Fermentation culture medium and application of fermentation culture medium in production of chitin degrading enzyme and chitosan oligosaccharide from fungicide

Abstract

The invention discloses a fermentation culture medium and application thereof in producing chitin degrading enzyme and chitosan oligosaccharide by a microbial agent, and belongs to the technical field of microorganisms. The fermentation culture medium comprises the following components: 10 to 20 g / L of edible fungus chaff, 8 to 12 g / L of shrimp shell powder, 1 to 5 g / L of lactose, 4 to 8 g / L of (NH4) 2SO4, 3 to 7 g / L of soybean meal, 3 to 6 g / L of NaCl, 5 to 15 g / L of MgSO4. 7H2O, 0.3 to 1 g / L of K2HPO4 and 0.4 to 1.2 g / L of KH2PO4, and the pH value of the fermentation culture medium is 6 to 7. According to the method, the edible fungus chaff and the shrimp shell powder are added into the fermentation culture medium, so that the production of the chitin degrading enzyme by the microbial inoculum can be remarkably promoted, and then the chitosan oligosaccharide with the remarkably improved content is obtained under the action of the chitin degrading enzyme; therefore, the fermentation culture medium has a relatively good application value in the production of chitin degrading enzyme and chitosan oligosaccharide by a microbial inoculum.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a fermentation medium and its application in producing chitin-degrading enzymes and chitooligosaccharides by microbial agents. Background Art

[0002] Chitooligosaccharide (COS) is an alkaline oligosaccharide with a degree of polymerization of 2-10 formed by N-acetylglucosamine (GlcN) linked by β-1,4 glycosidic bonds, which is depolymerized from chitosan obtained by deacetylation of chitin. Chitooligosaccharide has the advantages of high solubility, low molecular weight and good biocompatibility, and has special biological activities such as antioxidant, anti-inflammatory and antibacterial, anti-tumor, promoting plant growth, and promoting the growth of intestinal microorganisms. Therefore, it has good application prospects.

[0003] At present, there are mainly three production methods for chitooligosaccharide: chemical method, physical method and biological enzyme method. Chemical method and physical method are relatively common industrial technical means. However, considering product purity, production cost, environmental pollution and subsequent purification problems, enzymatic hydrolysis of chitin is usually used to obtain chitooligosaccharide. Specifically, microorganisms are first fermented and cultured with a fermentation medium to obtain chitinase and chitin deacetylase, and then the above chitinase and chitin deacetylase are used to degrade and deacetylate chitin respectively to obtain chitooligosaccharide. Compared with the chemical method and the physical method, the enzymatic preparation has strong specificity, mild reaction conditions, is easy to prepare, and is a more environmentally friendly production method.

[0004] However, the existing fermentation medium in the enzymatic preparation of chitooligosaccharide has problems such as complex components and low activities of chitinase and chitin deacetylase obtained by fermenting and culturing microorganisms. Summary of the Invention

[0005] The purpose of the present invention is to provide a fermentation medium and its application in producing chitin-degrading enzymes and chitooligosaccharides by microbial agents. It is used to solve the problems such as complex components of the existing fermentation medium in the enzymatic preparation of chitooligosaccharide and low activities of chitinase and chitin deacetylase obtained by fermenting and culturing microorganisms.

[0006] In the first aspect, the present invention provides a fermentation medium, which comprises the following components: edible mushroom residue 10-20 g / L, shrimp shell powder 8-12 g / L, lactose 1-5 g / L, (NH 4 ) 2 SO 4 4-8 g / L, soybean meal 3-7 g / L, NaCl 3-6 g / L, MgSO 4 ·7H 2 O 5-15 g / L, K 2 HPO 4 0.3-1 g / L, KH2 PO 4 0.4 - 1.2 g / L, and the pH value of the fermentation medium is 6 - 7.

[0007] In the present invention, the edible mushroom bran is the residue after cultivating edible mushrooms with raw materials such as straw, miscellaneous sawdust, cottonseed hulls, etc. to harvest fruiting body products. Its main components include the complex composed of the residual bodies of edible mushroom mycelia and the crude fiber and lignin whose structures have undergone qualitative changes after being enzymolyzed by edible mushrooms. Since the edible mushroom bran contains a large amount of mycelia and is rich in nutrients such as proteins, polysaccharides, and trace elements, therefore, after adding the edible mushroom bran to the fermentation medium, it can significantly promote the production of chitin-degrading enzyme by the microbial agent, and realize the resource utilization of the edible mushroom bran; in addition, after adding shrimp shell powder to the fermentation medium, the chitin-degrading enzyme produced by the microbial agent can further convert the shrimp shell powder into chitooligosaccharides, thereby realizing the comprehensive utilization of waste such as shrimp shell powder. Therefore, it has good application value.

