CN110616242A

CN110616242A - Method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae

Info

Publication number: CN110616242A
Application number: CN201911020275.0A
Authority: CN
Inventors: 孙建安; 毛相朝; 赵甜宇
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2019-12-27

Abstract

The invention discloses a method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae. The method comprises the steps of firstly activating Aspergillus oryzae strains stored in a test tube, transferring the activated Aspergillus oryzae strains to an erlenmeyer flask, then inoculating the activated Aspergillus oryzae strains to a solid culture medium, carrying out solid fermentation culture on the Aspergillus oryzae under the culture condition that the fermentation temperature is 30 ℃, and terminating the fermentation after 4 days of fermentation to obtain a fermentation product containing the chitosan oligosaccharide. The method optimizes 6 conditions of initial water content, inoculation amount, substrate addition amount, fermentation temperature, fermentation time and substrate treatment of the solid culture medium, and judges the optimal conditions by taking the chitosan oligosaccharide yield and the chitosan enzyme activity as indexes to finally obtain the chitosan oligosaccharide with the highest yield when the initial water content of the culture medium is 50%, the inoculation amount is 15%, the substrate addition amount is 2%, the fermentation temperature is 30 ℃ and the fermentation is carried out for four days. The method of the invention is suitable for industrial production of high-content chitosan oligosaccharide.

Description

Method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae

Technical Field

The invention belongs to the technical field of aspergillus oryzae fermentation, and particularly relates to a method for producing chitosan oligosaccharide by aspergillus oryzae solid state fermentation.

Background

Aspergillus oryzae (Aspergillus oryzae) belongs to the group of Aspergillus and Aspergillus flavus, belonging to the phylum Ascomycota, order Ascomycota, family Ascomycota, and is a widely used fungus in the brewing and fermentation industries. It has protease enzyme system with strong protein decomposition ability and saccharifying enzyme system with strong starch saccharification ability. Aspergillus oryzae is easy to culture, does not produce aflatoxin, and is a mould with high utilization value. Aspergillus oryzae is one of the safe strains approved by the U.S. food and drug administration. Aspergillus oryzae has been widely used in food industry, agriculture, animal husbandry, biotechnology, etc. Recent studies have found that fermentation of Aspergillus oryzae can produce chitosan oligosaccharide, a product of chitosan degradation. For the degradation of chitosan, there are two forms of specific enzyme degradation and non-specific enzyme degradation. The specific enzyme is chitosanase, and non-specific enzymes such as lipase and cellulase can also degrade chitosan.

Although chitosan has important biological activity, it has low solubility and is insoluble in water and organic solvents, thus limiting its application in food, pharmaceutical, and chemical products. The chitosan oligosaccharide which is the hydrolysate of chitosan has good water solubility due to the short molecular chain length and free amino in D-glucosamine, and can be widely applied to the fields of food, agriculture, biomedicine and the like. In the food industry, the chitosan oligosaccharide is a good natural preservative, has high safety and no toxic or side effect, can be used as a natural preservative, can lock water in food by a special structure, and also has a promotion effect on the absorption of human mineral substances. In the feed industry, the chitosan oligosaccharide has the advantages of no drug resistance, no drug residue, no damage and killing to beneficial bacteria and the like compared with other feed additives. It has stable physicochemical properties, can not be decomposed in gastrointestinal tract, can be directly absorbed by animals into blood to act on target cells, and can continuously and stably improve the immunity of animals. There have been many successful cases of chitosan oligosaccharide as an immunopotentiator added to aquatic feeds. In agriculture, the chitosan oligosaccharide can be used as a biological pesticide to exert insect resistance so as to improve the disease resistance of plants and accelerate the growth and development of the plants, has no residue in the using process, and is more environment-friendly.

