Probiotics for targeted degradation of rice-derived allergenic protein and application thereof

Abstract

The invention relates to the technical field of targeted protein degradation, and discloses a probiotic for targeted degradation of rice-derived allergenic protein and application, the probiotic is a new sphingomonas strain, the probiotic secretes an extracellular protease compound in the fermentation process, the rice allergenic protein with the molecular weight of 14 kDa and 26 kDa is specifically degraded, the degradation rate is larger than 90%, and the probiotic can be used for degrading the rice-derived allergenic protein in a targeted mode. By introducing a targeted degradation technology, in the process of intervening rice sensitization protein by probiotics, a specifically secreted protease compound is utilized to directly decompose key antigen epitopes of the sensitization protein and reduce immunogenicity of the sensitization protein, and meanwhile, by regulating intestinal immune balance, inhibiting excessive Th2-type immune response and enhancing regulatory T cell activity, the rice sensitization protein can be effectively degraded. The method can be used for blocking the allergy cascade reaction, realizing the dual synergy of high-efficiency elimination and immune tolerance of sensitization protein, breaking through the limitation that traditional probiotics only depend on indirect immune regulation, and improving the desensitization efficiency and safety.

Description

Technical Field

[0001] This invention relates to the field of targeted protein degradation technology, specifically to probiotics that target and degrade rice-derived allergenic proteins and their applications. Background Technology

[0002] Targeted protein degradation technology provides cells with a precise "navigation system" by designing special molecular devices, which can specifically identify and remove disease-related proteins.

[0003] Probiotics that target and degrade rice-derived allergenic proteins are an innovative therapy based on gut microbiota regulation, designed to address food allergies caused by rice protein. Rice, a major global food crop, is widely used in infant formula and special medical diets due to its high nutritional value and easy digestibility. However, many of its allergenic proteins can induce IgE-mediated immediate hypersensitivity reactions and T-cell-mediated delayed hypersensitivity reactions, seriously threatening the health of sensitive individuals. Probiotic therapy, by regulating gut microbiota structure, enhancing intestinal barrier function, and immune balance, has been proven to alleviate food allergy symptoms, making it one of the core strategies for current desensitization therapy.

[0004] Currently, existing probiotic strains used to alleviate food allergies mainly reduce allergic reactions indirectly by regulating the Th1 / Th2 immune balance and increasing the proportion of regulatory T cells. However, these strains have insufficient ability to target and degrade rice-specific allergenic proteins, resulting in limited allergen clearance efficiency.

[0005] Therefore, we propose a probiotic that targets and degrades rice-derived allergenic proteins and its application to address the aforementioned issues. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a probiotic that targets and degrades rice-derived allergenic proteins and its application, thus solving the problems mentioned in the background section.

[0007] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a probiotic that targets and degrades rice-derived allergenic proteins and its application, wherein the probiotic is a new species of Sphingosine monocytogenes; During fermentation, the probiotics secrete extracellular protease complexes that specifically degrade rice allergenic proteins with molecular weights of 14kDa and 26kDa, with a degradation rate of more than 90%. The extracellular protease complex comprises a metalloproteinase and a serine protease, with enzyme activities of 120-150 U / mg and 80-110 U / mg, respectively. The probiotics maintain targeted degradation activity under conditions of pH 5.0-8.0 and temperature 30-42℃, and can withstand the gastric acid environment with a survival rate of more than 85% for 2 hours.

[0008] Preferably, the fermentation medium comprises, by weight percentage: 3-5% rice protein hydrolysate, 1-2% glucose, 0.5-1.5% yeast extract, 0.1-0.3% dipotassium hydrogen phosphate, 0.05-0.1% magnesium sulfate, 0.01-0.03% calcium chloride, with the remainder being water; The rice protein hydrolysate is prepared by hydrolyzing rice gluten with alkaline protease, with a degree of hydrolysis of 15-20%.

[0009] Preferably, the culture conditions are: inoculum size 3-5%, fermentation temperature 35-37℃, initial pH 6.8-7.2, stirring speed 150-200 r / min, dissolved oxygen 20-30%, and fermentation time 18-24 hours; At the end of fermentation, the cell concentration reached 10^9-10^10 CFU / mL.

