A method for producing chitinase by fermentation of Aspergillus niger and its application

Abstract

The present invention provides a method for producing chitinase by fermenting Aspergillus niger and its application, comprising the following steps: step 1, crushing natural chitin; step 2, preparing a spore-producing medium containing natural chitin; step 3, culturing Aspergillus niger spores; step 4, preparing a chitinase-producing medium containing natural chitin; step 5, inoculating Aspergillus niger spores in the chitinase-producing medium for fermentation to produce chitinase, and after the fermentation is completed, obtaining a mixed solution containing Aspergillus niger mycelium and chitinase. The obtained Aspergillus niger mycelium and chitinase mixed solution is applied to the fermentation treatment of crayfish head waste. The present invention uses chitinase to enzymolyze chitin in the shrimp head, and the Aspergillus niger mycelium is metabolized to produce enzymes, further decomposing chitin, so that the effect of converting chitin into chito-oligosaccharide is optimal.

Description

Technical Field

[0001] The invention belongs to the technical field of lobster waste treatment, and particularly relates to a method for producing chitinase through Aspergillus niger fermentation and application thereof. Background Art

[0002] The production of crayfish in my country has been expanding. In recent years, the output has reached more than 2 million tons per year. Crayfish processing enterprises of varying sizes have emerged in many places. During the processing, the heads, which account for about 65% of the weight of the shrimp, are removed and thrown away as garbage, which not only pollutes the environment but also wastes resources. Fresh shrimp heads contain about 15% chitosan, 13% crude protein, 4.50% crude fat, 8.5% nitrogen-free extract, shrimp oil, astaxanthin, various amino acids, beneficial elements and vitamins.

[0003] At present, the effective ways to utilize shrimp heads are: (1) Extracting chitosan. Process flow: shrimp heads - soaking in hydrochloric acid - washing - soaking in alkali - washing - decolorization - washing - drying - crushing - packaging - finished product. (2) Producing glucosamine hydrochloride. It is an important derivative of chitosan and is used in medicine, food and light industry. (3) Extracting astaxanthin. Astaxanthin is a carotenoid that has a strong free radical scavenging effect, can resist oxidation, enhance immunity and prevent cancer. (4) Extracting nutrients. Extract lipids and recover proteins and inorganic salts. (5) Making seasoning products. It retains the inherent freshness and fragrance of shrimp heads and can be used as various convenience foods. (6) Making biological fertilizers.

[0004] In terms of fertilizers, dry crayfish shell powder (5-8%) is combined with white wine lees powder, rapeseed meal, and straw powder, and is composted with composite microbial agents (such as thermophilic myceliophthora, Bacillus subtilis, Trichoderma harzianum, Bacillus coagulans, Bacillus polymyxa, Aspergillus oryzae, and Lactobacillus plantarum) to produce biological organic fertilizers. Straw and crayfish processing waste are used as the main raw materials, and fermented with budding Aureobasidium and Bacillus to prepare functional all-element biological fertilizers. There are inventions that use lobster shell powder, tea seed shell powder, rice straw, etc. as raw materials, and add microbial agents (Bacillus licheniformis, Bacillus subtilis, lactic acid bacteria and yeast) to prepare biological organic fertilizers. Crayfish shell powder is mixed evenly with cow dung, horse dung, wood ash, wheat straw, etc., and microbial agents (Bacillus subtilis, yeast, lactic acid bacteria) are added to compost and ferment to prepare organic compound fertilizers.

[0005] In the above prior art methods of making shrimp head waste into biofertilizer, the microorganisms used in the method have limited decomposition of chitin during the fermentation cycle, and the generated chitosan oligosaccharides are relatively low, and the agricultural efficacy is not obvious. The present invention is different from most methods of making biofertilizer. It uses Aspergillus niger, which has strong chitinase production, to first grow and culture the bacteria and ferment the enzymes, then inoculate the fermentation mixture into shrimp head waste, and use chitinase to enzymolyze the chitosan in the shrimp head. At the same time, the Aspergillus niger mycelium metabolizes and produces enzymes to further decompose chitosan, so that the effect of converting chitosan into chitosan oligosaccharides is optimal. Summary of the invention

