ratio (%) | 3.1 | 4.6 | 92.3 |
Example 3
A method for preparing a microbial source chitosan oligosaccharide by a fermentation method comprises the following steps:
(1) fermenting by using aspergillus:
sucking 0.1-0.12mL of the preserved strain under aseptic operation, uniformly coating the strain in a slant culture medium, culturing at constant temperature of 30 ℃ for 6 days to obtain strain required for fermentation, scraping yellow spores under aseptic operation, transferring the yellow spores into aseptic normal saline, and preparing to obtain the spore with the spore concentration of 7.0 × 108spores/mL of spore suspension; transferring the spore suspension into a seed culture medium according to the inoculum size of 15 wt%, and culturing for 30h in a constant temperature shaking table at 30 ℃ and 200rpm to prepare a seed solution;
5L fermentation tank fermentation: the liquid loading amount is 3.5L, 525mL of the seed liquid is aseptically inoculated into 2975mL of fermentation medium, the culture temperature is 30 ℃, the rotation speed range is controlled to be 250rpm, the aeration ratio is 1.6v/v/min, after fermentation is carried out for 36h, the temperature of the fermentation liquid is raised to 60 ℃, the pH value is adjusted to 8 by using sodium hydroxide solution, and the fermentation liquid is kept warm and stirred for 24 h;
the slant culture medium comprises the following raw materials in parts by weight: peeling 220 parts of potato, 25 parts of glucose, 23 parts of agar, 1000 parts of water, pH6.8, and performing moist heat sterilization at 121 ℃ for 30 min;
the seed culture medium comprises the following raw materials in parts by weight: 15 parts of cane sugar, 15 parts of yeast powder, 8 parts of molasses, 7 parts of pH, 1000 parts of water and 30min of moist heat sterilization at 121 ℃; the fermentation medium comprises the following components in parts by weight: 30 parts of cane sugar, 20 parts of soluble starch, 10 parts of yeast powder, 15 parts of soybean meal, 4 parts of molasses, 1 part of sodium acetate, 1000 parts of water, 7 parts of pH value, and carrying out moist heat sterilization at 121 ℃ for 30 min;
(2) b, fermentation of the bacillus licheniformis:
adding 200mL of sterilized nutrient solution into the Aspergillus fermentation liquor obtained in the step (1), uniformly stirring, adjusting the pH value of the fermentation liquor to 7, cooling the temperature of the fermentation liquor to 39 ℃, adding 105g of Bacillus licheniformis powder into the fermentation liquor under aseptic operation, controlling the rotation speed to 350rpm and the aeration ratio to be 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 and K2HPO41.8 parts of MgSO 24·7H20.9 part of O in FeSO4·7H20.04 part of O, CaCl21.0 part of water and 200 parts of water, and sterilizing for 30min at 121 ℃ by moist heat after the nutrient solution is prepared.
(3) And (3) fermenting lactic acid bacteria:
and (3) inoculating 70g of lactic acid bacteria powder into the fermentation liquor in the step (2) under the aseptic operation, maintaining the positive pressure in the tank to be 0.04MPa by using aseptic nitrogen, standing for fermentation, intermittently stirring for 1 time every 12 hours for 5.0min at the stirring speed of 80rpm, and stopping fermentation after 240 hours of fermentation to obtain the liquid fermentation product containing the chitosan oligosaccharide.
(4) Deslagging and degerming treatment:
adjusting the pH of the liquid fermentation product containing the chitosan oligosaccharide obtained in the step (3) to 6.5 by using calcium hydroxide, and removing slag and bacteria by a four-stage filter device; the aperture of the filter medium of the four-stage filter device is as follows: 620 mesh, 1.2 μm, 0.47 μm and 0.25 μm, a clear transparent chitosan oligosaccharide filtrate was obtained.
(5) Adsorption treatment:
adjusting the pH value of the filtrate obtained in the step (4) to 10.5, adding activated weak base resin according to the solid-to-liquid ratio of 1:30, and stirring for 8 hours under the conditions of maintaining the system temperature at 40 ℃ and 220 rpm; and sampling at regular time during the period to detect the change of the protein concentration in the solution, filtering to remove the resin after the protein content in the solution cannot be detected, and adjusting the pH value of the solution to about 7.0 to obtain the chitosan oligosaccharide solution, wherein the concentration of the chitosan oligosaccharide is 8.3 g/L.
(6) Carrying out alcohol precipitation drying treatment:
and (3) adding 4 times of absolute ethyl alcohol into the chitosan oligosaccharide solution obtained in the step (5), uniformly stirring, standing overnight, filtering, taking a precipitate, washing with a 75% ethanol solution for 6 times, and drying in a freeze dryer for 36 hours to obtain 24.5g of white snowflake-shaped chitosan oligosaccharide solid powder.
TABLE 3 Chitosan oligosaccharide molecular weight distribution
| DP value of Chitosan oligosaccharide | 2 | 3 | 5 |
| Ratio (%) | 8.6 | 9.2 | 82.2 |
From the above examples, the present invention provides a method for preparing chitosan oligosaccharide of microbial origin by fermentation. The microbial chitosan oligosaccharide prepared by the method has concentrated molecular weight distribution, and the ratio of the chitosan pentasaccharide can reach 92.3% under the optimal condition.