The chitosan is prepared by using materials with rich chitin content, such as shrimp and crab shells, as main raw materials, removing protein components through alkali treatment, removing calcium through acid treatment, and removing acetyl. Then pass throughHydrolysis converts chitosan into chitosan oligosaccharides, and degradation can be accomplished by chemical, physical, enzymatic methods. The chemical methods include acid hydrolysis using strong acids such as HCl, and H₂O₂And the like. The chemical method is to obtain low molecular weight polymer by breaking beta-1, 4 glycosidic bond, the reaction speed is fast, the yield is high, but the reaction condition is difficult to control, the conversion efficiency is low, the purer product is difficult to obtain, and the product is mainly glucosamine monosaccharide. The chemical method uses a large amount of chemical reagents, is easy to cause pollution damage to the environment, generates toxic and harmful substances, and has violent reaction which is difficult to control. The physical method is simple and convenient and easy to operate, and comprises ultrasonic degradation, microwave degradation and gamma-ray degradation. However, the method has low yield, the molecular weight of the product is difficult to control, and the product with the same molecular weight is difficult to obtain. The enzymolysis method is a relatively mild degradation method compared with a chemical method, the reaction is efficient and controllable, the operation is simple and convenient, the product conversion rate is high, the yield is high, the product structure cannot be changed, byproducts are few, the environmental pollution cannot be caused, and the safety is higher. Chitosan can be hydrolyzed by various types of enzymes. Including non-specific enzymes: lipase, lysozyme, cellulase, etc., and the specific enzyme is chitosanase. The two enzymes have different effects in degradation, non-specific enzyme hydrolysis is not thorough, the product activity is low, the reaction speed is slow, and the product is complex and the condition is difficult to control to obtain the product with specific molecular weight. As can be seen by comparison, the effect of the chitosan enzyme method is better than that of other methods in various methods, and the product is easier to control. However, the chitosan enzyme method has low yield of chitosan, and needs to optimize fermentation conditions.

Disclosure of Invention

The invention aims to provide a method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae, which improves the yield of the chitosan oligosaccharide.

A method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae comprises the following steps:

(1) activating Aspergillus oryzae strain stored in a test tube, transferring to an Erlenmeyer flask, inoculating to a solid culture medium according to 15% of inoculation amount, and adjusting the water content of the solid culture medium to 50%;

(2) carrying out solid state fermentation culture on Aspergillus oryzae under the culture condition that the fermentation temperature is 30 ℃, and terminating the fermentation after fermenting for 4 days to obtain a fermentation product containing chitosan oligosaccharide.

The aspergillus oryzae strain activation method comprises the following operation steps: aspergillus oryzae stored in a test tube was inoculated on PDA medium, inverted and left to stand in an incubator at 30 ℃ for 4 days for activation, then the plate was washed with sterile physiological saline, the Aspergillus oryzae on the surface of the agar was gently scraped off using a spreader, and the eluate was transferred to a sterilized 100mL Erlenmeyer flask using a 5mL pipette gun, and 80mL of the eluate was collected from each tube of Aspergillus oryzae.

The solid culture medium contains 2% of chitosan.

The chitosan is subjected to tartaric acid immersion treatment for 12-24 hours before being added to the solid medium.

The chitosan degrading effect of the solid fermentation aspergillus oryzae on chitosan powder is not obvious, so that certain pretreatment needs to be carried out on chitosan, the chitosan becomes relatively soluble in the fermentation process, and the reaction is promoted. Chitosan is insoluble in water and alkali solutions, but soluble in dilute hydrochloric, nitric and most organic acid solutions. The chitosan is dissolved in dilute acid, and basically, a plurality of free amino groups on the molecular chain of the chitosan can be combined with a hydrogen ion from the solution, so that the chitosan becomes polyelectrolyte with positive charges, and hydrogen bonds among and in the molecules of the chitosan are destroyed, and the chitosan is dissolved in water. The adoption of hydrochloric acid and acetic acid is too strong, the fermentation process in a system is influenced, the degradation effect is lower, weak acids such as citric acid are adopted, the weak acids are easily decomposed and consumed in the fermentation process, and the inventor conducts repeated experiments to finally confirm that the tartaric acid immersion treatment is selected, so that the effect is optimal.

The preparation method of the PDA culture medium comprises the following steps: dissolving 4.01g potato glucose agar powder in 100ml distilled water, autoclaving at 121 deg.C for 15min, and pouring into plate.