[0006] In view of the problems existing in the prior art, the technical solutions adopted by the present invention to solve the problems existing in the prior art are as follows:

[0007] A method for producing chitinase by fermentation of Aspergillus niger, characterized in that it comprises the following steps:

[0008] Step 1, crushing natural chitin; step 2, preparing a spore-producing medium containing natural chitin; step 3, culturing Aspergillus niger spores; step 4, preparing a chitinase-producing medium containing natural chitin; step 5, inoculating Aspergillus niger spores in the chitinase-producing medium for fermentation to produce chitinase, and after the fermentation is completed, obtaining a mixed solution containing Aspergillus niger mycelium and chitinase.

[0009] The natural chitin in step 1 comes from commercially available shrimp shells, and the particle size after crushing is 100-160 meshes.

[0010] The spore-producing medium in step 2 comprises, by mass fraction, 1-2% of crushed natural chitin, 2-4% of ammonium nitrate, 0.04% of K2HPO4·3H2O, 0.05% of KH2PO4, 0.005% of FeSO4·7H2O, 0.01% of MnSO4, 0.02% of MgSO4·7H2O, 2% of agar powder, and the rest is water.

[0011] In the step 3, the specific process of spore culture of Aspergillus niger is as follows: based on the spore culture medium prepared in step 2, the culture medium is sterilized, cooled, and poured into a plate, a ring of Aspergillus niger JS01 slant strain is picked, and the spore suspension is evenly dispersed in sterile water to obtain a spore suspension; the spore suspension is sucked and evenly coated on the surface of the plate culture medium; the plate is inverted and cultured at a temperature of 26° C. to 30° C. for 3d to 5d until the plate is covered with spores.

[0012] The chitinase production culture medium prepared in step 4 includes, by mass fraction, 1% to 5% of crushed natural chitin, 2% to 8% of ammonium nitrate, 0.02% to 0.05% of FeSO4·7H2O, 0.05% to 0.1% of ZnSO4·H2O, 0.05% to 0.1% of KH2PO4, 0.01% to 0.05% of MgSO4·7H2O, 0.2% of NaCl, and the rest is water, and the pH is adjusted to 5 to 6.

[0013] The specific process of step 5 is as follows: adding sterile water to the plate full of Aspergillus niger spores obtained in step 3 to wash off all the spores; inoculating all the washed spore seed liquid into a chitinase production culture medium; after inoculation, fermenting at a temperature of 22° C. to 26° C. and 250 rpm to 300 rpm for 3 d to 5 d; after the fermentation is completed, a mixed solution containing Aspergillus niger mycelium and chitinase is obtained.

[0014] The Aspergillus niger in step 3 is Aspergillus niger JS01, which is obtained by the following process: collecting soil from 8 points in the shrimp-rice field, mixing evenly, and taking 40 grams of mixed samples; enriching the strain with natural chitin as the only carbon source, and preparing the enrichment medium according to the following mass fractions: 1% natural chitin, 1% sodium nitrate, 0.01% FeSO4·7H2O, 0.01% MnSO4, 0.02% KH2PO4, 0.02% MgCl2, and the rest is water; adding 40 grams of the mixed soil sample to 300 mL of the enrichment medium, and culturing at 25°C and 220rpm for 5 days; preparing the screening medium according to the following mass fractions: 0.5% natural chitin, 1% potassium nitrate, 0.01% FeSO4·7H2O, 0.02% KH2PO4, 0.02% MgCl2 0.02%, agar powder 2%, and the rest is water. After sterilization, pour the plate for later use; take the enriched cultured bacterial solution, perform a series of dilutions, and spread the bacterial solutions of different dilutions on the screening culture medium plates for separation, and culture them at 25°C for 4 days. Microorganisms that can use chitin as a carbon source grow on the plates to form single colonies; select single colonies for identification, and screen out Aspergillus niger that can use chitin as a carbon source, recorded as Aspergillus niger JS01. The strain was sent to the China Center for Type Culture Collection for preservation on June 30, 2022, and the classification name is: Aspergillus niger JS01 (Aspergillus niger JS01), the preservation number is: CCTCC NO: M20221000, address: Wuhan University, Wuhan, China.

