Geobacillus stearothermophilus cKM-Geo and application thereof, shrimp shell oligosaccharide peptide compound and preparation method thereof

Abstract

The invention relates to the technical field of food, in particular to geobacillus stearothermophilus cKM-Geo, application of the geobacillus stearothermophilus cKM-Geo, a shrimp shell oligosaccharide peptide compound and a preparation method of the shrimp shell oligosaccharide peptide compound. The invention provides geobacillus stearothermophilus cKM-Geo, the strain can grow in an oxygen-free environment, ventilation is not needed in the process, and proteolytic enzyme, cellulase, lipase and chitin deacetylase can be highly secreted and expressed; the invention further provides a preparation method for preparing the shrimp shell oligosaccharide peptide by utilizing geobacillus stearothermophilus cKM-Geo fermentation coupling steam explosion treatment, chitin and protein in the shrimp shell are converted into water-soluble oligopeptide and oligosaccharides, the water-soluble oligopeptide and oligosaccharides are separated from calcium carbonate in the shrimp shell, acid and alkali do not need to be used, in the process, waste water is little, pollution is little, and the method is suitable for industrial production. The resource utilization rate is high.

Description

Technical Field

[0001] This invention relates to the field of food technology, and in particular to a thermophilic Bacillus stearothermophilus cKM-Geo and its applications, a shrimp shell oligosaccharide peptide complex and its preparation method. Background Technology

[0002] Chitin is the most abundant naturally occurring nitrogen-containing organic compound on Earth besides protein. It is a polymer composed of 1,000 to 3,000 acetylglucosamine residues linked together by p1,4 glycoside chains. It is widely found in the shells or cuticles of invertebrates, especially shrimp and crabs. The annual total output of crayfish farming in China reaches 2.6336 million tons. The inedible parts such as the head and shell account for about 70% to 80% of the crayfish's body weight. The main components are calcium carbonate (55%), protein (25%), chitin (19%), and small amounts of fat, astaxanthin, etc. The combination of protein, calcium carbonate, and chitin forms a dense layered crystal structure, making chitin difficult to dissolve in water and stable in acid, alkali, and heat.

[0003] Chitosan is obtained by removing more than 55% of the N-acetyl groups from chitin. It has the effects of improving immunity, activating cells, preventing cancer, lowering blood lipids, lowering blood pressure, anti-aging, and regulating the body environment. It can be used in the fields of medicine, health care, food and environmental protection. However, chitosan can only be dissolved in dilute acid solutions, which limits its application.

[0004] Chitosan, after enzymatic treatment, yields chitosan oligosaccharides, with a molecular weight of approximately 3000 Da and a degree of polymerization of 2-20. Chitosan oligosaccharides are completely soluble in water and are the only known basic, positively charged oligosaccharides. They can maintain their structural integrity in the gastrointestinal tract, enter the bloodstream, and reach the whole body to exert their numerous biological functions. The Ministry of Agriculture and Rural Affairs' "Catalogue of Feed Additives" (2013 edition) approved chitosan oligosaccharides and chitosan oligosaccharides as feed additive ingredients. The National Health and Family Planning Commission's Announcement No. 6 of 2014 also approved chitosan oligosaccharides as a new food ingredient.

[0005] Currently, the preparation of chitosan oligosaccharides from chitosan mostly employs bio-enzymatic catalysis, a mild and environmentally friendly process. However, the preparation of chitin and chitosan often uses chemical methods, which involve high concentrations of alkali, long reaction times, unstable product quality, large amounts of wastewater, severe environmental pollution, and protein degradation that prevents recycling. Some innovative processes combine fermentation and enzyme catalysis to achieve simultaneous extraction of calcium, protein, and chitosan (chitosan oligosaccharides). For example, Chinese patent CN104046666A discloses a method for preparing chitosan through fermentation and enzymatic hydrolysis, which uses lactic acid bacteria fermentation for decalcification, protease hydrolysis of protein, and hot alkaline solution for deacetylation to prepare chitosan; and Chinese patent CN118389333B discloses a strain of *Lactobacillus plantarum* CJL-LP4 and its application, which discloses the application of fermentation to remove calcium carbonate using the patented *Lactobacillus plantarum* strain CJL-LP4. Chinese Patent CN110093387A discloses a method for preparing chitosan oligosaccharides by synergistic degradation of chitin in shrimp and crab shells using Bacillus and Paecilomyces. The method utilizes decalcified shrimp shells and Bacillus and Paecilomyces to synergistically degrade chitin in the shells to prepare chitosan oligosaccharides. Chinese Patent CN115261430A discloses a method for preparing chitosan oligosaccharides based on enzymatic hydrolysis. This method employs keratinase hydrolysis for targeted release, multi-strain co-fermentation, and combined bacterial-enzyme use to efficiently recover remaining protein and activate soluble chitosan oligosaccharides in the byproducts.

