A method for preparing chitooligosaccharides by enzymatic hydrolysis

Abstract

The present invention discloses a method for preparing chitooligosaccharides by enzymatic hydrolysis, belonging to the technical field of chitooligosaccharide production. In view of the relatively large proportion of by-products such as small shrimps, shrimp heads, and shrimp shells, the present invention first adopts a physical peeling method, uses a roller extrusion method to recover the surface organic matter in the shrimp shells, and preliminarily crushes the chitin to make its structure loose, which is beneficial to subsequent biological fermentation; then adopts specific enzymatic hydrolysis for directional release, multi-strain combined fermentation, and the combination of bacteria and enzymes to efficiently recover the remaining proteins and activate the soluble chitooligosaccharides in the by-products, without the use of a large amount of acids and alkalis, while improving the processing precision and resource utilization efficiency, and greatly reducing the environmental pollution during the processing process.

Description

Technical Field

[0001] The present invention belongs to the technical field of chitooligosaccharide production, and specifically relates to a method for preparing chitooligosaccharide based on enzymatic hydrolysis, and also relates to chitooligosaccharide prepared by this method. Background Art

[0002] Freshwater shrimps in Jiangsu mainly include Macrobrachium nipponense (including green shrimps and river shrimps), Macrobrachium rosenbergii, and freshwater crayfish (Procambarus clarkii). The annual output is nearly 3 million tons, and the current annual processing volume is 500,000 - 800,000 tons. During the processing of shrimp meat, a large amount of by-products such as shrimp heads, shrimp shells, and substandard small-sized products and scraps are generated, accounting for about 35% - 40%, with a very large proportion. Some of these by-products have been appropriately recycled, but generally speaking, they are of low quality and low efficiency, resulting in a large waste of resources.

[0003] Chitooligosaccharide (N-acetyl chito oligosaccharides, NACOS) is a degradation product of chitin. Due to its excellent properties such as safety, biocompatibility, and biodegradability, NACOS has been widely used in fields such as medicine, food, agriculture, cosmetics, and biomaterials. Currently, relatively in-depth research has been carried out on its preparation, extraction, and activity evaluation at home and abroad. In terms of preparation, some use snail enzyme for hydrolysis (Xie Xiaolan et al., 2015), some use exochitinase produced by Aspergillus fumigatus for preparation (Wu Yuanyuan et al., 2008), and some use genetically engineered recombinant chitinase for preparation (Ohnuma Takayuki et al., 2019). The core tool for preparing chitooligosaccharide is chitinase. Although there is a rich gene resource, the actual degradation effect is poor, mainly because of the poor solubility of chitin. Hydrolysis through colloidal chitin cannot form large-scale preparation, which becomes a technical bottleneck that cannot be promoted industrially. Summary of the Invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing chitooligosaccharide based on enzymatic hydrolysis.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A method for preparing chitooligosaccharide based on enzymatic hydrolysis, comprising the following steps:

[0007] (1) Select fresh shrimp shells, clean them, drain them, and then use a roller extrusion device to extrude and crush them to separate the surface organic matter and chitin in the shrimp shells, obtaining preliminarily crushed chitin;

[0008] (2) Add the obtained preliminarily crushed chitin to sterile water and make a slurry together, control the slurry concentration at 25%, and adjust the pH value of the slurry;

[0009] (3) Use keratinase to biochemically hydrolyze the protein components in the slurry, so that they are fully dissociated from chitin, and directionally hydrolyze to obtain polypeptides with anti-stress activity. The keratinase is obtained by fermentation and culture of Bacillus pumilus K9. Under the conditions of pH value of 8 - 11 and temperature of 55 - 65 °C, enzymatically hydrolyze for 2 - 4 h to obtain an enzymatic hydrolysis product, and then inactivate by heating;

[0010] (4) Configure Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus into a compound bacterial solution according to a mass ratio of 1∶1 - 2; Under the conditions of pH value of 6.8 - 8.0 and temperature of 30 - 45 °C, enzymatically hydrolyze for 2 - 4 h to obtain an enzymatic hydrolysis product, and then inactivate by heating;

[0011] (5) Use Lactobacillus reuteri to further enzymatically hydrolyze the enzymatic hydrolysis product obtained in step (4). Under the airtight conditions of pH value of 2.0 - 6.0 and temperature of 30 - 50 °C, enzymatically hydrolyze for 0.5 - 2 h to obtain an enzymatic hydrolysis product, and then inactivate by heating;

[0012] (6) Filter to remove the flocs in the enzymatic hydrolysis extract, and spray dry to obtain chitooligosaccharide powder.