The preparation method of the solid culture medium comprises the following steps: 25g of bran and 8% of chitosan are added into a 250ml conical flask, the initial water content is set to be 50%, the pH is naturally adjusted, and the conical flask is autoclaved for 20min at 121 ℃.

The invention has the beneficial effects that: the invention optimizes 6 conditions of initial water content, inoculation amount, substrate addition amount, fermentation temperature, fermentation time and substrate treatment of the solid culture medium, and determines the optimal conditions by taking the chitosan oligosaccharide yield and the chitosan enzyme activity as indexes. Finally, the chitosan oligosaccharide yield is the highest when the initial water content of the culture medium is 50%, the inoculation amount is 15%, the substrate addition amount is 2%, the fermentation temperature is 30 ℃ and the fermentation is carried out for four days. And TLC results showed that the chitosan oligosaccharide in the medium was chitosan. Under the optimal condition, the enzyme activity of the cellulase is 49.07U, the enzyme activity of the protease is 31.5956U, the enzyme activity of the chitosanase is 33.944U, and the enzyme activity of the lipase is 292.616U.

Drawings

FIG. 1 shows the change rule of the content of chitosan oligosaccharide in the fermentation process in the experiment for optimizing the content of water in the culture medium.

FIG. 2 is the change rule of chitosanase relative enzyme activity in fermentation process in the experiment of optimizing the water content in the culture medium.

FIG. 3 is a change rule of the chitosan oligosaccharide content in the fermentation process in the inoculation amount optimization experiment.

FIG. 4 is the change rule of chitosanase relative enzyme activity in fermentation process in the inoculation amount optimization experiment.

FIG. 5 is a change rule of the content of chitosan oligosaccharide in the fermentation process in the substrate addition amount optimization experiment.

FIG. 6 shows the variation of chitosanase relative to enzyme activity during fermentation in the experiment for optimizing the addition of substrate.

FIG. 7 is the change rule of the content of chitooligosaccharide in the fermentation process in the culture temperature optimization experiment.

FIG. 8 shows the variation of chitosanase relative to enzyme activity during fermentation in culture temperature optimization experiment.

FIG. 9 shows the results of TLC thin plate chromatography;

in the figure, 1:1g/L glucose solution, 2: chitosan oligosaccharide standard, 3: fermenting for 0h, and 4: fermenting for 96h, namely fermentation liquor-1, 5: fermenting for 96h to obtain fermentation liquor-2.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Fermentation experiment operation flow:

aspergillus oryzae strains stored in test tubes were inoculated onto PDA medium, inverted and left to stand in an incubator at 30 ℃ for 4 days, then the plates were rinsed with sterile physiological saline, the Aspergillus oryzae on the agar surface was gently scraped off using an applicator, and the eluate was transferred to a sterilized 100ml Erlenmeyer flask using a 5ml pipette. Inoculating the uniformly mixed strains into the prepared solid culture medium according to the inoculation amount of 10%, stirring the solid culture medium uniformly by using a spoon, periodically taking 2g of the solid culture medium components every 24 hours, transferring the solid culture medium components into 20ml of sterile water added with marbles, and placing a sampling bottle in a shaking table with a constant-temperature water bath at 30 ℃ for 30 min.

The enzymatic activity determination method of the chitosanase comprises the following steps:

definition of chitosanase enzyme activity: the amount of enzyme required for the hydrolysis of the substrate chitosan by chitosanase to produce 1. mu. mol of glucosamine.

And (3) uniformly mixing 200 mu l of fermentation supernatant with 200 mu l of 2% chitosan substrate solution, and placing in a constant-temperature water bath at 55 ℃ for heat preservation for 15 min. Adding 200ul of the reaction solution into 300ul of DNS reagent to terminate enzymatic reaction, (deactivating 200ul of fermentation liquor in boiling water bath for 10min in a control group, adding 2% chitosan substrate solution, uniformly mixing, adding 200ul of reaction solution into 300ul of DNS reagent to terminate enzymatic reaction), developing in boiling water bath for 5min, cooling, centrifuging at 12000rpm for 90s, and taking supernatant fluid to measure absorbance value under 540 nm.