[0015] The application process of chitinase in fermentation treatment of crayfish head waste comprises the following steps: step S1, crushing the crayfish head; step S2, adding auxiliary materials and sterilizing; step S3, inoculating a mixed solution containing Aspergillus niger mycelium and chitinase; step S4, fermentation; step S5, filter pressing, and collecting the filtrate and the filter residue respectively.

[0016] After the shrimp heads are crushed in step S1, the diameter of the fragments is less than 2 mm; in step S2, the auxiliary materials added are corn starch and rice protein powder; the mass ratio of the shrimp heads (dry weight) in step S1 to the corn starch and rice protein powder in step S2 is 6-10:0.5-1.0:2-4, and the rest is water.

[0017] In step S3, the inoculation conditions are: inoculation amount 5-10% (v / v), and the fermentation conditions in step S4 are: fermentation temperature 30-35°C, rotation speed 400-600rpm, ventilation ratio 0.3-0.6v / v·m, and fermentation time 2-4d.

[0018] The present invention has the following advantages:

[0019] The present invention solves the problem that the decomposition of chitin is limited and the generated chitosan oligosaccharides are low in the process of treating shrimp head waste in the prior art. Aspergillus niger with strong chitinase production is used to first perform bacterial growth culture and enzyme production fermentation, and then the fermentation mixture is inoculated into the shrimp head waste, and the chitin in the shrimp head is enzymatically hydrolyzed by chitinase. At the same time, the Aspergillus niger mycelium is metabolized to produce enzymes, and the chitosan is further decomposed, so that the effect of converting chitosan into chitosan oligosaccharides is optimal. After the shrimp head waste is fermented according to the method of the present invention, the functional chitosan oligosaccharide content obtained in the collected filtrate reaches 65-72 g / L. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further specifically described below through embodiments.

[0021] The raw material shrimp heads come from the waste of crayfish food processing plants, with a moisture content of about 40%; other substances are commercially available or self-prepared, and are of analytical grade; the instruments are conventional equipment.

[0022] 1. Isolation of Aspergillus niger JS01 strain:

[0023] (1) Soil was collected from 8 points in the shrimp-rice field, mixed evenly, and 40 g of mixed sample was taken;

[0024] (2) Preparation of enrichment medium: Use natural chitin as the sole carbon source for bacterial enrichment. Prepare the enrichment medium according to the following mass fractions: natural chitin 1%, sodium nitrate 1%, FeSO4·7H2O 0.01%, MnSO4 0.01%, KH2PO4 0.02%, MgCl2 0.02%, and the rest is water;

[0025] (3) Add 40 g of the mixed soil sample into 300 mL of enrichment medium and culture at 25 °C and 220 rpm for 5 days;

[0026] (4) Preparation of screening medium: Prepare the screening medium according to the following mass fractions: 0.5% natural chitin, 1% potassium nitrate, 0.01% FeSO4·7H2O, 0.02% KH2PO4, 0.02% MgCl2, 2% agar powder, and the rest is water. After sterilization, pour the medium into a plate for later use;

[0027] (5) Take the enriched cultured bacterial solution and perform serial dilutions. Spread the bacterial solution with different dilutions on the screening culture medium plate for separation. Culture at 25°C for 4 days. Microorganisms that can use chitin as a carbon source can grow on the plate and form single colonies.

[0028] (6) Single colonies were selected for identification, and a strain of Aspergillus niger that can use chitin as a carbon source was screened out and recorded as JS01. The Aspergillus niger JS01 strain was deposited in the China Center for Type Culture Collection (Wuhan University) on June 30, 2022, with the deposit number CCTCC NO: M20221000.

[0029] 2. Slant culture of Aspergillus niger JS01:

[0030] (1) Prepare culture medium: 1% natural chitin, 2% peptone, 0.01% FeSO4·7H2O, 0.02% KH2PO4, 0.02% MgCl2, 2% agar powder, and the rest water. Dissolve evenly and dispense into test tubes, with the filling volume being 1 / 3 of the height of the test tubes. Sterilize by plugging. After sterilization, place the culture medium in the test tubes into an inclined surface and cool.