[0006] In existing production technologies, chemical methods cause significant pollution and are unsustainable; commercial enzymes are expensive and difficult to obtain; the dense layered crystal structure and stable structure caused by intermolecular hydrogen bonding of natural chitin make it difficult for enzymes or bacteria to function; most strains do not secrete chitin deacetylase, making it impossible to achieve efficient preparation of oligosaccharide peptides from shrimp shell resources; conventional fermentation temperatures are around 30℃, which easily leads to contamination by other microorganisms; the products must undergo strict separation and purification processes before they can be used in food or require strict control of the aseptic fermentation environment, resulting in increased production costs. Therefore, there is an urgent need for a green and efficient method for preparing shrimp shell oligosaccharide peptides. Summary of the Invention

[0007] The purpose of this invention is to provide a thermophilic Bacillus steatophilus cKM-Geo strain that can highly secrete and express proteolytic enzymes, cellulases, lipases, and chitin deacetylases. Using this strain, fermentation coupled with steam explosion treatment can be used to prepare shrimp shell oligosaccharide peptide complexes in a green and efficient manner.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a thermophilic Bacillus stearothermophilus cKM-Geo, the Latin name of which is... Geobacillus stearothermophiluscKM-Geo is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO:M20251776.

[0009] The present invention also provides the application of the aforementioned thermophilic Bacillus stearothermophilus cKM-Geo in the preparation of shrimp shell oligosaccharide peptides.

[0010] This invention also provides a method for preparing a shrimp shell oligosaccharide peptide complex, comprising the following steps: (1) Crush and wash the shrimp shells to obtain shrimp shell particles; (2) The shrimp shell particles are subjected to steam explosion treatment for 10~80s to obtain the treated shrimp shells; (3) The treated shrimp shells and thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution were mixed and fermented to obtain fermentation liquid; (4) After centrifuging the fermentation broth, the supernatant was filtered and dried to obtain the shrimp shell oligosaccharide peptide complex. The thermophilic terbinafine bacillus cKM-Geo bacterial solution is obtained by expanding the thermophilic terbinafine bacillus cKM-Geo.

[0011] Preferably, in step (1), the cleaning method is high-pressure spraying, with a water temperature of 50~60℃, a pressure of 3~4 bar, and a water volume of 2~3 times the weight of the shrimp shell.

[0012] Preferably, in step (1), the crushing is extrusion crushing; the particle size of the shrimp shell particles is 5~20mm.

[0013] Preferably, in step (2), the temperature of the steam explosion is 110~140℃.

[0014] Preferably, in step (3), the viable count of the *Bacillus stearothermophilus* cKM-Geo bacterial solution is 2.1~3.7×10⁻⁶. 9 cfu / ml; the amount of the thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution added is 4~10% of the weight of the treated shrimp shells.

[0015] Preferably, in step (3), the fermentation temperature is 50~60℃ and the fermentation time is 36~72h.

[0016] Preferably, in step (4), the centrifugation speed is 13000~17000 rpm; The filtration is either candle filtration or ceramic membrane filtration; the drying is spray drying.

[0017] The present invention also provides the preparation method described above for preparing shrimp shell oligosaccharide peptide complex.

[0018] Beneficial effects: This invention provides a thermophilic Bacillus stearothermophilus cKM-Geo strain that can grow in an anaerobic environment without the need for ventilation and can highly secrete and express proteolytic enzymes, cellulases, lipases, and chitin deacetylases. When applied to the preparation of oligosaccharide peptides, it has a stronger conversion ability than natural strains, making it possible to achieve large-scale and efficient production of oligosaccharide peptides. This invention also provides a method for preparing shrimp shell oligosaccharide peptide complexes. Extrusion processing removes most of the attached fat and protein, resulting in a final product with low fat content, good sensory properties, and a long shelf life. The particle size of the crushed shrimp shells is controlled to be 5-20 mm, which facilitates heat and mass transfer during fermentation. Steam explosion treatment is then performed, which breaks down the dense structure of chitin, protein, and calcium carbonate, promoting subsequent catalytic reactions without causing excessive denaturation of proteins and chitin. Fermentation is then carried out using *Bacillus stearothermophilus* cKM-Geo at 50-60°C, resulting in no contamination from other microorganisms and strong conversion ability, significantly higher than that of natural strains, making large-scale, efficient production possible. This invention, through fermentation coupled with steam explosion treatment, converts chitin and protein in shrimp shells into water-soluble oligopeptides and oligosaccharides, separating them from the calcium carbonate in the shrimp shells. No acids or alkalis are needed, resulting in less wastewater, less pollution, and high resource utilization.