And (3) drawing a standard curve: taking 200ul of glucose standard solution of 0mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1mg/ml, adding 300ul of DNS reagent, developing in boiling water bath for 5min, cooling, and measuring absorbance at 540 nm. And drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate.

Glucosamine content determination (Elson-Morgan method):

mixing 100ul of fermentation supernatant with 100ul of acetylacetone reagent, reacting in boiling water bath for 30min, cooling, adding 200ul of absolute ethanol solution and 100ul of p-dimethylaminobenzaldehyde reagent, shaking, adding 400ul of absolute ethanol solution, and keeping the temperature at 60 ℃ for 1 h. Absorbance values were measured at 530 nm.

And (3) drawing a standard curve: mixing 100ul of glucosamine solution with 0mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1mg/ml with 100ul of acetylacetone reagent, reacting in boiling water bath for 30min, cooling, adding 200ul of absolute ethanol solution and 100ul of p-dimethylaminobenzaldehyde reagent, shaking, adding 400ul of absolute ethanol solution, and keeping the temperature at 60 ℃ for 1 h. Absorbance values were measured at 530 nm. And (3) drawing a standard curve by taking the glucosamine concentration as an abscissa and the absorbance as an ordinate.

And (3) enzyme activity determination of cellulase:

definition of cellulase activity: under certain temperature conditions, the cellulase hydrolyzes the enzyme amount required by the carboxymethyl cellulose sodium solution to generate 1 mu mol of glucose.

200ul of fermentation supernatant is uniformly mixed with 200ul of 8.0g/L hydroxymethyl cellulose sodium substrate solution, the temperature is kept at 37 ℃ for 30min, 200ul of reaction solution is added into 300ul of DNS reagent to terminate the enzymatic reaction. The color was developed in a boiling water bath for 5min and then cooled, and the absorbance value was measured at 540 nm. The standard curve drawing process is the same as the chitosan enzyme activity determination.

And (3) protease activity determination:

definition of protease activity: the amount of enzyme required for the protease to hydrolyze the substrate casein to produce 1. mu. mol tyrosine.

Mixing 200ul of fermentation leaching liquor with 200ul of phosphate buffer solution with pH of 7.2, placing in 40 ℃ water bath for preheating for 5min, adding 200ul of 1% casein solution preheated in the same way, placing in 40 ℃ water bath for accurate timing reaction for 20min, adding 400ul of 0.4mol/L trichloroacetic acid solution for terminating the reaction, standing for 3min, and centrifuging at 12000rpm for 5 min. (in the control group, the order of adding trichloroacetic acid solution and 1% casein solution is reversed) absorbing 40ul of supernatant, adding 200ul of 0.4mol/L sodium carbonate and 40ul of Folin phenol reagent, shaking up, placing in a water bath at 40 ℃ for heat preservation and color development for 20min, cooling, and measuring the absorbance value at 680 nm.

And (3) drawing a standard curve: respectively taking 40ul tyrosine standard solutions of 0ug/ml, 10ug/ml, 20ug/ml, 30ug/ml, 40ug/ml and 50ug/ml, respectively adding 200ul 0.4mol/L sodium carbonate solution and 40ul forskol reagent, placing in a constant-temperature water bath at 40 ℃ for heat preservation and color development for 20min, cooling, and determining absorbance at 680 nm. And drawing a standard curve by taking the tyrosine concentration as an abscissa and the absorbance as an ordinate.

And (3) lipase activity determination:

definition of lipase activity: the lipase hydrolyzes the substrate at a pH at a temperature and in an amount required for producing 1. mu. mol of fatty acid.

100ul of the fermentation extract was mixed with 100ul of phosphate buffer at pH 7.2, 20ul of 20mM pNPD was added and reacted at 37 ℃ for 5min, 200ul of 1% SDS was added (buffer was used instead of substrate in control group) to terminate the reaction, and absorbance was measured at 405 nm.