Claims (10)
1. A method for preparing chitosan oligosaccharide of microbial source by a fermentation method is characterized by comprising the following steps:
step 1): fermenting by using aspergillus: transferring aspergillus slant spores into a seed solution for culture, transferring the cultured seed solution into a fermentation culture medium for culture, and performing aspergillus fermentation;
step 2): b, fermentation of the bacillus licheniformis: supplementing sterilized nutrient solution into the aspergillus fermentation liquor obtained in the step 1), uniformly stirring, adjusting the pH of the fermentation liquor to 5.5-7, adding 5-30g/L of bacillus licheniformis powder under aseptic operation, and continuing fermentation;
step 3): and (3) fermenting lactic acid bacteria: inoculating 5-20g/L of lactobacillus powder into the fermentation liquor obtained in the step 2) under aseptic operation, and continuing fermentation to obtain a liquid fermentation product containing chitosan oligosaccharide;
step 4): deslagging and degerming treatment: 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 of the liquid obtained in the step 4) to 8.0-10.5 by using a sodium hydroxide solution, adding a weakly basic ion exchange resin according to the solid-liquid mass ratio of 1:10-1:30, adsorbing, filtering to remove the resin after adsorption is finished, and adjusting the solution to be neutral to obtain a chitosan oligosaccharide solution;
step 6): carrying out alcohol precipitation drying treatment: adding 3-4 times of anhydrous ethanol into the chitosan oligosaccharide solution obtained in the step 5), uniformly stirring, standing overnight, taking a precipitate, washing the precipitate for 4-6 times by using the ethanol solution, and drying in a freeze dryer to obtain white snowflake-shaped chitosan oligosaccharide solid powder.
2. The method for preparing chitosan oligosaccharide derived from microorganisms by fermentation according to claim 1, wherein the Aspergillus used in the fermentation in step 1) is Aspergillus ochraceus, which is classified and named as Aspergillus ochraceus, and the Latin has the name of Aspergillus ochraceus, and is deposited in the China general microbiological culture Collection center, CGMCC, with the strain preservation number: CGMCC No.15668, the preservation date is: 25/04/2018, depository address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
3. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the seed solution in step 1) is prepared by: sucking 0.1-0.12mL of strain under aseptic condition, uniformly coating on slant culture medium, culturing at 27-30 deg.C for 4-6 days to obtain strain required for fermentation, scraping yellow spore under aseptic condition, transferring into aseptic normal saline to obtain spore with spore concentration of 4.0 × 108-7.0×108spores/mL of spore suspension; transferring the spore suspension into a seed culture medium according to the inoculum size of 10-15 wt%, and culturing for 24-30h in a constant temperature shaking table at 27-30 ℃ and 200 rpm; inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 10-15 wt% under aseptic operation, culturing for 36-48h under the conditions of 27-30 ℃, 100rpm and 450rpm of rotation speed and 0.4-1.6 v/v/min of aeration ratio, then heating to 45-65 ℃, adjusting the pH to 6.0-8.0 by using a sodium hydroxide solution, and preserving heat and stirring for 12-24 h.
4. The method for preparing chitosan oligosaccharide derived from microorganisms by fermentation as claimed in claim 3, wherein the raw materials of the slant culture medium comprise, in parts by weight, 200-220 parts of peeled potato, 20-25 parts of glucose, 20-23 parts of agar and 1000 parts of water, the pH value is 6-6.8, and the slant culture medium is sterilized by moist heat at 121 ℃ for 30 min; the raw materials of the seed culture medium comprise 8-15 parts of sucrose, 8-15 parts of yeast powder, 2-8 parts of molasses and 1000 parts of water by weight, the pH value is 6-7, and the seed culture medium is subjected to moist heat sterilization at 121 ℃ for 30 min; 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 meal, 1.5-4 parts of molasses, 0.1-1 part of sodium acetate and 1000 parts of water, the pH value of the fermentation medium is 5.5-7, and the fermentation medium is subjected to moist heat sterilization at 121 ℃ for 30 min.
5. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the nutrient solution supplemented in step 2) comprises, in parts by weight, 17.5 to 70 parts of glucose, 0.35 to 2.8 parts of NaCl, and K2HPO40.6 to 1.8 portions of MgSO4·7H20.4-0.9 part of O, FeSO4·7H20.01 to 0.04 portion of O and CaCl20.1 to 1.0 portion and 200 portions of water are added, and after the nutrient solution is prepared, the wet heat sterilization is carried out for 30min at the temperature of 121 ℃, and the cooling is carried out for standby.
6. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the conditions for fermentation of Bacillus licheniformis in step 2) are as follows: the fermentation time is 12-24h, the fermentation temperature is 33-39 ℃, the rotating speed is 100-.
7. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the conditions for the fermentation with lactic acid bacteria in step 3) are as follows: fermenting for 168-240 hr, maintaining the positive pressure of 0.01-0.04MPa with sterile nitrogen, standing for fermenting, and intermittently stirring for 1 time every 12 hr at stirring speed of 30-80 rpm for 5.0 min.
8. The method for preparing the microbial chitosan oligosaccharide by the fermentation method according to claim 1, wherein the degerming and deslagging treatment in the step 4) is specifically as follows: the liquid fermentation product of the chitosan oligosaccharide with the adjusted pH value is subjected to deslagging and degerming by a four-stage filtering device, and the aperture of a filtering medium of the four-stage filtering device is as follows in sequence: 600-620 mesh, 1-1.2 μm, 0.45-0.47 μm and 0.22-0.25 μm.
9. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the adsorption treatment of step 5) is specifically: maintaining the temperature of the system at 30-40 ℃, stirring at the speed of 200-220rpm, adsorbing for 1-8 h, sampling at regular time during the period to detect the concentration change of the protein in the solution, and finishing the adsorption step after the content of the protein in the solution cannot be detected.
10. The method for preparing chitosan oligosaccharide of microbial origin by fermentation according to claim 1, wherein the ethanol solution used for washing in step 6) has a volume fraction of 75%, and the freeze-drying time is 24-36 h.
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