[0008] The fermentation medium provided by the present invention comprises the following components: edible mushroom bran 10 - 20 g / L, for example, it can be 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L or other values within this range; shrimp shell powder 8 - 12 g / L, for example, it can be 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L or other values within this range; lactose 1 - 5 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L or other values within this range; (NH 4 ) 2 SO 4 4 - 8 g / L, for example, it can be 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or other values within this range; soybean meal 3 - 7 g / L, for example, it can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L or other values within this range; NaCl 3 - 6 g / L, for example, it can be 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L or other values within this range; MgSO 4 ·7H 2 O 5 - 15 g / L, for example, it can be 5 g / L, 7 g / L, 10 g / L, 13 g / L, 15 g / L or other values within this range; K 2 HPO 4 0.3 - 1 g / L, for example, it can be 0.3 g / L, 0.5 g / L, 0.7 g / L, 0.9 g / L, 1 g / L or other values within this range; KH 2 PO 40.4 - 1.2 g / L, for example, it can be 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.2 g / L or other values within this range; and the pH value of the fermentation medium is 6 - 7, for example, it can be 6, 6.2, 6.5, 6.8, 7 or other values within this range.

[0009] In some embodiments, the fermentation medium comprises the following components: edible mushroom bran 15 g / L, shrimp shell powder 10 g / L, lactose 3 g / L, (NH 4 ) 2 SO 4 6.782 g / L, soybean meal 5 g / L, NaCl 4.5 g / L, MgSO 4 ·7H 2 O 9 g / L, K 2 HPO 4 0.7 g / L, KH 2 PO 4 0.879 g / L, and the pH value of the fermentation medium is 6.5.

[0010] In a second aspect, the present invention provides the use of any of the above fermentation media in the production of chitin degrading enzyme and chitosan oligosaccharide by the microbial agent.

[0011] In some embodiments, the use comprises the following steps: S1, inoculating the microbial agent into a seed medium for culture to obtain a seed solution; S2, inoculating the seed solution into the fermentation medium for fermentation culture to obtain a fermentation broth; S3, centrifuging the fermentation broth to obtain a fermentation supernatant, and further subjecting the fermentation supernatant to ultrafiltration to obtain a chitin degrading enzyme solution and a chitosan oligosaccharide solution.

[0012] In the application method of the fermentation medium provided by the present invention for the production of chitin degrading enzyme and chitosan oligosaccharide by the microbial agent, it has advantages such as simple operation, and can use waste materials such as edible mushroom bran and shrimp shell powder in the fermentation medium as raw materials to ferment and obtain chitin degrading enzyme with significantly improved enzyme activity and chitosan oligosaccharide with significantly increased content, significantly improving the comprehensive utilization rate of waste materials such as edible mushroom bran and shrimp shell powder, and reducing resource waste and environmental pollution.

[0013] In some embodiments, in step S1, the microbial agent comprises bacteria producing chitin deacetylase and bacteria producing chitosanase; among them, the bacteria producing chitin deacetylase include Rhodococcus HQcdag, and the bacteria producing chitosanase include Bacillus cereus.

[0014] It is understandable that in the present invention, the bacteria producing chitin deacetylase and the bacteria producing chitosanase can be selected from the conventional bacteria in the prior art according to actual usage requirements, as long as the above-mentioned bacteria can produce chitin deacetylase and chitosanase. In the present invention, the bacteria producing chitin deacetylase preferably include Rhodococcus sp. HQcdag, and the bacteria producing chitosanase preferably include Bacillus cereus.

[0015] In some embodiments, the ratio of the viable cell count of the bacteria producing chitin deacetylase to the viable cell count of the bacteria producing chitosanase is (2 - 4):1. For example, it can be 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or other ratios within this range.