[0019] The shrimp shell oligosaccharide peptide complex prepared by this invention can significantly proliferate beneficial intestinal flora and inhibit the growth of harmful flora. It also has high solubility and good safety, and can be widely used in various dosage forms such as general foods, health foods and functional foods for intestinal regulation. The shrimp shell oligosaccharide peptide complex has good acid-base and heat stability, strong processing and application performance, and can significantly improve economic benefits. Attached Figure Description

[0020] Figure 1 The enzyme activity change curve during the fermentation process described in Example 2; Figure 2 The curve showing the change in enzyme activity during the fermentation process described in Example 3.

[0021] Preservation Instructions

[0022] Thermophilic Bacillus stearothermophilus cKM-Geo, Latin name Geobacillus stearothermophilus cKM-Geo is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO:M 20251776. Detailed Implementation

[0023] This invention provides a thermophilic Bacillus stearothermophilus cKM-Geo, the Latin name of which is... Geobacillus stearothermophilus cKM-Geo is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO:M20251776.

[0024] The present invention also provides the application of the aforementioned thermophilic Bacillus stearothermophilus cKM-Geo in the preparation of shrimp shell oligosaccharide peptides; Thermophilic Bacillus stearothermophilus cKM-Geo can grow in an anaerobic environment without the need for ventilation, and it can secrete and express proteolytic enzymes, cellulases, lipases, and chitin deacetylases at high rates. Compared with natural strains, it has a strong transformation ability, which makes it possible to achieve large-scale and efficient production of oligosaccharide peptides.

[0025] This invention also provides a method for preparing a shrimp shell oligosaccharide peptide complex, comprising the following steps: (1) Crush and clean the shrimp shells to remove most of the organic matter and impurities such as fat and protein attached to the surface of the shrimp shells, and obtain shrimp shell particles; (2) The shrimp shell particles are subjected to steam explosion treatment for 10~80s to destroy the dense layered crystal structure formed by the combination of protein, calcium carbonate and chitin in the shrimp shell, and the treated shrimp shell is obtained. (3) The treated shrimp shells and thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution were mixed and fermented to obtain fermentation liquid; (4) After centrifuging the fermentation broth, the supernatant was filtered and dried to obtain the shrimp shell oligosaccharide peptide complex. The thermophilic terbinafine bacillus cKM-Geo bacterial solution is obtained by expanding the thermophilic terbinafine bacillus cKM-Geo.

[0026] In this invention, in step (1), the cleaning method is high-pressure spraying, the water temperature is 50~60℃, preferably 53~57℃, more preferably 55℃, the pressure is 3~4 bar, preferably 3.3~3.7 bar, more preferably 3.5 bar, and the water volume is 2~3 times the weight of the shrimp shell, preferably 2.5 times; The crushing is performed by compression crushing; the particle size of the shrimp shell particles is 5~20mm.

[0027] In this invention, in step (2), the temperature of the steam explosion is 110~140℃, preferably 120~130℃, and more preferably 125℃; the retention time of the steam explosion is preferably 30~60s, and more preferably 45s.

[0028] In this invention, in step (3), before mixing and fermenting the treated shrimp shells and the thermophilic Bacillus stearothermophilus cKM-Geo bacterial liquid, the treated shrimp shells need to be cooled to 50~60℃, preferably 53~57℃, and more preferably 55℃. The viable count of the *Bacillus steatophilus* cKM-Geo bacterial suspension was 2.1–3.7 × 10⁻⁶. 9 The cfu / ml concentration is preferably 2.5~3.3×10⁻⁶. 9 The cfu / ml concentration is further optimized to be 2.9 × 10⁻⁶. 9 cfu / ml; the amount of the thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution added is 4-10% of the weight of the treated shrimp shells, preferably 6-8%, and more preferably 7%; The fermentation temperature is 50~60℃, preferably 53~57℃, and more preferably 55℃; the fermentation time is 36~72h, preferably 45~63h, and more preferably 54h.