[0013] Furthermore, in step (1), add water accounting for 20% - 25% of the total amount of shrimp shells during the extrusion process.

[0014] Furthermore, in steps (3), (4), and (5), after obtaining the enzymatic hydrolysis product, inactivate by heating at a temperature of 90 °C for 10 min.

[0015] Furthermore, in step (3), the viable count of Bacillus pumilus K9 is 10 7 cfu / mL.

[0016] Furthermore, in step (3), under the conditions of pH value of 9 and temperature of 60 °C, enzymatically hydrolyze for 2 h to obtain an enzymatic hydrolysis product.

[0017] Furthermore, in step (4), configure Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus into a compound bacterial solution according to a mass ratio of 1∶2, and the total viable count of the compound bacterial solution is 10 8 cfu / mL.

[0018] Further, in step (4), enzymatic hydrolysis is carried out at a pH value of 7.4 and a temperature of 40 °C for 3 h to obtain an enzymatic hydrolysis product.

[0019] Further, in step (5), the viable count of the Lactobacillus reuteri bacterial liquid is 10 7 cfu / mL.

[0020] Further, in step (5), enzymatic hydrolysis is carried out under airtight conditions at a pH value of 5.0 and a temperature of 40 °C for 0.5 h to obtain an enzymatic hydrolysis product.

[0021] Further, in step (6), spray drying is carried out under the conditions that the inlet air temperature is 140 °C and the outlet air temperature is 70 °C.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] In the present application, for the by-products such as small shrimps, shrimp heads, and shrimp shells with a relatively large proportion, the present invention first adopts a physical peeling method, uses a roller extrusion method to recover the surface organic matter in the shrimp shells, and preliminarily crushes chitin to make its structure loose, which is beneficial to subsequent biological fermentation; then adopts specific enzymatic hydrolysis for directional release and multi-strain co-fermentation, combines bacteria and enzymes, efficiently recovers the remaining proteins, activates the soluble chitooligosaccharides in the by-products, and does not require the use of a large amount of acids and alkalis. While improving the processing precision and resource utilization efficiency, it greatly reduces the environmental pollution during the processing process. The yield of chitooligosaccharides prepared by enzymatic hydrolysis using the method of the present invention is greater than 15%. Description of the Drawings

[0024] Figure 1 It is the technical flow chart of the present invention. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the method, steps or conditions of the present invention belongs to the scope of the present invention. In the following embodiments, unless otherwise specified, the technical means used in the embodiments are all conventional means well known to those skilled in the art.

[0026] The strains and culture conditions used in the following embodiments are as follows:

[0027] Bacillus pumilus K9 was deposited in the China General Microbiological Culture Collection Center on August 19, 2013, with the deposit number CGMCC No. 8046. The culture medium used for fermentation culture is as follows: 1 - 3% wool, 1 - 2% maltose, 1 - 2% yeast powder, 0.04% dipotassium hydrogen phosphate, 0.05% sodium chloride. The culture conditions are: initial pH 8.5, inoculum size 8%, culture temperature 35°C, rotation speed 220 r / min, liquid loading 25 ml / 250 ml, and 48 h in the late fermentation stage. See CN 103642735B.

[0028] Bacillus subtilis natto ND - 1 - A27 was deposited in the China Center for Type Culture Collection on March 14, 2018. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No: M 2018131. The composition of the fermentation medium is: 40 - 60 g / L glycerol, 40 - 60 g / L yeast powder, 180 - 200 g / L soy peptone, 0.5 - 0.8 g / L dipotassium hydrogen phosphate. Fermentation culture is carried out at 35 - 38°C for 3 - 6 days. See CN 108410775 B.