And (3) drawing a standard curve: an amount of the pNP stock solution was added to a 96-well plate and diluted with ultrapure water to give a standard solution with a gradient of 10uM, 0uM to 250 uM. Mixing well, standing for 2min, and detecting absorption wavelength at 405 nm. According to the measured values, the absorbance OD405 was plotted on the abscissa and the pNP concentration was plotted on the ordinate.

TLC thin plate chromatography:

the developing solvent is n-propanol: ammonia (volume 2: 1). The developer is 0.1% ninhydrin ethanol solution. The plate was gently drawn with a pencil 1cm from the bottom and spotted equidistantly with 2.5ul pipette tips three times per sample using the chitosan oligosaccharide mixture (1-6 sugar) as a reference. And (5) placing the paper chromatographic plate in an unfolding cylinder, and soaking the lower end of the paper chromatographic plate into a unfolding agent for unfolding. After the unfolding is finished, the paper chromatography plate is dried by a blower, and the paper chromatography plate is placed into the unfolding cylinder for secondary unfolding after a small amount of unfolding agent is supplemented into the cylinder. And (3) after the unfolding is finished, drying the paper chromatography plate by using a blower, soaking the paper chromatography plate into a developing solution, developing for 5min at 110 ℃, and developing spots of the chitosan oligosaccharide.

Example 1: optimization of moisture content in culture medium

On the basis of a fermentation solid medium, distilled water (the water content of the bran is 12%) is added into the medium according to the initial water content of 30%, 40%, 50%, 60% and 70%, and the mixture is cultured in an incubator at 30 ℃ for 120h, and a sample is taken every 24 h. Respectively measuring the glucosamine content, the relative enzyme activity of the chitosanase and the number of spores. The results are shown in FIG. 1-2, which are the change rule of the chitosan oligosaccharide content and the relative enzyme activity of the chitosanase in the fermentation process in sequence.

As can be seen from FIGS. 1-2, the production of chitooligosaccharides varied with time. Since a part of the chitosan oligosaccharide is consumed by the Aspergillus oryzae strain in the culture medium while the chitosan oligosaccharide is produced, the yield of the chitosan oligosaccharide is in a dynamic process. Most of the gradients showed a tendency to rise first, then fall and then rise, and all gradients of the medium reached a maximum at 24h of fermentation. The moisture content has a great influence on the yield of chitosan oligosaccharide, wherein the chitosan oligosaccharide yield of the culture medium with the initial moisture content of 50 percent is higher than that of other culture media with the initial moisture content, and the maximum value reaches 3.26mg/g at 24 h.

The relative enzyme activity of the chitosan is increased firstly and then decreased to be kept at a stable level along with the increase of time, and the culture medium with the water content of 40 percent reaches the highest enzyme activity within 48 hours, and the culture media with other water contents reach the maximum value within 24 hours. The highest enzyme activity was produced by fermentation of medium with 30% moisture content the first day.

Although the relative enzyme activity of chitosan oligosaccharide was high at an initial moisture content of 30%, the chitosan oligosaccharide production in the medium with a moisture content of 50% was superior to that in the medium with other gradients, and thus the initial moisture content of 50% was selected as the optimum value.

Example 2: inoculum size optimization

After the initial water content is determined to be 50%, inoculating according to the inoculum sizes of 5%, 10% and 15% respectively on the basis of a fermentation solid medium, culturing for 120h in an incubator at 30 ℃, and sampling once every 24 h. Respectively measuring the glucosamine content and the chitosanase activity. The results are shown in FIGS. 3-4, which are the change rules of the chitosan oligosaccharide content and the relative enzyme activity of the chitosanase in the fermentation process.

As can be seen from FIGS. 3-4, the production of chitooligosaccharides in the medium with an inoculum size of 5% increased gradually with time, with a maximum value of 2.55mg/ml, produced during 120h of fermentation; the inoculation amount of the culture medium with 10 percent is increased along with the increase of time and then decreased to increase, the maximum value is generated when the fermentation is carried out for 96 hours, and the maximum value is 2.36 mg/ml; the 15% medium production of endo-oligosaccharides increased first with time, then decreased, then increased and then decreased, with a maximum of 96h fermentation, 2.85 mg/ml. The maximum yield was 2.85mg/ml from the inoculum size of 15% of the medium at 96h of fermentation. It was observed that the yield of chitosan oligosaccharide at 15% inoculum size varied in a manner consistent with a 50% variation in moisture content cycle.