[0029] In this invention, during the fermentation process, the peak activity of proteolytic enzymes is ≥10500U / ml, the peak activity of cellulase is ≥1000U / ml, the peak activity of lipase is ≥400U / ml, and the peak activity of chitin deacetylase is ≥260U / ml.

[0030] In this invention, in step (4), the centrifugation speed is 13000~17000 rpm, preferably 14000~16000 rpm, and more preferably 15000 rpm; the centrifugation is performed using a high-speed tubular centrifuge.

[0031] The pH of the supernatant needs to be adjusted and the solution inactivated before filtration. Adjust the pH of the supernatant to 4.2-4.8, preferably 4.4-4.6, and more preferably 4.5; The acid used to adjust the pH is citric acid or lactic acid; The inactivation step is to keep the temperature at 90°C for 15-30 minutes, preferably 20-25 minutes, and more preferably 22.5 minutes. The filtration is either candle filtration or ceramic membrane filtration; The specific steps of the candle filtration are as follows: the filter pore size is 0.22 micrometers, a 1 mm thick diatomaceous earth is pre-coated as a filter cake before filtration, and the supernatant is filtered after the filtrate is clear and transparent. The ceramic membrane filter has a particle size of 100 nm. The drying process is spray drying. The inlet air temperature of the spray dryer is 190~200℃, more preferably 193~197℃, and more preferably 195℃; The outlet air temperature of the spray dryer is 80~92℃, more preferably 83~89℃, and more preferably 86℃; The homogenization pressure of the spray drying is 60-70 bar, more preferably 63-67 bar, and even more preferably 65 bar.

[0032] The present invention also provides the preparation method described above for preparing shrimp shell oligosaccharide peptide complex; The shrimp shell oligosaccharide peptide complex contains at least one of the following characteristics: ① The shrimp shell oligosaccharide peptide complex has a protein content of ≥40%, wherein the oligopeptide content is ≥90%, the content of peptides ≤1000Da is ≥75%, the ash content is ≤5%, and the fat content is ≤0.55%; ②The shrimp shell oligosaccharide peptide complex has an amino oligosaccharide content of ≥50%, of which the disaccharide-decapsulose content is ≥85% and the degree of deacetylation is ≥90%; ③The top five amino acids in the shrimp shell oligosaccharide peptide complex are histidine, glutamic acid, aspartic acid, leucine and glycine, in that order.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Isolation and screening of strains

[0035] 50g of crayfish shells were collected from the center of the crayfish shell collection site and placed in 250mL of sterile physiological saline. The mixture was then placed on a shaker at 150rpm for 30min, centrifuged to remove the precipitate, and the supernatant was placed in an 80℃ water bath for 1h. 1mL of the bacterial suspension was then inoculated into sterile and cooled LB liquid medium (5g / L yeast extract, 10g / L peptone, 10g / L sodium chloride) at an inoculation rate of 1% (v / v). The culture temperature was 60℃ and the shaker speed was 150rpm for 24h to obtain the enriched solution. The enrichment solution was serially diluted appropriately and spread on LB solid plates (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder). The plates were incubated upside down at 60°C for 24 h. Single colonies with different morphologies were picked and streaked on LB solid plates under the same conditions and incubated upside down for 24 h. Single colonies that grew after streaking were picked to prepare glycerol tube cultures and stored at 80°C. Single colonies that grew after streaking were selected and inoculated into LB liquid medium. The cultures were incubated at 60℃ and 120 rpm for 24 h to obtain the fermentation broth. The activities of proteolytic enzymes, lipases, cellulases, and chitin deacetylases in the fermentation broth were measured. A strain with high overall efficiency in producing proteolytic enzymes, lipases, cellulases, and chitin deacetylases was obtained and named cKM-Geo. Morphological, physiological, biochemical, and 16S rDNA identification confirmed it as *Bacillus stearothermophilus*. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO: M20251776. The method for determining protease activity was GB / T 28715-2012 Spectrophotometric Method for Determination of Acidic and Neutral Protease Activity in Feed Additives; the method for determining lipase activity was GB / T 23535-2009 Lipase Preparations; the method for determining cellulase activity was QB / T 2583-2023 Cellulase Preparations; and the method for determining chitin deacetylase was performed using a Plant Chitin Deacetylase (CDA) ELISA kit.