[0016] In the present invention, by controlling the ratio of the viable cell count of the bacteria producing chitin deacetylase to the viable cell count of the bacteria producing chitosanase within a specific range, the yield of chitosan oligosaccharide can be further increased.

[0017] In some embodiments, in step S1, the seed medium comprises the following components: peptone 5 - 15 g / L. For example, it can be 5 g / L, 7 g / L, 10 g / L, 13 g / L, 15 g / L or other values within this range; beef extract 1 - 5 g / L. For example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L or other values within this range; sodium chloride 3 - 7 g / L. For example, it can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L or other values within this range; and the pH value of the seed medium is 6.5 - 7.5. For example, it can be 6.5, 6.7, 7, 7.3, 7.5 or other values within this range. The culture specifically includes: culturing at a temperature of 25 - 35 °C (for example, it can be 25 °C, 27 °C, 30 °C, 33 °C, 35 °C or other values within this range) and a rotation speed of 180 - 220 r / min (for example, it can be 180 r / min, 190 r / min, 200 r / min, 210 r / min, 220 r / min or other values within this range) for 15 - 25 h. For example, it can be 15 h, 17 h, 20 h, 23 h, 25 h or other values within this range.

[0018] It is understandable that the seed medium can be selected from the conventional media in the prior art according to the type of the microbial agent, as long as it can meet the normal growth of the microbial agent.

[0019] In some embodiments, in step S2, the inoculation amount of the seed liquid is 3 - 7%, for example, it can be 3%, 4%, 5%, 6%, 7% or other values within this range; the fermentation culture specifically includes: at a temperature of 25 - 35°C (for example, it can be 25°C, 27°C, 30°C, 33°C, 35°C or other values within this range) and a rotation speed of 180 - 220 r / min (for example, it can be 180 r / min, 190 r / min, 200 r / min, 210 r / min, 220 r / min or other values within this range), culture for 30 - 40 h, for example, it can be 30 h, 32 h, 35 h, 38 h, 40 h or other values within this range.

[0020] It can be understood that the inoculation amount of the seed liquid and the specific parameters of the fermentation culture can be adjusted conventionally according to the growth conditions of the microbial agent, as long as the normal growth of the microbial agent can be satisfied.

[0021] In some embodiments, in step S3, the centrifugation treatment specifically includes: under the condition of a rotation speed of 6000 - 10000 r / min (for example, it can be 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min or other values within this range), centrifuge for 10 - 20 min, for example, it can be 10 min, 12 min, 15 min, 18 min, 20 min or other values within this range.

[0022] It can be understood that the specific parameters of the centrifugation treatment can be adjusted conventionally according to the amount of the microbial agent, as long as the separation of the thallus and the product can be achieved.

[0023] In some embodiments, in step S3, the ultrafiltration treatment specifically includes: concentrating and filtering using a 30 kDa ultrafiltration tube.

[0024] In some embodiments, in step S3, the chitin degrading enzyme solution includes chitin deacetylase and chitosanase, and the chitosan oligosaccharide solution includes chitobiose, chitotriose and chitotetraose.

[0025] The beneficial effects of the present invention are: different from the prior art, the present invention can significantly promote the production of chitin degrading enzyme by the microbial agent by adding edible mushroom bran and shrimp shell powder to the fermentation medium, and then under the action of this chitin degrading enzyme, chitosan oligosaccharide with a significantly increased content is obtained; the fermentation medium in the present invention significantly improves the comprehensive utilization rate of waste such as edible mushroom bran and shrimp shell powder, reduces resource waste and environmental pollution, and therefore has good application value. Detailed implementation manners

[0026] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] For the experimental methods without specific conditions noted in the embodiments, they are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in "Molecular Cloning: A Laboratory Manual" by M.R. Green, "Molecular Biology" by Robert·F·Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels without special instructions.

[0028] In the present invention, the preservation number of Rhodococcus HQcdag is CCTCC NO: M 2020336, which is derived from the description in the Chinese patent application document CN112553123A.

[0029] Bacillus cereus is derived from the description in the Chinese patent application document CN118291429A.

[0030] Example 1 This example provides a fermentation medium, which includes the following components: edible mushroom residue 10 g / L, shrimp shell powder 10 g / L, lactose 3 g / L, (NH 4 ) 2 SO 4 6.782 g / L, soybean meal 5 g / L, NaCl 4.5 g / L, MgSO 4 ·7H 2 O 9 g / L, K 2 HPO 4 0.7 g / L, KH 2 PO 4 0.879 g / L, and the pH value of the fermentation medium is 6.5.