The relative enzyme activity of the culture medium with three inoculation amounts reaches the maximum value within 24 hours, the enzyme activity is reduced rapidly within 24-48 hours, and the relative enzyme activity is kept within a certain range after 24 hours. The difference between the 15% of the inoculated amount of the enzyme activity and other two gradients is obvious, the 15% of the inoculated amount of the enzyme activity is the largest, the second is 5%, and the last is 10%.

In conclusion, the difference of the chitosan oligosaccharide yield of the culture medium with three inoculation amounts in 48h is not obvious, and the chitosan oligosaccharide yield of the culture medium with 15% of the inoculation amount after 48h is obviously higher than that of the culture medium with 5% and 10% of the inoculation amount, so that the 15% of the inoculation amount is selected as the optimal value.

Example 3: optimization of substrate addition

After the initial water content is determined to be 50% and the inoculation amount is 15%, chitosan is added according to the addition amount of 2%, 5%, 8% and 10% of substrate on the basis of a fermentation solid medium, the mixture is cultured in an incubator at 30 ℃ for 120 hours, and sampling is carried out once every 24 hours. Respectively measuring the glucosamine content and the chitosanase activity. The results are shown in FIGS. 5-6, which are the change rules of the chitosan oligosaccharide content and the relative enzyme activity of the chitosanase in the fermentation process.

As can be seen from FIGS. 5 to 6, the yields of chitooligosaccharides in the four media with different substrate addition levels varied with time and tended to increase, decrease, increase and decrease. The four culture mediums with different gradients reach the highest value after fermentation for 96 hours, and the conversion rates of the substrate addition amounts of 2%, 5%, 8% and 10% are 25.5%, 9.5%, 7.0% and 5.5% in sequence. It can be seen that the yield of product converted per 1g of substrate gradually decreases as the amount of substrate added increases.

The culture medium with the substrate addition amount of 2% reaches the maximum enzyme activity within 48 hours, and the rest culture medium reaches the maximum enzyme activity within 24 hours. The influence of the addition amount of the substrate on the enzyme activity is obvious, the highest enzyme activity with the addition amount of the substrate being 8 percent is obviously greater than the enzyme activity with the addition amount of the substrate being 5 percent, and the lowest enzyme activity is 2 percent and is 10 percent.

In summary, the most highly converted 2% was selected as the optimal substrate addition.

Example 4: culture temperature optimization

After determining that the initial moisture content is 50%, the inoculation amount is 15% and the substrate addition amount is 2%, respectively culturing the solid culture medium in a constant-temperature incubator at 27 ℃, 30 ℃ and 37 ℃ for 120h, and sampling every 24 h. Respectively measuring the glucosamine content and the chitosanase activity. The results are shown in FIGS. 7-8, which are the change rules of the chitosan oligosaccharide content and the relative enzyme activity of the chitosanase in the fermentation process.

As can be seen from FIGS. 7 to 8, the yields of chitooligosaccharides in the media with different culture temperatures varied with time and tended to increase, decrease and increase. Wherein the culture medium with the culture temperature of 25 ℃ reaches the maximum value of 2.42mg/g at 48 h; the maximum value of the culture medium with the culture temperature of 30 ℃ reaches 2.43mg/g at 24 h; the culture medium with the culture temperature of 37 ℃ reaches the maximum value of 2.37mg/g in 48 h.

The fermentation medium reaches the maximum enzyme activity within 48 hours when the culture temperature is 25 ℃ and 30 ℃, and the fermentation medium reaches the maximum enzyme activity within 24 hours when the culture temperature is 37 ℃. Wherein the culture medium with the culture temperature of 37 ℃ has the highest enzyme activity, the second 30 ℃ and the lowest 25 ℃.

In conclusion, the temperature has no significant effect on the yield of the chitosan oligosaccharide, but the culture temperature is relatively stable at 30 ℃. Therefore, 30 ℃ was selected as the culture temperature.