[0036] Example 2: A shrimp shell oligosaccharide peptide complex

[0037] Thermophilic Bacillus stearothermophilus cKM-Geo bacterial suspension: The strain described in Example 1 was inoculated into seed liquid (the seed liquid culture medium and expansion culture medium include the following components: glucose 1%, corn steep liquor 3%, soybean meal 2%, calcium sulfate 0.2%, magnesium sulfate 0.05%), and expanded at 58°C for 48 h to obtain thermophilic Bacillus stearothermophilus cKM-Geo bacterial suspension; (1) Fresh shrimp shells are crushed by a tracked extrusion crusher and simultaneously washed with 50°C hot water at 3 bar pressure, which is 3 times the weight of the shrimp shells. After washing, there are no obvious attached organic matter and impurities such as fat and protein on the surface of the shrimp shells, and shrimp shell particles are obtained. The particle size of the shrimp shell particles is between 5 and 10 mm. (2) The shrimp shell particles were subjected to steam explosion treatment at a temperature of 110°C and a retention time of 80s to obtain the treated shrimp shells. (3) After cooling the treated shrimp shells to 60℃, inoculate them with 4% of the weight of the treated shrimp shells with a bacterial suspension of Bacillus stearothermophilus cKM-Geo (with a viable count of 2.7 × 10⁻⁶). 9 Mix (cfu / ml) and ferment at 60℃ for 72 hours to obtain fermentation broth; (4) The fermentation broth was centrifuged at 15,000 rpm using a high-speed tubular centrifuge, and the supernatant was collected. The pH of the supernatant was adjusted to 4.2 with citric acid solution, and then inactivated at 90°C for 30 min. The supernatant was then filtered using a candle filter with a pore size of 0.22 micrometers. Before filtration, a 1 mm thick diatomaceous earth was pre-coated as a filter cake. The supernatant was filtered after the filtrate was refluxed until it was clear and transparent. The clarified filtrate was spray-dried with an inlet air temperature of 190°C, an outlet air temperature of 92°C, and a homogenization pressure of 60 bar to obtain the shrimp shell oligosaccharide peptide complex.

[0038] Example 3: A shrimp shell oligosaccharide peptide complex

[0039] The preparation of thermophilic Bacillus steatis cKM-Geo bacterial suspension is described in Example 2; (1) Fresh shrimp shells are crushed by a tracked extrusion crusher and simultaneously washed with 50°C hot water at 4 bar pressure with 3 times the weight of the shrimp shells. After washing, there are no obvious attached organic matter and impurities such as fat and protein on the surface of the shrimp shells, and shrimp shell particles are obtained with a particle size of 10~20mm. (2) The shrimp shell particles were subjected to steam explosion treatment at a temperature of 140°C and a holding time of 10s to obtain the treated shrimp shells. (3) After cooling the treated shrimp shells to 50℃, inoculate them with 10% of the weight of the treated shrimp shells with a bacterium tumefaciens cKM-Geo bacterial solution (with a viable count of 2.7 × 10⁻⁶). 9 Mix (cfu / ml) and ferment at 50℃ for 36 hours to obtain fermentation broth; (4) The fermentation broth was centrifuged at 15,000 rpm using a high-speed tubular centrifuge, and the supernatant was collected. The pH of the supernatant was adjusted to 4.8 with lactic acid solution, and then inactivated at 90°C for 15 min. The supernatant was then filtered using a candle filter with a pore size of 0.22 micrometers. Before filtration, a 1 mm thick diatomaceous earth was pre-coated as a filter cake. The supernatant was filtered after the filtrate was refluxed until it was clear and transparent. The clarified filtrate was spray-dried with an inlet air temperature of 195°C, an outlet air temperature of 85°C, and a homogenization pressure of 65 bar to obtain the shrimp shell oligosaccharide peptide complex.