[0029] Bacillus mucilaginosus, with the deposit number CGMCC 5766. Fermentation medium (g / L): glucose 60, urea 0.3, CaCO3 5, MgSO4·7H2O 0.6, K2HPO4·3H2O 0.2, NaCl 0.4, pH 7.0 - 7.2, sterilized at 121°C for 20 min. The culture conditions are: 30°C, 150 r / min for 72 h. See CN 105712493 B.

[0030] Lactobacillus reuteri was deposited in the China General Microbiological Culture Collection Center at No. 3, Yard 1, Beichen West Road, Beijing on December 6, 2017, with the deposit number CGMCC No. 15021. The medium is MRS liquid medium, and the formula is: glucose 20, beef extract 10, peptone 10, yeast extract 5, sodium acetate anhydrous 5, diammonium citrate 2, potassium dihydrogen phosphate 2, manganese sulfate 0.19, magnesium sulfate 0.58, Tween 80 mL, anaerobically cultured at 37°C for 48 h. See CN110564638 A.

[0031] Example 1

[0032] A method for preparing chitooligosaccharides based on enzymatic hydrolysis, comprising the following steps:

[0033] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then use a roller extrusion device to crush them. During the extrusion process, add water accounting for 25% of the total amount of shrimp shells to separate the surface organic matter (protein, fat) from chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells, obtaining preliminarily broken chitin.

[0034] (2) Add the obtained preliminarily broken chitin to sterile water and beat it into a slurry. Control the slurry concentration at about 25%, and adjust the pH value of the slurry for later use.

[0035] (3) Use keratinase to biochemically hydrolyze the protein component in the slurry, making it fully free from chitin, and directionally hydrolyze to obtain polypeptides with anti - stress activity. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9. The viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Enzymatically hydrolyze for 2 - 4 h under the conditions of pH value 8 - 11 and temperature 55 - 65 °C to obtain the enzymatic hydrolysis product. Then inactivate at a temperature of 80 - 95 °C for 10 min.

[0036] (4) Configure Bacillus subtilis natto ND - 1 - A27 and Bacillus mucilaginosus into a compound bacterial liquid according to a mass ratio of 1∶1 - 2. The total viable count of the compound bacterial liquid is 10 8 cfu / mL. Enzymatically hydrolyze for 2 - 4 h under the conditions of pH value 6.8 - 8.0 and temperature 30 - 45 °C to obtain the enzymatic hydrolysis product. Through the growth of the compound microorganisms, further promote the hydrolysis of the surface chitosan and weaken the natural structure of chitosan. Then inactivate at a temperature of 80 - 95 °C for 10 min.

[0037] (5) Use Lactobacillus reuteri to further enzymatically hydrolyze the enzymatic hydrolysis product obtained in step (4). The viable count of the Lactobacillus reuteri bacterial liquid is 10 7 cfu / mL. Enzymatically hydrolyze for 0.5 - 2 h under the closed conditions of pH value 2.0 - 6.0 and temperature 30 - 45 °C to obtain the enzymatic hydrolysis product. Then inactivate at a temperature of 80 - 95 °C for 10 min.

[0038] (6) Filter to remove the flocculants in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain chitooligosaccharide powder. The yield of chitooligosaccharide is greater than 15%.

[0039] Example 2

[0040] By optimizing the enzymatic hydrolysis temperature, time, and pH value in Example 1, it was found that the optimal reaction conditions were as follows:

[0041] A method for preparing chitooligosaccharides by enzymatic hydrolysis, comprising the following steps:

[0042] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then crush them using a roller extrusion device. During the extrusion process, add water accounting for 25% of the total amount of shrimp shells to separate the surface organic matter (protein, fat) and chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells, obtaining preliminarily crushed chitin.

[0043] (2) Add the obtained preliminarily crushed chitin to sterile water and make a slurry together. Control the slurry concentration at about 25%, and adjust the pH of the slurry to 9 for standby.