[0010] Preferably, the extracellular protease complex is extracted by the following method: the supernatant is collected after centrifugation of the fermentation broth, concentrated by ultrafiltration membrane, and then purified by anion exchange chromatography column, with the eluent being a Tris-HCl buffer of 0.1-0.3M NaCl.

[0011] Preferably, it can be prepared as freeze-dried bacterial powder and liquid bacterial agent; The preparation of the freeze-dried bacterial powder includes: mixing bacterial cells and a freeze-drying protectant at a mass ratio of 1:1 to 1:3, wherein the freeze-drying protectant is composed of 5-10% trehalose, 8-12% skim milk powder, and 3-5% glycerol; The number of viable bacteria after freeze-drying is greater than 10^11 CFU / g.

[0012] Preferably, it is applied to any of the following fields: In the food processing industry: Probiotics and their produced protease complexes are added to rice raw materials and treated at 35-40℃ for 1-2 hours to achieve a degradation rate of more than 90% for allergenic proteins; In the pharmaceutical field: it is used as an active ingredient in the preparation of oral desensitizing agents, with a daily intake of 10^8-10^10 CFU; In the feed industry: Add to aquatic animal feed at a rate of 0.1-0.5% to reduce feed allergenicity.

[0013] Preferably, in the food processing field, it is specifically used to prepare hypoallergenic infant rice cereal: rice slurry is mixed with probiotics at a ratio of 100:1 to 100:3, fermented at 37°C for 2-3 hours to inactivate the enzymes, and then spray-dried to obtain the finished rice cereal product; The residual amount of allergenic protein in the finished product is less than 0.5 μg / g.

[0014] Preferably, in the pharmaceutical field, the oral desensitizing preparation is an enteric-coated capsule, comprising: 30-50% probiotic lyophilized powder, 20-30% prebiotics, 5-10% mucosal adhesive, and the remainder being filler; The capsule disintegrates within 30 minutes in simulated intestinal fluid, releasing more than 90% of the live bacteria.

[0015] Preferably, in the feed industry, specifically for desensitization treatment of aquatic feed: the probiotic fermentation liquid is sprayed onto the surface of feed pellets at a rate of 1-2 mL / kg, and the mixture is sealed at 30-35°C for 4-6 hours to reduce the activity of α-amylase inhibitors of allergenic proteins in the feed by 85-95%.

[0016] Preferably, the probiotics and Lactobacillus plantarum CICC23121 are compounded in a live bacteria ratio of 1:1 to 1:2 to synergistically enhance the intestinal barrier function. The combined bacterial flora increased the expression level of tight junction protein in intestinal epithelial cells by 2.5-3.0 times in an in vitro model.

[0017] (III) Beneficial Effects Compared with existing technologies, this invention provides probiotics that target and degrade rice-derived allergenic proteins and their applications, offering the following beneficial effects: 1. In this invention, by introducing targeted degradation technology, during the process of probiotic intervention on rice allergenic proteins, the key antigenic epitopes of the allergenic proteins are directly decomposed by the specifically secreted protease complex, thereby reducing their immunogenicity. At the same time, by regulating the intestinal immune balance, excessive Th2 immune responses are inhibited and regulatory T cell activity is enhanced, thereby blocking the allergic cascade reaction. This achieves a dual synergy of efficient clearance of allergenic proteins and immune tolerance, breaking through the limitations of traditional probiotics that rely solely on indirect immune regulation, and improving desensitization efficiency and safety. 2. In this invention, by constructing a multi-scenario application system, in the food processing field, probiotic fermentation is used to degrade allergenic proteins in rice raw materials in situ, retaining nutrition while eliminating allergy risks. In the oral formulation field, gastric acid protection technology and enteric delivery system are combined to ensure that active bacteria colonize the intestines efficiently and continuously exert desensitization function. In the feed field, surface spraying process is used to achieve precise pretreatment of allergenic proteins, reducing the incidence of allergic diseases in aquatic animals. This forms a full-chain solution covering "food-pharmaceutical-feed", solving the technical bottlenecks of existing technologies such as single application scenarios and incomplete degradation. 3. In this invention, a tiered protection mechanism is designed to optimize the combination of probiotic strains and protease expression activity for different age groups and allergy severity. In the infant stage, the focus is on the deep hydrolysis of allergenic proteins in hypoallergenic complementary foods and the rapid repair of the intestinal barrier. In the adult stage, the induction of immune tolerance and the regulation of chronic inflammation are strengthened. For special populations, a complex microbiota synergistic strategy is adopted to simultaneously degrade allergenic proteins and regulate the microbiota-immune axis homeostasis, thereby achieving personalized desensitization intervention and avoiding the problems of insufficient efficacy and poor adaptability caused by "one-size-fits-all" solutions.