[0040] Example 4: A shrimp shell oligosaccharide peptide complex

[0041] The preparation of thermophilic Bacillus steatis cKM-Geo bacterial suspension is described in Example 2; (1) Fresh shrimp shells are crushed by a tracked extrusion crusher and simultaneously washed with 50°C hot water at 3.6 bar pressure, twice the weight of the shrimp shells. After washing, there are no obvious attached organic matter and impurities such as fat and protein on the surface of the shrimp shells, and shrimp shell particles are obtained. The particle size of the shrimp shell particles is between 8 and 15 mm. (2) The shrimp shell particles were subjected to steam explosion treatment at a temperature of 120°C and a retention time of 65s to obtain the treated shrimp shells. (3) After cooling the treated shrimp shells to 55℃, inoculate them with 6% of the weight of the treated shrimp shells with a bacterial suspension of Bacillus stearothermophilus cKM-Geo (viable count of 2.7×10⁻⁶). 9 Mix (cfu / ml) and ferment at 55℃ for 60 hours to obtain fermentation broth; (4) The fermentation broth was centrifuged at 15,000 rpm using a high-speed tubular centrifuge, and the supernatant was collected. The pH of the supernatant was adjusted to 4.5 with lactic acid solution, and then inactivated at 90℃ for 20 min. After filtration through a 100 nm ceramic membrane, it was spray-dried with an inlet air temperature of 200℃, an outlet air temperature of 80℃, and a homogenization pressure of 70 bar to obtain the shrimp shell oligosaccharide peptide complex.

[0042] Comparative Example 1: A method for preparing chitosan oligosaccharides based on enzymatic hydrolysis (CN115261430A)

[0043] (1) Select fresh shrimp shells and wash them 2-3 times. After draining, use a roller extrusion device to crush them. Add water accounting for 25% of the total amount of shrimp shells during the extrusion process to separate the surface organic matter (protein, fat) and chitin (chitin, calcium, and a small amount of organic matter) in the shrimp shells to obtain the initially crushed chitin. (2) Add the obtained pre-crushed chitin to sterile water and slurry together. The slurry concentration is controlled at about 25%, and the pH value of the slurry is adjusted for later use. (3) The protein components in the slurry were bio-enzymatically hydrolyzed using keratinase to fully release them from the chitin. Targeted hydrolysis yielded stress-resistant polypeptides. The keratinase was obtained through fermentation culture of Bacillus pumilus K9, with a viable count of 10-1. 7 The enzyme was hydrolyzed at a concentration of cfu / mL for 2-4 hours at a pH of 8-11 and a temperature of 55-65℃ to obtain the hydrolysate; then it was inactivated by heating at a temperature of 80-95℃ for 10 minutes. (4) A compound bacterial solution was prepared by mixing Bacillus subtilis natto ND-1-A27 and Bacillus mucilaginosus at a mass ratio of 1:1~2. The total number of viable bacteria in the compound bacterial solution was 10. 8CFU / mL; enzymatic hydrolysis was performed at pH 6.8–8.0 and temperature 30–45℃ for 2–4 h to obtain the enzymatic hydrolysis product; the growth of the complex microorganisms further promoted the hydrolysis of the surface chitin, weakening the natural structure of chitin. Then, the product was inactivated by heating at 80–95℃ for 10 min. (5) The enzymatic hydrolysate obtained in step (4) was further enzymatically hydrolyzed using Lactobacillus reuteri. The viable count of the Lactobacillus reuteri culture was 10. 7 The enzyme was enzymatically hydrolyzed at a concentration of cfu / mL under sealed conditions at pH 2.0-6.0 and a temperature of 30-45℃ for 0.5-2 hours to obtain the enzymatic hydrolysate; then it was heated at a temperature of 80-95℃ for 10 minutes to inactivate the enzyme. (6) Filter to remove flocculent matter from the enzymatic extract, and spray dry under the conditions of inlet temperature of 140℃ and outlet temperature of 70℃ to obtain chitosan oligosaccharide powder. The yield of chitosan oligosaccharide is greater than 15%.

[0044] Comparative Example 2: A method for preparing chitosan oligosaccharides by synergistic degradation of chitin in shrimp and crab shells using Bacillus and Paecilomyces (CN110093387A)