[0031] When preparing this fermentation medium, the edible mushroom residue needs to be dried in the sun and then crushed by a pulverizer and passed through a 60-mesh sieve; the shrimp shell powder is prepared by crushing the crayfish shells with a pulverizer and passing through a 60-mesh sieve.

[0032] A method for fermenting a microbial agent to produce chitin degrading enzyme and chitosan oligosaccharide using this fermentation medium includes the following steps: S1. Inoculate Rhodococcus sp. HQcdag and Bacillus cereus into a seed medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7) respectively, and culture at 30 °C with a rotation speed of 200 r / min for 20 h to obtain a Rhodococcus sp. HQcdag seed solution and a Bacillus cereus seed solution; S2. Mix the Rhodococcus sp. HQcdag seed solution and the Bacillus cereus seed solution prepared in step S1 according to a viable cell count ratio of 4:1, then inoculate into the fermentation medium at an inoculation amount of 7%, and culture at 30 °C with a rotation speed of 200 r / min for 36 h to obtain a fermentation broth; S3. Centrifuge the fermentation broth prepared in step S2 at a rotation speed of 8000 r / min for 15 min to obtain a fermentation supernatant, and continue to concentrate and filter the fermentation supernatant with a 30 kDa ultrafiltration tube to obtain a chitin degrading enzyme solution and a chitosan oligosaccharide solution.

[0033] Example 2 This example provides a fermentation medium, which comprises the following components: 15 g / L edible mushroom residue, 10 g / L shrimp shell powder, 3 g / L lactose, (NH 4 ) 2 SO 4 6.782 g / L, 5 g / L soybean meal, 4.5 g / L NaCl, MgSO 4 ·7H 2 O 9 g / L, K 2 HPO 4 0.7 g / L, KH 2 PO 4 0.879 g / L, and the pH value of the fermentation medium is 6.5.

[0034] When preparing the fermentation medium, the edible mushroom residue needs to be dried in the sun, then crushed with a crusher and passed through a 60-mesh sieve; the shrimp shell powder is prepared by crushing crayfish shells with a crusher and passing through a 60-mesh sieve.

[0035] A method for fermenting a microbial agent to produce chitin degrading enzyme and chitosan oligosaccharide using the fermentation medium comprises the following steps: S1. Inoculate Rhodococcus sp. HQcdag and Bacillus cereus into a seed medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7) respectively, and culture at 30 °C with a rotation speed of 200 r / min for 20 h to obtain a Rhodococcus sp. HQcdag seed solution and a Bacillus cereus seed solution; S2. Mix the Rhodococcus HQcdag seed liquid and Bacillus cereus seed liquid prepared in step S1 according to the viable count ratio of 4:1, and then inoculate them into the fermentation medium at an inoculation amount of 7%. Incubate at 30 °C and a rotation speed of 200 r / min for 36 h to obtain a fermentation broth. S3. Centrifuge the fermentation broth prepared in step S2 at a rotation speed of 8000 r / min for 15 min to obtain a fermentation supernatant. Then, concentrate and filter the fermentation supernatant using a 30 kDa ultrafiltration tube to obtain a chitin-degrading enzyme solution and a chitosan oligosaccharide solution.

[0036] Example 3 This example provides a fermentation medium, which includes the following components: edible mushroom residue 20 g / L, shrimp shell powder 10 g / L, lactose 3 g / L, (NH 4 ) 2 SO 4 6.782 g / L, soybean meal 5 g / L, NaCl 4.5 g / L, MgSO 4 ·7H 2 O 9 g / L, K 2 HPO 4 0.7 g / L, KH 2 PO 4 0.879 g / L, and the pH value of the fermentation medium is 6.5.

[0037] When preparing the fermentation medium, the edible mushroom residue needs to be dried in the sun and then crushed using a crusher and passed through a 60-mesh sieve; the shrimp shell powder is prepared by crushing crayfish shells using a crusher and passing through a 60-mesh sieve.