Example 5: fermentation time optimization

As can be seen from the optimization period of the substrate addition amount, when the substrate addition amount is 2%, the difference between the chitosan oligosaccharide yield of 48h and the chitosan oligosaccharide yield of 96h in fermentation is large. Although 48h can save time cost, ending the fermentation at 48h and starting a new fermentation cycle can cause great waste of material. Therefore, 96h with higher chitosan oligosaccharide yield was selected as the optimal fermentation time.

From the above, the aspergillus oryzae solid state fermentation medium was finally selected to be fermented under the culture conditions of 15% inoculum size, 2% substrate addition, 50% medium initial water content and 30 ℃ fermentation temperature, and the fermentation was terminated at the time of the fourth day of fermentation when the chitosan oligosaccharide yield was the highest. The enzyme activities of protease, cellulase, lipase and chitosanase were measured on the culture medium under the above conditions. And analyzing the oligosaccharide products in the culture medium by adopting a TLC thin plate chromatography method.

The results of the enzyme activity assay are shown in table 1 below, and the results of the enzyme activity assay of lipase indicate that lipase has activity on p-nitrophenol hexanoate, p-nitrophenol octanoate, p-nitrophenol decanoate and p-nitrophenol palmitate, but has the highest activity and stable activity on p-nitrophenol octanoate.

TABLE 1

The experiment group takes 1g/L glucose solution and chitosan oligosaccharide standard as the control. The samples are fermentation liquor which is fermented for 0h and 96 h. Comparison of the two fermentation broths revealed that chitobiose was produced after fermentation (FIG. 9). And a certain amount of glucose is present in the medium itself.

Example 6: substrate processing optimization

The chitosan was immersion treated with tartaric acid for 18 hours prior to addition to the solid media, as a control untreated chitosan substrate.

Fermenting the Aspergillus oryzae solid fermentation culture medium under the culture conditions that the inoculation amount is 15%, the substrate addition amount is 2%, the initial water content of the culture medium is 50% and the fermentation temperature is 30 ℃, and stopping fermentation at the moment when the chitosan oligosaccharide yield is highest on the fourth day of fermentation.

Finally, the yield of the chitosan oligosaccharide in the solid fermentation product treated by the tartaric acid substrate is 3.38mg/g, the yield of the chitosan oligosaccharide in the solid fermentation product not treated by the substrate is 2.66mg/g, and the yield increasing effect is obvious. If the substrate is immersed and treated by hydrochloric acid with the same molar concentration for 18 hours, the yield of the chitosan oligosaccharide in the final solid fermentation product is 1.38mg/g, and the inhibition effect is obvious.

Claims

1. A method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae is characterized by comprising the following steps:

2. The method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae according to claim 1, wherein the step of activating aspergillus species is: aspergillus oryzae stored in a test tube was inoculated on PDA medium, inverted and left to stand in an incubator at 30 ℃ for 4 days for activation, then the plate was washed with sterile physiological saline, the Aspergillus oryzae on the surface of the agar was gently scraped off using a spreader, and the eluate was transferred to a sterilized 100mL Erlenmeyer flask using a 5mL pipette gun, and 80mL of the eluate was collected from each tube of Aspergillus oryzae.

3. The method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae according to claim 1, wherein chitosan is contained in an amount of 2% in the solid state culture medium.

4. The method for producing chitosan oligosaccharide by solid state fermentation of Aspergillus oryzae as claimed in claim 3, wherein said chitosan is subjected to tartaric acid immersion treatment for 12-24 hours before being added to the solid state culture medium.

5. The method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae according to claim 1, wherein the PDA medium is configured by: dissolving 4.01g potato glucose agar powder in 100ml distilled water, autoclaving at 121 deg.C for 15min, and pouring into plate.

6. The method for producing chitosan oligosaccharide by solid state fermentation of aspergillus oryzae according to claim 1, wherein the solid state culture medium is prepared by the following steps: 25g of bran and 8% of chitosan are added into a 250ml conical flask, the initial water content is set to be 50%, the pH is naturally adjusted, and the conical flask is autoclaved for 20min at 121 ℃.