[0045] a. Raw material processing: Shrimp and crab shells are ground in a pulverizer, the pulverized material is decalcified, the precipitate is collected by centrifugation, and the precipitate containing chitin is used as the carbon and nitrogen source for fermentation production. Trace elements are added to make a fermentation culture medium. The fermentation medium consists of: 40g shrimp and crab shell powder, 2g yeast, 0.4g KH2PO4, 0.1g MgSO4, 0.005g FeSO4, 0.25mg ZnSO4, 1000mL water, and pH=5. b. Preparation of bacterial strains: (1) Plate culture: Bacillus was streaked on a double-layer plate medium with chitin as the sole carbon source and cultured at 28°C for 24 hours. After culture, the plate was stored in a refrigerator for later use. Paecilomyces was streaked on a double-layer plate medium with chitin as the sole carbon source and cultured at 23°C for 72 hours. After culture, the plate was stored in a refrigerator for later use. (2) Liquid culture: Pick up the Bacillus strain cultured in (1) with an inoculation loop and inoculate it into 50 mL of liquid culture medium with chitin as the sole carbon source. Culture on a shaker at 28℃ and 150 rpm for 16 h. The cultured strain is ready for use. Pick up the Paecilomyces strain cultured in (1) with an inoculation loop and inoculate it into 50 mL of liquid culture medium with chitin as the sole carbon source. Culture on a shaker at 23℃ and 150 rpm for 24 h. The cultured strain is ready for use. c. Fermentation production The Bacillus and Paecilomyces liquid cultures prepared in step (2) were inoculated into 200 mL of the fermentation medium prepared in step a, with inoculation amounts of 0.8 mL and 0.2 mL, respectively. The cultures were cultured on a shaker at 25 °C and 150 rpm for 36 h. After the chitin was completely degraded, the fermentation was terminated. The cultures were centrifuged and the supernatant was collected to obtain the fermentation product containing chitosan oligosaccharides. d. Purification of chitosan oligosaccharides First, add 1 / 5 column volume of distilled water to the glass chromatography column, open the stopcock at the bottom of the column to expel air bubbles, and then slowly pour the polyacrylamide gel resin soaked in distilled water into the chromatography column while stirring, adding a total of about 340 mL of resin. After the column is packed, turn on the constant flow pump and wash the resin with 2-3 times the volume of 0.1 mol / L NH4HCO3 until the resin is compacted. Dissolve the chitosan oligosaccharide product obtained above in 2 mL of 0.1 mol / L NH4HCO3 solution. After complete dissolution, filter through a 0.22 μm filter membrane, and then slowly add the fermentation product solution along the wall. After the sample is completely added to the column, open the stopcock to allow the solution to flow down. When the sample solution is level with the column surface, turn on the constant flow pump and elute with 0.1 mol / L NH4HCO3 solution. Turn on the automatic fraction collector and collect one tube every 10 min at a certain flow rate. After rotary evaporation and concentration, analyze by TLC and combine the same components. The degree of deacetylation of chitosan trisaccharide was 92.3%, that of chitosan tetrasaccharide was 93.7%, that of chitosan pentasaccharide was 93.4%, and that of chitosan hexasaccharide was 89.2%.

[0046] Experimental Example 1: Enzyme Activity Detection

[0047] Referring to the method described in Example 1, the enzyme activity during the fermentation process in Examples 2 and 3 was detected, and the results are as follows: Figure 1 , Figure 2 As shown; The results showed that when the inoculum size of *Bacillus stearothermophilus* cKM-Geo was 4%, the activities of protease and chitin deacetase reached their highest values ​​after 60 hours of fermentation, and the activities of cellulase and lipase reached their highest values ​​after 66 hours of fermentation. When the inoculum size of *Bacillus stearothermophilus* cKM-Geo was 10%, the activities of protease, cellulase, and lipase reached their highest values ​​after 18 hours of fermentation, and the activities of chitin deacetase reached their highest value after 21 hours of fermentation.

[0048] Experimental Example 2: Component Detection of Shrimp Shell Oligosaccharide Peptide Complex

[0049] The components and contents of the shrimp shell oligosaccharide peptide complex prepared in Example 2 were determined, and the results are shown in Table 1. Table 1. Components of Shrimp Shell Oligosaccharide Peptide Complex

[0050] The results showed that the shrimp shell oligosaccharide peptide complex had a protein content of ≥40%, an ash content of ≤5%, a fat content of ≤0.55%, and an amino oligosaccharide content of ≥50%, of which the disaccharide-decapsulose content was ≥85%, the degree of deacetylation was ≥90%, and the top five amino acids were histidine, glutamic acid, aspartic acid, leucine and glycine. It can be widely used in general foods, health foods and functional foods for intestinal regulation.

[0051] Experimental Example 3: Determination of the molecular weight of protein peptides in shrimp shell oligosaccharide peptide complex

[0052] The molecular weight distribution of the shrimp shell oligosaccharide peptide complex prepared in Example 3 was determined according to Appendix A of QB / T 4588—2013 Freshwater Fish Protein Peptides. The results are shown in Table 2. Table 2. Protein peptide molecular weight distribution of shrimp shell oligosaccharide peptide complex.

[0053] The results showed that the oligopeptide content in the protein of the shrimp shell oligosaccharide peptide complex was ≥90%, and the content of peptides ≤1000Da was ≥75%. The product had a small molecular weight, was easily absorbed, and had high bioavailability.