[0018] 4. In this invention, by constructing a gut microbiota-immune axis homeostasis regulatory network, the gut microbiota structure is reshaped and the immune tolerance pathway is activated while targeting and degrading sensitizing proteins. Probiotic metabolites are used to activate dendritic cell GPR43 / GPR109a receptors, promoting regulatory T cell differentiation and IL-10 secretion, inhibiting Th2 immune responses and excessive IgE production, and simultaneously blocking antigen presentation of sensitizing proteins and the allergic inflammatory cascade. By specifically enriching tolerant bacteria such as Akkermania, the expression of tight junction proteins in the intestinal barrier is enhanced, and intestinal mucosal permeability is reduced, forming a long-term desensitization protection mechanism integrating "degradation-barrier-immunity" to reduce the risk of allergy recurrence. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Probiotics that target and degrade rice-derived allergenic proteins and their application; the probiotic is a new species of Sphingosine monocytogenes. During fermentation, probiotics secrete extracellular protease complexes that specifically degrade rice allergenic proteins with molecular weights of 14kDa and 26kDa, with a degradation rate greater than 90%. The extracellular protease complex contains metalloproteinases and serine proteases with enzyme activities of 120 U / mg and 80 U / mg, respectively. The probiotics maintain targeted degradation activity at pH 5.0 and 30℃, and can withstand the acidic environment of the stomach for 2 hours with a survival rate of more than 85%. The fermentation medium consists of the following components by weight percentage: 3% rice protein hydrolysate, 1% glucose, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.01% calcium chloride, and the remainder is water. Rice protein hydrolysate was prepared by hydrolyzing rice gluten with alkaline protease, with a degree of hydrolysis of 15%.

[0021] The culture conditions were: inoculum size 3%, fermentation temperature 35℃, initial pH 6.8, stirring speed 150 r / min, dissolved oxygen 20%, and fermentation time 18 hours. At the end of fermentation, the cell concentration reached 10^9 CFU / mL; Extracellular protease complexes were extracted by the following method: the supernatant was collected after centrifugation of the fermentation broth, concentrated by ultrafiltration, and then purified by anion exchange chromatography column with 0.1M NaCl in Tris-HCl buffer as the eluent. It can be prepared as freeze-dried bacterial powder and liquid bacterial agent; The preparation of freeze-dried bacterial powder includes: mixing bacterial cells and freeze-drying protectant at a mass ratio of 1:1. The freeze-drying protectant is composed of 5% trehalose, 8% skim milk powder, and 3% glycerol. After freeze-drying, the viable bacterial count is greater than 10^11 CFU / g; Applicable to any of the following fields: In the food processing field: adding probiotics and their produced protease complex to rice raw materials and treating them at 35°C for 1 hour resulted in a degradation rate of more than 90% for allergenic proteins. In the pharmaceutical field: it is used as an active ingredient in the preparation of oral desensitizing agents, with a daily intake of 10^8 CFU. In the feed industry: Add to aquatic animal feed at a dosage of 0.1% to reduce feed allergenicity; In the food processing field, it is specifically used to prepare hypoallergenic infant rice cereal: rice slurry is mixed with probiotics at a ratio of 100:1, fermented at 37°C for 2 hours to inactivate the enzymes, and then spray-dried to obtain the finished rice cereal product; The residual amount of allergenic protein in the finished product is less than 0.5 μg / g; In the pharmaceutical field, oral desensitizing preparations are enteric-coated capsules containing: 30% probiotic lyophilized powder, 20% prebiotics, 5% mucosal adhesive, and the remainder being fillers. The capsules disintegrated within 30 minutes in simulated intestinal fluid, with an amplification of over 90% live bacteria. In the feed industry, specifically for desensitization treatment of aquatic feed: the probiotic fermentation liquid is sprayed onto the surface of feed pellets at a rate of 1 mL / kg, and the mixture is sealed at 30°C for 4 hours to reduce the activity of α-amylase inhibitors of allergenic proteins in the feed by 85%. Probiotics and Lactobacillus plantarum CICC23121 are combined in a 1:1 live bacteria ratio to synergistically enhance intestinal barrier function; The combined bacterial flora increased the expression level of tight junction protein in intestinal epithelial cells by 2.5 times in an in vitro model.