[0044] (3) Use keratinase to biochemically hydrolyze the protein component in the slurry, enabling it to fully dissociate from chitin and directionally hydrolyze to obtain polypeptides with anti - stress activity. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9. The viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Perform enzymatic hydrolysis for 2 h under the conditions of pH value 9 and temperature 60°C to obtain an enzymatic hydrolysis product. Then inactivate at 90°C for 10 min.

[0045] (4) Configure Bacillus subtilis natto ND - 1 - A27 and Bacillus mucilaginosus into a compound bacterial solution according to a mass ratio of 1:2. The total viable count of the compound bacterial solution is 10 8 cfu / mL. Perform enzymatic hydrolysis for 3 h under the conditions of pH value 7.4 and temperature 40°C to obtain an enzymatic hydrolysis product. Then inactivate at 90°C for 10 min.

[0046] (5) Further enzymatically hydrolyze the enzymatic hydrolysis product obtained in step (4) using Lactobacillus reuteri. The viable count of the Lactobacillus reuteri bacterial solution is 10 7 cfu / mL. Perform enzymatic hydrolysis for 0.5 h under the conditions of pH value 5.0 and temperature 40°C in a closed environment to obtain an enzymatic hydrolysis product. Then inactivate at 90°C for 10 min.

[0047] (6) Filter to remove the flocculants in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140°C and an outlet air temperature of 70°C to obtain chitooligosaccharide powder. The yield of chitooligosaccharides is 18.6%.

[0048] Comparative Example 1

[0049] A method for preparing chitooligosaccharides by enzymatic hydrolysis, comprising the following steps:

[0050] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then crush them using a roller extrusion device. During the extrusion process, add water accounting for 25% of the total amount of shrimp shells to separate the surface organic matter (protein, fat) from the chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells, obtaining preliminarily crushed chitin.

[0051] (2) Add the obtained preliminarily crushed chitin to sterile water and make a slurry, control the slurry concentration at about 25%, and adjust the pH of the slurry to 9 for standby.

[0052] (3) Use keratinase to biochemically hydrolyze the protein component in the slurry, making it fully free from chitin, and directionally hydrolyze to obtain polypeptides with anti - stress activity. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9. The viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Enzymatically hydrolyze for 2 h under the conditions of pH value of 9 and temperature of 60 °C to obtain an enzymatic hydrolysis product. Then inactivate at a temperature of 90 °C for 10 min.

[0053] (4) Configure Bacillus subtilis natto ND - 1 - A27 and Bacillus mucilaginosus into a compound bacterial solution according to a mass ratio of 1:2. The total viable count of the compound bacterial solution is 10 8 cfu / mL. Enzymatically hydrolyze for 3 h under the conditions of pH value of 7.4 and temperature of 40 °C to obtain an enzymatic hydrolysis product. Then inactivate at a temperature of 90 °C for 10 min.

[0054] (5) Filter to remove the floccules in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain chitooligosaccharide powder, and the yield of chitooligosaccharides is 13.9%.

[0055] Comparative Example 2

[0056] A method for preparing chitooligosaccharides by enzymatic hydrolysis, comprising the following steps:

[0057] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then crush them using a roller extrusion device. During the extrusion process, add water accounting for 25% of the total amount of shrimp shells to separate the surface organic matter (protein, fat) from the chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells, obtaining preliminarily crushed chitin.

[0058] (2) Add the obtained preliminarily crushed chitin to sterile water and beat it into a pulp. Control the pulp concentration at about 25%, and adjust the pH of the pulp to 9 for standby.

[0059] (3) Use keratinase to biochemically hydrolyze the protein components in the pulp, so that they are fully freed from chitin, and directionally hydrolyze to obtain polypeptides with anti-stress activity. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9. The viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Under the conditions of pH value of 9 and temperature of 60 °C, enzymatically hydrolyze for 2 h to obtain an enzymatic hydrolysis product. Then heat inactivate at a temperature of 90 °C for 10 min.