[0038] A method for fermenting a bacterial agent to produce chitin-degrading enzyme and chitosan oligosaccharide using this fermentation medium includes the following steps: S1. Inoculate Rhodococcus HQcdag and Bacillus cereus into a seed medium (peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, pH value 7) respectively, and incubate at 30 °C and a rotation speed of 200 r / min for 20 h to obtain Rhodococcus HQcdag seed liquid and Bacillus cereus seed liquid. S2. Mix the Rhodococcus HQcdag seed liquid and Bacillus cereus seed liquid prepared in step S1 according to the viable count ratio of 4:1, and then inoculate them into the fermentation medium at an inoculation amount of 7%. Incubate at 30 °C and a rotation speed of 200 r / min for 36 h to obtain a fermentation broth. S3. Centrifuge the fermentation broth obtained in step S2 at 8000 r / min for 15 min to obtain the fermentation supernatant. Then, concentrate and filter the fermentation supernatant using a 30 kDa ultrafiltration tube to obtain chitin-degrading enzyme solution and chitosan oligosaccharide solution.

[0039] Comparative Example 1 This comparative example provides a fermentation medium, which includes the following components: yeast extract powder 2.5 g / L, peptone 2.5 g / L, colloidal chitin 5 g / L, NaCl 5 g / L, K 2 HPO 4 1 g / L, KH 2 PO 4 1 g / L, MgSO 4 0.5 g / L, and the pH value of the fermentation medium is 6.5.

[0040] A method for fermenting a bacterial agent to produce chitin-degrading enzyme and chitosan oligosaccharide using this fermentation medium includes the following steps: S1. Inoculate Rhodococcus sp. HQcdag and Bacillus cereus into a seed medium (peptone 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, pH value 7) respectively, and culture at 30 °C and 200 r / min for 20 h to obtain Rhodococcus sp. HQcdag seed liquid and Bacillus cereus seed liquid; S2. Mix the Rhodococcus sp. HQcdag seed liquid and Bacillus cereus seed liquid prepared in step S1 according to the viable cell count of 4:1, and then inoculate them into this fermentation medium at an inoculation amount of 3%, and culture at 35 °C and 160 r / min for 24 h to obtain the fermentation broth; S3. Centrifuge the fermentation broth obtained in step S2 at 8000 r / min for 15 min to obtain the fermentation supernatant. Then, concentrate and filter the fermentation supernatant using a 30 kDa ultrafiltration tube to obtain chitin-degrading enzyme solution and chitosan oligosaccharide solution.

[0041] Performance Test Test the activities of the chitin-degrading enzyme solutions (chitin deacetylase and chitosanase) prepared in Examples 1 - 3 and Comparative Example 1 and the content of chitosan oligosaccharide in the chitosan oligosaccharide solution.

[0042] Specifically, the determination method of chitin deacetylase activity is as follows: Take a test tube, and successively add 1 mL of 200 mg / L p-nitroacetanilide solution, 1 mL of chitin-degrading enzyme solution with an appropriate concentration, and 3 mL of 0.05 mol / L phosphate buffer solution pre-warmed at 50 °C to make the total reaction volume 5 mL. React in a water bath at 50 °C for 30 min, terminate the enzymatic reaction in a boiling water bath, centrifuge, and measure the absorbance value of the supernatant (A400 ). Add 1 mL of inactivated chitinase solution with the same concentration to the blank control system, and measure the absorbance (A 0 ) of the supernatant. Each sample corresponds to a blank. One enzyme activity unit (U) is defined as: using p-nitroacetanilide as the substrate, the amount of enzyme required to produce 1 μg of p-nitroaniline per hour at 45 °C is defined as one enzyme activity unit.

[0043] The method for determining the chitosanase activity is as follows: Use the DNS colorimetric method to determine the chitosanase activity. Mix 0.2 mL of appropriately diluted chitinase solution and 0.8 mL of 1.0% colloidal chitosan (chitosan dissolved in 0.05 mol / L acetic acid and sodium acetate buffer with a pH value of 5.4), and react at 50 °C for 20 min. After the reaction, terminate the reaction in a boiling water bath for 10 min. Add 1.5 mL of DNS solution, immediately cool it after boiling water bath for 10 min, and make up the volume to 5 mL with deionized water. Use the inactivated chitinase solution as the blank control, and detect the absorbance at 540 nm. Set parallel experiments, with three parallels in each group, and take the average value. Use glucosamine hydrochloride as the standard curve to calculate the amount of reducing sugar in the reaction of the enzyme and the substrate; the enzyme activity unit (U) is defined as: under certain conditions, the amount of enzyme required to produce 1 μmol of reducing sugar per minute is defined as 1 U.