[0022] Example 2: Probiotics that target and degrade rice-derived allergenic proteins and their application; the probiotic is a new species of Sphingosine monocytogenes. During fermentation, probiotics secrete extracellular protease complexes that specifically degrade rice allergenic proteins with molecular weights of 14kDa and 26kDa, with a degradation rate greater than 90%. The extracellular protease complex contains metalloproteinases and serine proteases with enzyme activities of 13 U / mg and 90 U / mg, respectively. The probiotics maintained their targeted degradation activity at pH 7.0 and 36°C, and could withstand the acidic environment of the stomach for 2 hours with a survival rate of more than 85%. The fermentation medium consists of the following components by weight percentage: 4% rice protein hydrolysate, 1.5% glucose, 1.0% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.07% magnesium sulfate, 0.02% calcium chloride, and the remainder is water. Rice protein hydrolysate was prepared by hydrolyzing rice gluten with alkaline protease, with a degree of hydrolysis of 18%.

[0023] The culture conditions were: inoculum size 4%, fermentation temperature 36℃, initial pH 7.0, stirring speed 180 r / min, dissolved oxygen 25%, and fermentation time 22 hours. At the end of fermentation, the cell concentration reached 10^9 CFU / mL; Extracellular protease complexes were extracted by the following method: the supernatant was collected after centrifugation of the fermentation broth, concentrated by ultrafiltration, and then purified by anion exchange chromatography column with 0.2M NaCl in Tris-HCl buffer as the eluent. It can be prepared as freeze-dried bacterial powder and liquid bacterial agent; The preparation of freeze-dried bacterial powder includes: mixing bacterial cells and freeze-drying protectant at a mass ratio of 1:2. The freeze-drying protectant is composed of 8% trehalose, 10% skim milk powder, and 4% glycerol. After freeze-drying, the viable bacterial count is greater than 10^11 CFU / g; Applicable to any of the following fields: In the food processing field: adding probiotics and their produced protease complex to rice raw materials and treating them at 38°C for 1 hour resulted in a degradation rate of more than 90% for allergenic proteins. In the pharmaceutical field: it is used as an active ingredient in the preparation of oral desensitizing agents, with a daily intake of 10^9 CFU; In the feed industry: Add to aquatic animal feed at a dosage of 0.3% to reduce feed allergenicity; In the food processing field, it is specifically used to prepare hypoallergenic infant rice cereal: rice slurry is mixed with probiotics at a ratio of 100:2, fermented at 37°C for 2 hours to inactivate the enzymes, and then spray-dried to obtain the finished rice cereal product; The residual amount of allergenic protein in the finished product is less than 0.5 μg / g; In the pharmaceutical field, oral desensitizing preparations are enteric-coated capsules containing: 40% probiotic lyophilized powder, 25% prebiotics, 8% mucosal adhesive, and the remainder being fillers. The capsules disintegrated within 30 minutes in simulated intestinal fluid, with an amplification of over 90% live bacteria. In the feed industry, specifically for desensitization treatment of aquatic feed: probiotic fermentation broth is sprayed onto the surface of feed pellets at a rate of 1.5 mL / kg, and the mixture is sealed at 32°C for 5 hours to reduce the activity of α-amylase inhibitors, which are allergenic proteins, in the feed by 90%. Probiotics and Lactobacillus plantarum CICC23121 are combined in a live bacteria ratio of 1:1.5 to synergistically enhance intestinal barrier function. The combined bacterial flora increased the expression level of tight junction protein in intestinal epithelial cells by 2.8 times in an in vitro model.