[0060] (4) The viable count of Bacillus mucilaginosus is 10 8 cfu / mL. Under the conditions of pH value of 7.4 and temperature of 40 °C, enzymatically hydrolyze for 3 h to obtain an enzymatic hydrolysis product. Then heat inactivate at a temperature of 90 °C for 10 min.

[0061] (5) Further enzymatically hydrolyze the enzymatic hydrolysis product obtained in step (4) with Lactobacillus reuteri. The viable count of the Lactobacillus reuteri bacterial liquid is 10 7 cfu / mL. Under the closed conditions of pH value of 5.0 and temperature of 40 °C, enzymatically hydrolyze for 0.5 h to obtain an enzymatic hydrolysis product. Then heat inactivate at a temperature of 90 °C for 10 min.

[0062] (6) Filter to remove the flocs in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain chito-oligosaccharide powder. The yield of chito-oligosaccharide is 12.5%.

[0063] Comparative Example 3

[0064] A method for preparing chito-oligosaccharide based on enzymatic hydrolysis, comprising the following steps:

[0065] (1) Select fresh shrimp shells, wash them 2-3 times, drain them, and use a roller extrusion device to extrude and crush them. Add 25% of the total amount of shrimp shells of water during the extrusion process to separate the surface organic matter (protein, fat) and chitin (chitin, calcium, a small amount of organic matter) in the shrimp shells to obtain preliminarily crushed chitin.

[0066] (2) Add the obtained preliminarily crushed chitin to sterile water and beat it into a pulp. Control the pulp concentration at about 25%, and adjust the pH of the pulp to 9 for standby.

[0067] (3) Use keratinase to biochemically hydrolyze the protein components in the slurry, enabling them to fully dissociate from chitin and directionally hydrolyze to obtain polypeptides with anti-stress activity. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9, and the viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Hydrolyze for 2 h under the conditions of pH 9 and a temperature of 60 °C to obtain a hydrolyzate. Then, inactivate at 90 °C for 10 min by heating.

[0068] (4) Bacillus subtilis natto ND-1-A27 is hydrolyzed for 3 h under the conditions of pH 7.4 and a temperature of 40 °C to obtain a hydrolyzate. Then, inactivate at 90 °C for 10 min by heating.

[0069] (5) Lactobacillus reuteri is used to further hydrolyze the hydrolyzate obtained in step (4). The viable count of the Lactobacillus reuteri bacterial solution is 10 7 cfu / mL. Hydrolyze for 0.5 h under the airtight conditions of pH 5.0 and a temperature of 40 °C to obtain a hydrolyzate. Then, inactivate at 90 °C for 10 min by heating.

[0070] (6) Filter to remove the flocculants in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain chito-oligosaccharide powder, and the yield of chito-oligosaccharide is 10.8%.

[0071] Comparative Example 4

[0072] A method for preparing chito-oligosaccharide based on enzymatic hydrolysis, comprising the following steps:

[0073] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then use a roller extrusion device to extrude and crush them. Add 25% of the total amount of shrimp shells of water during the extrusion process to separate the surface organic matter (protein, fat) and chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells to obtain preliminarily crushed chitin.

[0074] (2) Add the obtained preliminarily crushed chitin to sterile water and beat into a slurry. Control the slurry concentration at about 25%, and adjust the pH of the slurry to 9 for standby.

[0075] (3) Use keratinase to biochemically hydrolyze the protein components in the slurry, enabling them to fully dissociate from chitin and directionally hydrolyze to obtain polypeptides with anti-stress activity. The keratinase is obtained by fermenting and culturing Bacillus pumilus K9. The viable count of Bacillus pumilus K9 is 10 7 cfu / mL. Under the conditions of a pH value of 9 and a temperature of 60 °C, enzymatically hydrolyze for 2 h to obtain an enzymatic hydrolysate. Then, heat inactivate at a temperature of 90 °C for 10 min.

[0076] (4) Use Lactobacillus reuteri to further enzymatically hydrolyze the enzymatic hydrolysate obtained in step (3). The viable count of the Lactobacillus reuteri bacterial solution is 10 7 cfu / mL. Under the conditions of a pH value of 5.0, a temperature of 40 °C, and a closed environment, enzymatically hydrolyze for 0.5 h to obtain an enzymatic hydrolysate. Then, heat inactivate at a temperature of 90 °C for 10 min.