[0044] The method for determining the content of chitosan oligosaccharide is as follows: The content of chitosan oligosaccharide is determined by high performance liquid chromatography (HPLC). The high performance liquid chromatograph (produced by Shimadzu Corporation, Japan) is equipped with an NH2P-50 4E amino column. The mobile phase is acetonitrile: water = 70:30, the flow rate is 1 mL / min, the column temperature is 30 °C, and it is detected by a RID-10A differential refractive index detector. The test sample passes through a 0.22 µm organic filter membrane, and the external standard method is used and the Labsloutions software is used for data processing to obtain the content of chitosan oligosaccharide.

[0045] Among them, the test results of the activities of the chitinase solutions (chitin deacetylase and chitosanase) prepared in Examples 1-3 and Comparative Example 1 and the content of chitosan oligosaccharide in the chitosan oligosaccharide solution are shown in Table 1 below.

[0046] Table 1 Test results of the activities of chitinase solutions and the content of chitosan oligosaccharide

[0047] As can be seen from Table 1, in Example 1, using 10 g / L of edible mushroom residue and 10 g / L of shrimp shell powder as the main fermentation substrates, that is, the concentration ratio of edible mushroom residue to shrimp shell powder is 1:1, the chitin deacetylase activity of the prepared chitinase solution is 562.6 U / ml, the chitosanase activity is 79.3 U / ml, and the final content of chitosan oligosaccharide obtained is 1.54 g / L. The chitosan oligosaccharide includes chitobiose (GlcN) 2, chitotriose (GlcN) 3 and chitotetraose (GlcN) 4 , and their proportional relationship is 1:1.58:0.61. Compared with Comparative Example 1, both enzyme activities in Example 1 were significantly increased, and the content of chitosan oligosaccharide prepared was also significantly increased. The results showed that using edible mushroom residue and shrimp shell powder as the fermentation substrate had a good promoting effect on the enzyme production of Bacillus cereus and Rhodococcus sp. HQcdag, thereby increasing the yield of chitosan oligosaccharide. In addition, the proportion of chitobiose (GlcN) 2 , chitotriose (GlcN) 3 and chitotetraose (GlcN) 4 did not change significantly, which was determined by the substrate specificity of chitosanase.

[0048] In Example 2, using 15 g / L of edible mushroom residue and 10 g / L of shrimp shell powder as the main fermentation substrate, that is, the concentration ratio of edible mushroom residue to shrimp shell powder was 1.5:1, the enzyme activity of chitin deacetylase prepared was 617.9 U / ml (1.47 times that of Comparative Example 1), and the enzyme activity of chitosanase was 88.2 U / ml (2.04 times that of Comparative Example 1). Finally, the content of chitosan oligosaccharide obtained was 1.70 g / L. The chitosan oligosaccharide included chitobiose (GlcN) 2 , chitotriose (GlcN) 3 and chitotetraose (GlcN) 4 , and their proportional relationship was 1:1.62:0.58. Compared with Example 1, the concentration ratio of edible mushroom residue to shrimp shell powder was further increased, effectively promoting the enzyme production of Bacillus cereus and Rhodococcus sp. HQcdag, and further increasing the enzyme activities of the two enzymes and the yield of chitosan oligosaccharide.

[0049] In Example 3, using 20 g / L of edible mushroom residue and 10 g / L of shrimp shell powder as the main fermentation substrate, that is, the concentration ratio of edible mushroom residue to shrimp shell powder was 2:1, the enzyme activity of chitin deacetylase prepared was 652.7 U / ml (1.55 times that of Comparative Example 1), and the enzyme activity of chitosanase was 82.7 U / ml. Finally, the content of chitosan oligosaccharide obtained was 1.68 g / L. The chitosan oligosaccharide included chitobiose (GlcN) 2 , chitotriose (GlcN) 3 and chitotetraose (GlcN) 4 , and their proportional relationship was 1:1.61:0.59. Although the content of chitosan oligosaccharide remained at a relatively high level, it decreased slightly compared with Example 2. It was speculated that when the concentration ratio of edible mushroom residue to shrimp shell powder was continuously increased, the nutrition of the fermentation substrate was beneficial to the growth of Rhodococcus sp. HQcdag, but it was no longer suitable for the cultivation of Bacillus cereus, resulting in a decrease in its enzyme activity and a decrease in the prepared chitosan oligosaccharide.