[0031] (2) After the culture medium in the test tube cools down to form a slant, pick up the Aspergillus niger colonies growing on the screening plate, streak inoculate on the slant, and culture at 26°C for about 5 days. When black spores are formed, this is the slant culture and is ready for use.

[0032] Embodiment 1:

[0033] 1. The spore culture of Aspergillus niger comprises the following steps:

[0034] (1) Preparation of spore-forming culture medium

[0035] The spore-producing medium was prepared according to the following mass fractions: 1% natural chitin, 2% ammonium nitrate, 0.04% K2HPO4·3H2O, 0.05% KH2PO4, 0.005% FeSO4·7H2O, 0.01% MnSO4, 0.02% MgSO4·7H2O, 2% agar powder, and the rest was water.

[0036] (2) After sterilizing the culture medium at 121°C for 30 min, cool it and pour it into a plate;

[0037] (3) Pick a loopful of Aspergillus niger JS01 slant strain, add it to 1 mL of sterile water, shake well and disperse it to obtain a spore suspension;

[0038] (4) Pipette 200 μl of spore suspension and spread it evenly on the surface of the plate culture medium;

[0039] (5) The plate was incubated upside down at 30°C for 5 days until it was covered with spores.

[0040] 2. Fermentation to produce chitinase and obtain mycelium, comprising the following steps:

[0041] (1) Prepare chitinase production medium (200 mL of liquid in a 1000 mL Erlenmeyer flask);

[0042] The chitinase production culture medium was prepared according to the following mass fractions: 1% natural chitin, 2% ammonium nitrate, 0.02% FeSO4·7H2O, 0.05% ZnSO4·H2O, 0.05% KH2PO4, 0.01% MgSO4·7H2O, 0.2% Nacl, and the rest was water, and the pH was adjusted to 5.5. Sterilize at 121°C for 30 min and cool.

[0043] (2) Preparation of seed solution

[0044] Add 5 mL of sterile water to the plate full of Aspergillus niger spores to wash off all the spores;

[0045] (3) Vaccination

[0046] Inoculate all 5 mL of spore seed solution into chitinase-producing medium;

[0047] (4) Fermentation enzyme production

[0048] After inoculation, the mixture was fermented at 26°C and 250 rpm for 3 days. After fermentation, a mixture containing chitinase and Aspergillus niger mycelium was obtained for use, wherein the enzyme activity was 32 U / mL and the wet weight of mycelium was 250 mg / mL.

[0049] 3. Fermenting the crayfish head waste, comprising the following steps:

[0050] (1) Crushing shrimp heads

[0051] 9.6 kg of shrimp head waste left over from a crayfish food processing plant was taken and its moisture content was 38%. The shrimp heads were crushed by a crusher to a diameter of less than 2 mm.

[0052] (2) Add auxiliary materials and sterilize

[0053] Add auxiliary materials to the crushed shrimp heads, namely 0.5 kg of corn starch and 2 kg of rice protein powder, add 15 L of water, stir evenly, put into a 30 L mechanical stirring fermentation tank, pass steam to maintain 121 ° C for 30 minutes, and cool.

[0054] (3) Vaccination

[0055] 1.2 L of a mixture containing Aspergillus niger mycelium and chitinase was inoculated into a 50 L fermentation tank.

[0056] (4) Fermentation

[0057] The fermentation temperature was controlled at 30°C, the stirring speed was 400 rpm, the ventilation ratio was 0.3 v / v·m, and the time was 2 days.

[0058] (5) Filtration to obtain filtrate and filter residue

[0059] After the fermentation is completed, the product is filtered through a filter press to obtain a filtrate and a residue respectively. The filtrate is 12L and contains functional chitosan oligosaccharides at a content of 65g / L. It can be appropriately diluted and then applied to crops by flushing, drip irrigation or foliar spraying to promote crop growth and enhance immune resistance. 3.8kg of the residue is obtained and can be used as an organic fertilizer or mixed with an inorganic compound fertilizer.