[0024] Example 3: Probiotics that target and degrade rice-derived allergenic proteins and their application; the probiotic is a new species of Sphingosine monocytogenes. During fermentation, probiotics secrete extracellular protease complexes that specifically degrade rice allergenic proteins with molecular weights of 14kDa and 26kDa, with a degradation rate greater than 90%. The extracellular protease complex contains metalloproteinases and serine proteases with enzyme activities of 150 U / mg and 110 U / mg, respectively. The probiotics maintained their targeted degradation activity at pH 8.0 and 42°C, and could withstand the acidic environment of the stomach for 2 hours with a survival rate of more than 85%. The fermentation medium consists of the following components by weight percentage: 5% rice protein hydrolysate, 2% glucose, 1.5% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.1% magnesium sulfate, 0.03% calcium chloride, and the remainder is water. Rice protein hydrolysate was prepared by hydrolyzing rice gluten with alkaline protease, with a degree of hydrolysis of 20%.

[0025] The culture conditions were: inoculum size 5%, fermentation temperature 37℃, initial pH 7.2, stirring speed 200 r / min, dissolved oxygen 30%, and fermentation time 24 hours. At the end of fermentation, the cell concentration reached 10^10 CFU / mL; Extracellular protease complexes were extracted by the following method: the supernatant was collected after centrifugation of the fermentation broth, concentrated by ultrafiltration, and then purified by anion exchange chromatography column with 0.3M NaCl in Tris-HCl buffer as the eluent. It can be prepared as freeze-dried bacterial powder and liquid bacterial agent; The preparation of freeze-dried bacterial powder includes: mixing bacterial cells and freeze-drying protectant at a mass ratio of 1:3. The freeze-drying protectant is composed of 10% trehalose, 12% skim milk powder, and 5% glycerol. After freeze-drying, the viable bacterial count is greater than 10^11 CFU / g; Applicable to any of the following fields: In the food processing field: adding probiotics and their produced protease complex to rice raw materials and treating them at 40°C for 2 hours resulted in a degradation rate of more than 90% for allergenic proteins. In the pharmaceutical field: it is used as an active ingredient in the preparation of oral desensitizing agents, with a daily intake of 10^10 CFU. In the feed industry: Add to aquatic animal feed at a rate of 0.5% to reduce feed allergenicity; In the food processing field, it is specifically used to prepare hypoallergenic infant rice cereal: rice slurry is mixed with probiotics at a ratio of 100:3, fermented at 37°C for 3 hours to inactivate enzymes, and then spray-dried to obtain the finished rice cereal product; The residual amount of allergenic protein in the finished product is less than 0.5 μg / g; In the pharmaceutical field, oral desensitizing preparations are enteric-coated capsules, which contain: 50% probiotic lyophilized powder, 30% prebiotics, 10% mucosal adhesive, and the remainder being fillers. The capsules disintegrated within 30 minutes in simulated intestinal fluid, with an amplification of over 90% live bacteria. In the feed industry, specifically for desensitization treatment of aquatic feed: probiotic fermentation broth is sprayed onto the surface of feed pellets at a rate of 2 mL / kg, and the mixture is sealed at 35°C for 6 hours to reduce the activity of α-amylase inhibitors of allergenic proteins in the feed by 95%. Probiotics and Lactobacillus plantarum CICC23121 are combined in a 1:2 live bacteria ratio to synergistically enhance intestinal barrier function; The combined bacterial flora increased the expression level of tight junction protein in intestinal epithelial cells by 3.0 times in an in vitro model.

[0026] Comparative Example 1 differs from Example 1 in that the probiotic used in this comparative example is conventional Lactobacillus rhamnosus CICC23121, which does not secrete a specific protease complex that targets and degrades rice allergenic proteins.

[0027] Comparative Example 2 differs from Example 2 in that the probiotics in this comparative example did not contain a metalloproteinase activator during fermentation, resulting in a decrease in metalloproteinase activity to less than 50 U / mg, while the serine protease activity remained at 80 U / mg.

[0028] Comparative Example 3 differs from Example 3 in that: this comparative example did not use Lactobacillus plantarum CICC23121, but only used a single Sphingomonas strain, and the strain ratio was not optimized.

[0029] Comparative Example 4 differs from Example 3 in that: no mucosal adhesive and prebiotics were added when preparing oral enteric-coated capsules in this comparative example; only freeze-dried bacterial powder and microcrystalline cellulose were added.

[0030] The probiotic preparations and their application effects in Examples 1-3 and Comparative Examples 1-4 were tested. The items and methods are as follows: Allergenic protein degradation rate: The probiotic fermentation broth and rice slurry were mixed at a ratio of 100:2 and reacted at 37°C for 2 hours. The residual amount of 14kDa / 26kDa allergenic protein was detected by SDS-PAGE and ELISA, and the degradation rate was calculated.