[0050] In Comparative Example 1, edible mushroom bran and shrimp shell powder were not added. The chitin deacetylase activity of the prepared chitin deacetylase was 421.4 U / ml, the chitosanase activity was 43.2 U / ml, and the final content of chitosan oligosaccharide was 1.13 g / L. The chitosan oligosaccharide included chitobiose (GlcN) 2 , chitosan trisaccharide (GlcN) 3 and chitosan tetrasaccharide (GlcN) 4 , and their proportional relationship was 1:1.60:0.60. The enzyme activities of the two enzymes and the yield of chitosan oligosaccharide decreased significantly.

[0051] In summary, by adding edible mushroom bran and shrimp shell powder to the fermentation medium, the present invention can significantly promote the production of chitin-degrading enzymes by the microbial agent. Then, under the action of the chitin-degrading enzymes, chitosan oligosaccharide with a significantly increased content can be obtained.

[0052] The above-described embodiments merely represent the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A fermentation medium, characterized in that: The fermentation medium comprises the following components: 10-20 g / L of edible fungus dregs, 8-12 g / L of shrimp shell powder, 1-5 g / L of lactose, 4-8 g / L of (NH4)2SO4, 3-7 g / L of soybean meal, 3-6 g / L of NaCl, 5-15 g / L of MgSO4·7H2O, 0.3-1 g / L of K2HPO4 and 0.4-1.2 g / L of KH2PO4, and the pH value of the fermentation medium is 6-7.

2. The fermentation medium according to claim 1, characterized in that The invention comprises the following components: 15 g / L edible fungus dregs, 10 g / L shrimp shell powder, 3 g / L lactose, 6.782 g / L (NH4)2SO4, 5 g / L soybean meal, 4.5 g / L NaCl, 9 g / L MgSO4·7H2O, 0.7 g / L K2HPO4 and 0.879 g / L KH2PO4, and the pH value of the fermentation medium is 6.

5.

3. Use of the fermentation medium according to any one of claims 1 to 2 in producing chitin-degrading enzymes and chitosan oligosaccharides in bacterial agents.

4. The use according to claim 3, characterized in that: The steps include: S1, inoculating the bacterial agent into a seed culture medium for culturing to obtain a seed solution; S2, inoculating the seed liquid into the fermentation medium for fermentation culture to obtain a fermentation liquid; S3, centrifuging the fermentation liquid to obtain a fermentation supernatant, and further ultrafiltration the fermentation supernatant to obtain a chitin degrading enzyme solution and a chitosan oligosaccharide solution.

5. The use according to claim 4, characterized in that: In step S1, the bacterial agent includes bacteria producing chitin deacetylase and bacteria producing chitosanase; Wherein, the bacteria producing chitin deacetylase include Rhodococcus sp. HQcdag, and the bacteria producing chitosanase include Bacillus cereus.

6. The use according to claim 5, characterized in that: The ratio of the number of live bacteria of the chitin deacetylase-producing bacteria to the number of live bacteria of the chitosanase-producing bacteria is (2-4):

1.

7. The use according to claim 4, characterized in that: In step S1, the seed culture medium comprises the following components: 5-15 g / L peptone, 1-5 g / L beef extract, 3-7 g / L sodium chloride, and the pH value of the seed culture medium is 6.5-7.5; The culturing specifically includes: culturing for 15-25 hours at a temperature of 25-35° C. and a rotation speed of 180-220 r / min.

8. The use according to claim 4, characterized in that: In step S2, the inoculation amount of the seed liquid is 3-7%, and the fermentation culture specifically includes: culturing for 30-40 hours at a temperature of 25-35°C and a rotation speed of 180-220 r / min.

9. The use according to claim 4, characterized in that: In step S3, the centrifugal treatment specifically includes: centrifuging for 10-20 minutes at a rotation speed of 6000-10000 r / min.

10. The use according to claim 4, characterized in that: In step S3, the chitosan degrading enzyme solution includes chitosan deacetylase and chitosanase, and the chitosan oligosaccharide solution includes chitobiose, chitotriose and chitotetraose.

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