[0077] (5) Filter to remove the flocculants in the enzymatic hydrolysis extract, and perform spray drying under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C to obtain chito-oligosaccharide powder. The yield of chito-oligosaccharide is 3.4%.

[0078] Comparative Example 5

[0079] A method for preparing chito-oligosaccharide based on enzymatic hydrolysis, comprising the following steps:

[0080] (1) Select fresh shrimp shells, wash them 2 - 3 times, drain, and then use a roller extrusion device to extrude and crush them. During the extrusion process, add water accounting for 25% of the total amount of shrimp shells to separate the surface organic matter (protein, fat) from chitin (chitosan, calcium, a small amount of organic matter) in the shrimp shells to obtain preliminarily crushed chitin.

[0081] (2) Add the obtained preliminarily crushed chitin to sterile water and make a slurry together. Control the slurry concentration at about 25%, and adjust the pH of the slurry to 9 for standby.

[0082] (3) Prepare a compound bacterial solution by mixing Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus in a mass ratio of 1:2. The total viable count of the compound bacterial solution is 10 8 cfu / mL. Under the conditions of a pH value of 7.4 and a temperature of 40 °C, enzymatically hydrolyze the slurry for 3 h to obtain an enzymatic hydrolysate. Then, heat inactivate at a temperature of 90 °C for 10 min.

[0083] (4) Further enzymatically hydrolyze the enzymatic hydrolysate obtained in step (3) with Lactobacillus reuteri. The viable cell count of the Lactobacillus reuteri solution is 10 7 cfu / mL. Enzymatically hydrolyze for 0.5 h under airtight conditions at a pH value of 5.0 and a temperature of 40 °C to obtain an enzymatic hydrolysate. Then, inactivate at 90 °C for 10 min.

[0084] (5) Filter to remove the flocculants in the enzymatic hydrolysis extract, and perform spray drying under the conditions that the inlet air temperature is 140 °C and the outlet air temperature is 70 °C to obtain chitooligosaccharide powder. The yield of chitooligosaccharide is 15.2%.

[0085] It can be seen from Example 2 and Comparative Example 5 that the present invention uses specific keratinase to biochemically hydrolyze the protein components in the slurry, making them fully free from chitin, and directionally hydrolyzing to obtain polypeptides with anti-stress activity, obtaining a hydrolyzate with high activity. Through deep hydrolysis, it provides a good nutritional environment for subsequent multi-strain co-fermentation, and the combined action of bacteria and enzymes. Comparing Example 2 with Comparative Examples 2, 3, and 4, it can be seen that the present invention adopts a multi-strain fermentation process, uses Bacillus subtilis natto ND-1-A27 that can secrete a high level of extracellular protease and produce a large amount of vitamin K and has probiotic effects, and at the same time uses Bacillus mucilaginosus to increase the hydrolysis of colloidal chitin. Through the coordinated fermentation of different Bacillus strains, it promotes the proliferation of microorganisms and the generation of various biological enzymes. Utilizing the coordinated growth and enzyme-producing ability of multiple microorganisms, on the one hand, it can remove the residual biomass in the shrimp shell and ferment the remaining chitin in situ, and on the other hand, through the growth of microorganisms, it can weaken the natural structure of chitin, and through the chitinase, chitosanase, and oxidase secreted by microorganisms, it can promote the hydrolysis of the surface chitin to obtain substances such as chitooligosaccharide with high immunological activity. It can be seen from Example 2 and Comparative Example 1 that after the coordinated aerobic fermentation of Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus in this application, anaerobic fermentation of Lactobacillus reuteri is carried out. In the anaerobic state, the previous Bacillus strains enter the spore resting state or dissolve, releasing more biological enzymes. Through the fermentation of lactic acid bacteria, the pH of the system decreases, producing organic acids to promote the dissolution of calcium, and at the same time promoting the further release of chitooligosaccharide. The fermentation of lactic acid bacteria also plays a role in correcting the taste of the system and providing various probiotic factors.