[0060] Embodiment 2:

[0061] 1. The spore culture of Aspergillus niger comprises the following steps:

[0062] (1) Preparation of spore-forming culture medium

[0063] The spore-producing medium was prepared according to the following mass fractions: 1.5% natural chitin, 3% ammonium nitrate, 0.04% K2HPO4·3H2O, 0.05% KH2PO4, 0.005% FeSO4·7H2O, 0.01% MnSO4, 0.02% MgSO4·7H2O, 2% agar powder, and the rest was water.

[0064] (2) After sterilizing the culture medium at 121°C for 30 min, cool it and pour it into a plate;

[0065] (3) Pick a loopful of Aspergillus niger JS01 slant strain, add it to 3 mL of sterile water, shake well and disperse it to obtain a spore suspension;

[0066] (4) Pipette 150 μl of spore suspension and spread it evenly on the surface of the plate culture medium;

[0067] (5) The plates were incubated upside down at 26°C for 4 days until they were covered with spores.

[0068] 2. Fermentation to produce chitinase and obtain mycelium, comprising the following steps:

[0069] (1) Prepare chitinase production medium (200 mL of liquid in a 1000 mL Erlenmeyer flask);

[0070] The chitinase production medium was prepared according to the following mass fractions: 3% natural chitin, 5% ammonium nitrate, 0.03% FeSO4·7H2O, 0.08% ZnSO4·H2O, 0.07% KH2PO4, 0.03% MgSO4·7H2O, 0.2% Nacl, and the rest was water, and the pH was adjusted to 5.8. Sterilize at 121°C for 30 min and cool.

[0071] (2) Preparation of seed solution

[0072] Add 8 mL of sterile water to the plate full of Aspergillus niger spores to wash off all the spores;

[0073] (3) Vaccination

[0074] Inoculate 8 mL of spore seed solution into chitinase-producing medium;

[0075] (4) Fermentation enzyme production

[0076] After inoculation, the mixture was fermented at 24°C and 280 rpm for 4 days. After fermentation, a mixture containing chitinase and Aspergillus niger mycelium was obtained for use, wherein the enzyme activity was 58 U / mL and the mycelium wet weight was 410 mg / mL.

[0077] 3. Fermenting the crayfish head waste, comprising the following steps:

[0078] (1) Crushing shrimp heads

[0079] 12.3 kg of shrimp head waste left over from a crayfish food processing plant was taken and its moisture content was 35%. The shrimp heads were crushed with a crusher to a diameter of less than 2 mm.

[0080] (2) Add auxiliary materials and sterilize

[0081] Add auxiliary materials to the crushed shrimp heads, including 0.75 kg of corn starch and 3 kg of rice protein powder, add 20 L of water, stir evenly, put into a 50 L mechanical stirring fermentation tank, sterilize with steam at 121 ° C for 30 min, and cool.

[0082] (3) Vaccination

[0083] Inoculate 2.5 L of a mixture containing Aspergillus niger mycelium and chitinase into a 50 L fermentation tank.

[0084] (4) Fermentation

[0085] The fermentation temperature was controlled at 32°C, the stirring speed was 500 rpm, the ventilation ratio was 0.4 v / v·m, and the fermentation time was 3 days.

[0086] (5) Filtration to obtain filtrate and filter residue

[0087] After the fermentation is completed, the product is filtered through a filter press to obtain a filtrate and a residue respectively. The filtrate is 18L and contains functional chitosan oligosaccharides with a content of 72g / L. It can be appropriately diluted and then applied to crops by flushing, drip irrigation or foliar spraying to promote crop growth and enhance immune resistance. The residue is 5.1kg and can be used as an organic fertilizer or mixed with inorganic compound fertilizers.

[0088] Example 3

[0089] 1. The spore culture of Aspergillus niger comprises the following steps:

[0090] (1) Preparation of spore-forming culture medium

[0091] The spore-producing medium was prepared according to the following mass fractions: 2% natural chitin, 4% ammonium nitrate, 0.04% K2HPO4·3H2O, 0.05% KH2PO4, 0.005% FeSO4·7H2O, 0.01% MnSO4, 0.02% MgSO4·7H2O, 2% agar powder, and the rest was water.