[0054] Experiment Example 4

[0055] The degree of deacetylation of the amino oligosaccharides in the products prepared by Comparative Example 2, Comparative Example 1, and Comparative Example 2, as well as the preparation process, were compared. The results showed that Comparative Example 1 used multiple enzymes and strains for fermentation and enzymatic hydrolysis, while Comparative Document 2 required decalcification and further separation and purification processes. It is evident that the processes of Comparative Example 1 and Comparative Example 2 are cumbersome. The shrimp shells treated by fermentation and steam explosion of Bacillus stearothermophilus cKM-Geo in this application have a degree of deacetylation of amino oligosaccharides greater than or equal to 91%. The higher the degree of deacetylation, the more amino content of chitosan oligosaccharides, the higher the biological activity, and the better the absorption and utilization rate by the human body. The product is rich in oligopeptides and oligosaccharides, and can be used as a compound prebiotic in general foods, health foods and functional foods for intestinal regulation. Moreover, this application uses shrimp shells fermented and steam-exploded by Bacillus stearothermophilus cKM-Geo to co-produce oligopeptides and amino oligosaccharides. The deacetylation values ​​of the amino oligosaccharides are similar and there is no significant difference. However, the method described in Example 2 does not require chemical decalcification treatment and further separation and purification processes. The protein content of the product is greater than or equal to 42%, the amino oligosaccharide content is greater than or equal to 53%, and the proportion of aminobiose-aminodecanose in the amino oligosaccharides is greater than or equal to 87%. The raw material utilization rate is high, the process is simple and the cost is low, and it is easy to industrialize.

[0056] As can be seen from the above embodiments, the present invention provides *Bacillus stearothermophilus* cKM-Geo and its applications, a shrimp shell oligosaccharide peptide complex and its preparation method. The present invention utilizes *Bacillus stearothermophilus* cKM-Geo fermentation coupled with steam explosion treatment to convert chitin and protein in shrimp shells into water-soluble oligopeptides and oligosaccharides, which are then separated from calcium carbonate in the shrimp shells. This process requires no acid or alkali, generates less wastewater, causes less pollution, and has a high resource utilization rate.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thermophilic Bacillus steatophilus cKM-Geo, characterized in that, The Latin name of the thermophilic strobilurinary spores cKM-Geo is Geobacillus stearothermophilus cKM-Geo is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 5, 2025, with accession number CCTCC NO:M20251776.

2. The application of the thermophilic Bacillus stearothermophilus cKM-Geo as described in claim 1 in the preparation of shrimp shell oligosaccharide peptides.

3. A method for preparing a shrimp shell oligosaccharide peptide complex, characterized in that, Includes the following steps: (1) Crush and wash the shrimp shells to obtain shrimp shell particles; (2) The shrimp shell particles are subjected to steam explosion treatment for 10~80s to obtain the treated shrimp shells; (3) The treated shrimp shells and thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution were mixed and fermented to obtain fermentation liquid; (4) After centrifuging the fermentation broth, the supernatant was filtered and dried to obtain the shrimp shell oligosaccharide peptide complex. The thermophilic geosporin cKM-Geo bacterial culture is obtained by expanding the thermophilic geosporin cKM-Geo as described in claim 1.

4. The preparation method according to claim 3, characterized in that, In step (1), the cleaning method is high-pressure spraying, with a water temperature of 50~60℃, a pressure of 3~4 bar, and a water volume of 2~3 times the weight of the shrimp shell.

5. The preparation method according to claim 3, characterized in that, In step (1), the crushing is extrusion crushing; the particle size of the shrimp shell particles is 5~20mm.

6. The preparation method according to claim 3, characterized in that, In step (2), the temperature of the steam explosion is 110~140℃.

7. The preparation method according to claim 3, characterized in that, In step (3), the viable count of the *Bacillus steatophilus* cKM-Geo bacterial solution is 2.1~3.7×10⁻⁶. 9 cfu / ml; the amount of the thermophilic Bacillus stearothermophilus cKM-Geo bacterial solution added is 4~10% of the weight of the treated shrimp shells.

8. The preparation method according to claim 3, characterized in that, In step (3), the fermentation temperature is 50~60℃ and the fermentation time is 36~72h.

9. The preparation method according to claim 3, characterized in that, In step (4), the centrifugation speed is 13000~17000 rpm; The filtration is either candle filtration or ceramic membrane filtration; the drying is spray drying.

10. The shrimp shell oligosaccharide peptide complex is prepared by the preparation method according to any one of claims 3 to 9.

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