Claims

1. A method for preparing chitooligosaccharides by enzymatic hydrolysis, characterized in that, It includes the following steps: (1) Select fresh shrimp shells, clean them, drain them, and then use a roller extrusion device to extrude and crush them to separate the surface organic matter and chitin in the shrimp shells, obtaining preliminarily crushed chitin; (2) Add the obtained preliminarily crushed chitin to sterile water and make a pulp together, control the pulp concentration at 25%, and adjust the pH value of the pulp; (3) Keratinase is used to biochemically hydrolyze the protein components in the slurry, enabling them to fully dissociate from chitin and obtaining polypeptides with anti-stress activity through directional hydrolysis. The keratinase is obtained by fermentation and cultivation of Bacillus pumilus K9. Under the conditions of pH value of 8 - 11 and temperature of 55 - 65 °C, enzymatic hydrolysis is carried out for 2 - 4 h to obtain an enzymatic hydrolysis product, and then heat inactivation is performed; the viable count of the Bacillus pumilus K9 is 10 7 cfu / mL; (4)Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus are configured into a compound bacterial liquid according to a mass ratio of 1:2, and the total viable count of the compound bacterial liquid is 10 8 cfu / mL; enzymolysis is carried out for 2-4 h under the conditions of pH value of 6.8-8.0 and temperature of 30-45 °C to obtain an enzymolysis product, and then heat inactivation is carried out; (5) Use Lactobacillus reuteri to further enzymatically hydrolyze the enzymatic hydrolysate obtained in step (4), enzymatically hydrolyze for 0.5 - 2 h under airtight conditions at a pH value of 2.0 - 6.0 and a temperature of 30 - 50 °C to obtain an enzymatic hydrolysate, and then heat to inactivate; (6) Filter to remove the flocculants in the enzymatic hydrolysis extract, and spray dry to obtain chitooligosaccharide powder; The Bacillus pumilus K9, Bacillus subtilis natto ND-1-A27, Bacillus mucilaginosus, and Lactobacillus reuteri are Bacillus pumilus K9 with a preservation number of CGMCC No. 8046, Bacillus subtilis natto ND-1-A27 with a preservation number of CCTCC No: M 2018131, Bacillus mucilaginosus with a preservation number of CGMCC 5766, and Lactobacillus reuteri with a preservation number of CGMCC No. 15021.

2. The method for preparing chitooligosaccharides by enzymatic hydrolysis according to claim 1, wherein In step (1), 20% - 25% of the total amount of water in the shrimp shells is added during the extrusion process.

3. The method for preparing chito-oligosaccharide based on enzymatic hydrolysis according to claim 1, wherein In steps (3), (4), and (5), after obtaining the enzymatic hydrolysate, heat inactivation is carried out at a temperature of 90 °C for 10 min.

4. The method for preparing chitooligosaccharide based on enzymatic hydrolysis according to claim 1, wherein In step (3), enzymatic hydrolysis is carried out for 2 h under the conditions of a pH value of 9 and a temperature of 60 °C to obtain an enzymatic hydrolysate.

5. The method for preparing chitooligosaccharides by enzymatic hydrolysis according to claim 1, wherein In step (4), enzymatic hydrolysis is carried out for 3 h under the conditions of a pH value of 7.4 and a temperature of 40 °C to obtain an enzymatic hydrolysate.

6. The method for preparing chitooligosaccharides based on enzymatic hydrolysis according to claim 1, characterized in that, In step (5), the viable count of the Lactobacillus reuteri bacterial liquid is 10 7 cfu / mL.

7. The method for preparing chitooligosaccharides by enzymatic hydrolysis according to claim 1, wherein In step (5), enzymatic hydrolysis is carried out for 0.5 h under airtight conditions at a pH value of 5.0 and a temperature of 40 °C to obtain an enzymatic hydrolysate.

8. The method for preparing chito-oligosaccharides by enzymatic hydrolysis according to claim 1, wherein In step (6), spray drying is carried out under the conditions of an inlet air temperature of 140 °C and an outlet air temperature of 70 °C.

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