[0092] (2) After sterilizing the culture medium at 121°C for 30 min, cool it and pour it into a plate;

[0093] (3) Pick a loopful of Aspergillus niger JS01 slant strain, add it to 2 mL of sterile water, shake well and disperse it to obtain a spore suspension;

[0094] (4) Pipette 100 μl of spore suspension and spread it evenly on the surface of the plate culture medium;

[0095] (5) Incubate the plate upside down at 28°C for 3 days until the plate is covered with spores.

[0096] 2. Fermentation to produce chitinase and obtain mycelium, comprising the following steps:

[0097] (1) Prepare chitinase production medium (200 mL of liquid in a 1000 mL Erlenmeyer flask);

[0098] The chitinase production culture medium was prepared according to the following mass fractions: 5% natural chitin, 8% ammonium nitrate, 0.05% FeSO4·7H2O, 0.1% ZnSO4·H2O, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.2% Nacl, and the rest was water, and the pH was adjusted to 6.0. Sterilize at 121°C for 30 min and cool.

[0099] (2) Preparation of seed solution

[0100] Add 10 mL of sterile water to the plate full of Aspergillus niger spores to wash off all the spores;

[0101] (3) Vaccination

[0102] Inoculate all 10 mL of spore seed solution into chitinase-producing medium;

[0103] (4) Fermentation enzyme production

[0104] After inoculation, the mixture was fermented at 22°C and 300 rpm for 5 days. After fermentation, a mixture containing chitinase and Aspergillus niger mycelium was obtained for use, wherein the enzyme activity was 44 U / mL and the mycelium wet weight was 320 mg / mL.

[0105] 3. Fermentation of crayfish head waste, comprising the following steps:

[0106] (1) Crushing shrimp heads

[0107] 16.5 kg of shrimp head waste left over from a crayfish food processing plant was taken and its moisture content was 40%. The shrimp heads were crushed with a crusher to a diameter of less than 2 mm.

[0108] (2) Add auxiliary materials and sterilize

[0109] Add auxiliary materials to the crushed shrimp heads, namely 1.0 kg of corn starch and 4 kg of rice protein powder, add 25 L of water, stir evenly, put into a 50 L mechanical stirring fermentation tank, sterilize with steam at 121 ° C for 30 min, and cool.

[0110] (3) Vaccination

[0111] 3.5 L of a mixture containing Aspergillus niger mycelium and chitinase was inoculated into a 50 L fermentation tank.

[0112] (4) Fermentation

[0113] The fermentation temperature was controlled at 35°C, the stirring speed was 600 rpm, the ventilation ratio was 0.5 v / v·m, and the fermentation time was 4 days.

[0114] (5) Filtration to obtain filtrate and filter residue

[0115] After the fermentation is completed, the product is filtered through a filter press to obtain a filtrate and a residue respectively. The filtrate is 24L and contains functional chitosan oligosaccharides with a content of 69g / L. It can be appropriately diluted and then applied to crops by flushing, drip irrigation or foliar spraying to promote crop growth and enhance immune resistance. 6.9kg of the residue is obtained and can be used as an organic fertilizer or mixed with inorganic compound fertilizers.

[0116] The protection scope of the present invention is not limited to the above-mentioned embodiments. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the scope and spirit of the present invention. If these changes and modifications fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include these changes and modifications.

Claims

1. An application of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger, characterized in that: The method for producing chitinase by fermentation of Aspergillus niger comprises the following steps: Step 1, crushing natural chitin; Step 2, preparing a conidia-producing medium containing natural chitin, wherein the conidia-producing medium comprises, by mass fraction, 1-2% of crushed natural chitin, 2-4% of ammonium nitrate, 0.04% of K2HPO4·3H2O, 0.05% of KH2PO4, 0.005% of FeSO4·7H2O, 0.01% of MnSO4, 0.02% of MgSO4·7H2O, 2% of agar powder, and the remainder is water; Step 3, spore culture of Aspergillus niger: based on the spore culture medium prepared in step 2, the culture medium is sterilized, cooled, and poured into a plate, a ring of Aspergillus niger slant strains is picked up, and the spore suspension is evenly dispersed in sterile water to obtain a spore suspension; the spore suspension is aspirated and evenly coated on the surface of the plate culture medium; the plate is inverted and cultured at a temperature of 26° C. to 30° C. for 3 to 5 days until the plate is covered with spores; Step 4, preparing a chitinase-producing culture medium containing natural chitin; Step 5, inoculating Aspergillus niger spores into a chitinase-producing medium for fermentation to produce chitinase, and after the fermentation is completed, obtaining a mixed solution containing Aspergillus niger mycelium and chitinase; Fermentation treatment of crayfish head waste comprises the following steps: The process of applying the mixed solution containing Aspergillus niger mycelium and chitinase obtained by the above steps to fermentation treatment of crayfish head waste includes the following steps: step S1, crushing the shrimp heads; step S2, adding auxiliary materials and sterilizing; step S3, inoculating the mixed solution containing Aspergillus niger mycelium and chitinase; step S4, fermentation; step S5, filter pressing, and collecting the filtrate and the filter residue respectively; The Aspergillus niger is recorded as Aspergillus niger JS01, and its preservation number is CCTCC NO: M20221000; the natural chitin in step 1 comes from commercially available shrimp shells.

2. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 1, characterized in that: The natural chitin in step 1 comes from commercially available shrimp shells with a particle size of 100 to 160 meshes after crushing.

3. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 1, characterized in that: The chitinase production culture medium prepared in step 4 includes, by mass fraction, 1% to 5% of crushed natural chitin, 2% to 8% of ammonium nitrate, 0.02% to 0.05% of FeSO4·7H2O, 0.05% to 0.1% of ZnSO4·H2O, 0.05% to 0.1% of KH2PO4, 0.01% to 0.05% of MgSO4·7H2O, 0.2% of NaCl, and the rest is water, and the pH is adjusted to 5 to 6.

4. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 3, characterized in that: The specific process of step 5 is as follows: adding sterile water to the plate full of Aspergillus niger spores JS01 obtained in step 3 to wash off all the spores; inoculating all the washed spore seed liquid into the chitinase production culture medium; after inoculation, fermenting at a temperature of 22° C. to 26° C. and 250 rpm to 300 rpm for 3 d to 5 d; after the fermentation is completed, a mixed solution containing Aspergillus niger JS01 mycelium and chitinase is obtained.

5. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 1, characterized in that: In step 3, Aspergillus niger JS01 is obtained by the following process: soil from 8 points in the shrimp-rice field is collected in a dispersed manner, mixed evenly, and 40 grams of mixed sample is taken; natural chitin is used as the only carbon source for strain enrichment, and enrichment medium is prepared according to the following mass fractions: natural chitin 1%, sodium nitrate 1%, FeSO4·7H2O 0.01%, MnSO4 0.01%, KH2PO4 0.02%, MgCl2 0.02%, and the rest is water; 40 grams of the mixed soil sample is added to 300 mL of enrichment medium, and cultured at 25°C 220 rpm for 5 days; The screening culture medium was prepared according to the following mass fractions: 0.5% natural chitin, 1% potassium nitrate, 0.01% FeSO4·7H2O, 0.02% KH2PO4, 0.02% MgCl2, 2% agar powder, and the rest was water. After sterilization, the cultured bacteria solution was poured into a plate for later use. A series of dilutions were performed on the enriched culture medium solution, and the bacteria solutions with different dilutions were spread on the screening culture medium plate for separation. The culture was cultured at 25°C for 4 days, and the microorganisms that could use chitin as a carbon source grew on the plate to form single colonies. The single colonies were selected for identification, and Aspergillus niger that could use chitin as a carbon source was screened.

6. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 1, characterized in that: After the shrimp heads are crushed in step S1, the diameter of the fragments is less than 2 mm; in step S2, the auxiliary materials added are corn starch and rice protein powder; the mass ratio of the shrimp heads in step S1 to the corn starch and rice protein powder in step S2 is 6-10: 0.5-1.0: 2-4, and the rest is water.

7. The use of producing chitosan oligosaccharides by fermenting shrimp head waste with Aspergillus niger as claimed in claim 1, characterized in that: In step S3, the inoculation conditions are: inoculation amount 5-10% v / v, and the fermentation conditions in step S4 are: fermentation temperature 30-35° C., rotation speed 400-600 rpm, ventilation ratio 0.3-0.6 v / v·m, and fermentation time 2-4 d.