[0031] Gastric acid tolerance: The survival rate of live bacteria was determined by plate count after 2 hours of simulated gastric juice treatment.

[0032] Intestinal colonization rate: An in vitro intestinal model was used to detect the number of viable bacteria and colonization stability in the colonic segment 48 hours after drug administration.

[0033] Immunomodulatory activity: Serum IgE levels and intestinal tight junction protein expression were detected by ELISA using a mouse allergy model.

[0034]

[0035] Comparison and analysis of the data in the table show that the probiotics using methods 1-3 to target and degrade rice-derived allergenic proteins exhibit significantly superior performance compared to the probiotics using methods 1-4. This indicates that by introducing targeted degradation technology, during the probiotic intervention with rice-derived allergenic proteins, the key antigenic epitopes of the allergenic proteins are directly decomposed using specifically secreted protease complexes, reducing their immunogenicity. Simultaneously, by regulating intestinal immune balance, inhibiting excessive Th2 immune responses, and enhancing regulatory T cell activity, the allergic cascade reaction is blocked, achieving efficient clearance and immune tolerance of the allergenic proteins. This dual-synergistic approach overcomes the limitations of traditional probiotics that rely solely on indirect immune regulation, enhancing desensitization efficiency and safety. By constructing a multi-scenario application system, in the food processing field, probiotic fermentation degrades allergenic proteins in rice raw materials in situ, preserving nutrients while eliminating allergy risks. In the oral formulation field, it combines gastric acid protection technology with an enteric delivery system to ensure efficient colonization of active bacteria in the gut and sustained desensitization function. In the feed field, surface spraying technology enables precise pretreatment of allergenic proteins, reducing the incidence of allergic diseases in aquatic animals. This forms a complete chain solution covering "food—pharmaceuticals—feed," effectively addressing allergens. To overcome the limitations of existing technologies, such as limited application scenarios and incomplete degradation, this approach designs a tiered protection mechanism. Targeting different age groups and allergy severity, it optimizes probiotic strain combinations and protease expression activity. For infants, the focus is on deep hydrolysis of allergenic proteins in hypoallergenic complementary foods and rapid repair of the intestinal barrier. For adults, it strengthens immune tolerance induction and chronic inflammation regulation. For special populations, a synergistic strategy involving a complex microbiota is employed to simultaneously degrade allergenic proteins and regulate the microbiota-immune axis homeostasis, achieving personalized desensitization intervention. This avoids the insufficient efficacy and poor adaptability issues caused by a "one-size-fits-all" approach, and achieves this by constructing a microbiota-immune axis homeostasis regulation mechanism. This biosynthetic network, while targeting and degrading sensitizing proteins, restructures the gut microbiota and activates the immune tolerance pathway. It utilizes probiotic metabolites to activate dendritic cell GPR43 / GPR109a receptors, promoting regulatory T cell differentiation and IL-10 secretion, inhibiting Th2 immune responses and excessive IgE production, and simultaneously blocking antigen presentation of sensitizing proteins and the allergic inflammatory cascade. Through specific enrichment of tolerant bacteria such as Akkermansia, it enhances the expression of tight junction proteins in the intestinal barrier, reduces intestinal mucosal permeability, and forms a long-lasting desensitization and protection mechanism integrating degradation, barrier, and immunity, thereby reducing the risk of allergy recurrence. By comparing and analyzing the relevant data in the table, it can be seen that the probiotics of the present invention that target and degrade rice-derived allergenic proteins have good degradation, applicability and desensitization properties. This indicates that the probiotics of the present invention that target and degrade rice-derived allergenic proteins have a broader market prospect and are more suitable for promotion.

[0036] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A probiotic that targets and degrades rice-derived allergenic proteins, characterized in that: The probiotics are a new species of the genus Sphingosine Monoclonal; During fermentation, the probiotics secrete extracellular protease complexes that specifically degrade rice allergenic proteins with molecular weights of 14kDa and 26kDa, with a degradation rate of more than 90%. The extracellular protease complex comprises a metalloproteinase and a serine protease, with enzyme activities of 120-150 U / mg and 80-110 U / mg, respectively. The probiotics maintain targeted degradation activity under conditions of pH 5.0-8.0 and temperature 30-42℃, and can withstand the gastric acid environment with a survival rate of more than 85% for 2 hours.

2. The probiotic for targeted degradation of rice-derived allergenic proteins according to claim 1, characterized in that, The fermentation medium consists of the following components by weight percentage: 3-5% rice protein hydrolysate, 1-2% glucose, 0.5-1.5% yeast extract, 0.1-0.3% dipotassium hydrogen phosphate, 0.05-0.1% magnesium sulfate, 0.01-0.03% calcium chloride, with the remainder being water. The rice protein hydrolysate is prepared by hydrolyzing rice gluten with alkaline protease, with a degree of hydrolysis of 15-20%.

3. The probiotic for targeted degradation of rice-derived allergenic proteins according to claim 1, characterized in that, The cultivation conditions are as follows: inoculum size 3-5%, fermentation temperature 35-37℃, initial pH 6.8-7.2, stirring speed 150-200 r / min, dissolved oxygen 20-30%, and fermentation time 18-24 hours. At the end of fermentation, the cell concentration reached 10^9-10^10 CFU / mL.

4. The probiotic for targeted degradation of rice-derived allergenic proteins according to claim 1, characterized in that, The extracellular protease complex was extracted by the following method: the supernatant was collected after centrifugation of the fermentation broth, concentrated by ultrafiltration, and then purified by anion exchange chromatography column with Tris-HCl buffer containing 0.1-0.3M NaCl as the eluent.

5. The probiotic for targeted degradation of rice-derived allergenic proteins according to claim 1, characterized in that, It can be prepared as freeze-dried bacterial powder and liquid bacterial agent; The preparation of the freeze-dried bacterial powder includes: mixing bacterial cells and a freeze-drying protectant at a mass ratio of 1:1 to 1:3, wherein the freeze-drying protectant is composed of 5-10% trehalose, 8-12% skim milk powder, and 3-5% glycerol; The number of viable bacteria after freeze-drying is greater than 10^11 CFU / g.

6. The application of the probiotics as described in claims 1-5 in the degradation of rice allergenic proteins, characterized in that, Applicable to any of the following fields: In the food processing industry: Probiotics and their produced protease complexes are added to rice raw materials and treated at 35-40℃ for 1-2 hours to achieve a degradation rate of more than 90% for allergenic proteins. In the pharmaceutical field: it is used as an active ingredient in the preparation of oral desensitizing agents, with a daily intake of 10^8-10^10 CFU; In the feed industry: Add to aquatic animal feed at a rate of 0.1-0.5% to reduce feed allergenicity.

7. The application of the probiotics according to claim 6 in the degradation of rice allergenic proteins, characterized in that, In the food processing field, it is specifically used to prepare hypoallergenic infant rice cereal: rice slurry is mixed with probiotics at a ratio of 100:1 to 100:3, fermented at 37°C for 2-3 hours to inactivate the enzymes, and then spray-dried to obtain the finished rice cereal. The residual amount of allergenic protein in the finished product is less than 0.5 μg / g.

8. The application of the probiotics according to claim 6 in the degradation of rice allergenic proteins, characterized in that, In the pharmaceutical field, the oral desensitizing preparation is an enteric-coated capsule containing: 30-50% probiotic lyophilized powder, 20-30% prebiotics, 5-10% mucosal adhesive, and the remainder being filler. The capsule disintegrates within 30 minutes in simulated intestinal fluid, releasing more than 90% of the live bacteria.

9. The application of the probiotics according to claim 6 in the degradation of rice allergenic proteins, characterized in that, In the feed industry, specifically for desensitization treatment of aquatic feed: the probiotic fermentation liquid is sprayed onto the surface of feed pellets at a rate of 1-2 mL / kg, and the mixture is sealed at 30-35℃ for 4-6 hours to reduce the activity of α-amylase inhibitors of allergenic proteins in the feed by 85-95%.

10. The application of the probiotics according to claim 6 in the degradation of rice allergenic proteins, characterized in that: The probiotics and Lactobacillus plantarum CICC23121 are combined in a live bacteria ratio of 1:1 to 1:2 to synergistically enhance the intestinal barrier function. The combined bacterial flora increased the expression level of tight junction protein in intestinal epithelial cells by 2.5-3.0 times in an in vitro model.

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