Preparation method and application of deinococcus radiodurans metagen

Abstract

The invention discloses a preparation method and application of a deinococcus radiodurans metagen. The preparation method comprises the following steps: (1) preparing liquid seeds; (2) solid state fermentation: 5-8% (v / w) of the liquid seed liquid obtained in the step (1) is inoculated to a solid fermentation culture medium, the solid fermentation culture medium is prepared from, by mass, 50-60% of soybean meal, 30-40% of sunflower seeds, 4-12% of flaxseed meal and 2-8% of palm pomace, and constant-temperature fermentation is performed at 28-32 DEG C for 48-60 h; and (3) post-treatment: after the fermentation is finished, drying the fermented material at a low temperature of 55-65 DEG C until the moisture is less than or equal to 5% to obtain the deinococcus radiodurans metagen powder. The microencapsulation embedding method comprises the following steps: (1) preparing a wall material solution, (2) dispersing a core material, (3) forming microcapsules, and (4) carrying out post-treatment.The technical problems that active substances in deinococcus radiodurans metabiotics are easy to degrade and low in bioavailability in the pet food processing and digestion process and cannot stably exert the functions of oxidation resistance and flora regulation are solved.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and functional pet food technology, and in particular relates to a microencapsulation technology for metabiotics fermented with *Radiata resistant* and its functional food for regulating the weight of dogs and cats. Background Technology

[0002] Obesity can trigger systemic, mild chronic inflammation. Excessive adipose tissue, especially visceral fat, secretes various pro-inflammatory factors and leads to a large accumulation of reactive oxygen species, causing oxidative stress. Simultaneously, it further damages the integrity of the intestinal barrier, leading to increased intestinal permeability (i.e., the "leaky gut" phenomenon). Changes in intestinal permeability can cause gut microbiota dysbiosis, which in turn produces harmful substances such as endotoxins, exacerbating systemic inflammation and oxidative stress, forming a vicious cycle of "obesity-oxidative stress-gut microbiota imbalance-inflammation," driving further deterioration of metabolic function. In addition to controlling diet and increasing exercise, it is also necessary to alleviate oxidative stress, regulate gut microbiota, and suppress chronic inflammation.

[0003] Deinococcus radiodurans, a microorganism with extremely strong environmental resistance, is widely used in bioremediation and radiation protection research. In recent years, studies have found that this bacterium and its metabolite, Deinococcus radiodurans postbiotics (DRP), have broad application potential in the pharmaceutical, food, and cosmetic fields. Its core component is a unique carotenoid produced by the bacterium—Deinoxanthin (DX). Studies have confirmed that DX has significantly higher antioxidant activity than common antioxidants such as β-carotene and astaxanthin, possessing a highly efficient ability to scavenge free radicals. However, DX, a key component of Deinococcus radiodurans postbiotics (DRP), has extremely poor physicochemical stability. This substance is highly sensitive to heat, oxygen, and acidic environments. In the routine processing of pet food, such as the extrusion process (high temperature and high pressure) of cat food, the steaming and sterilization process of wet food, and the long-term baking process of various snacks, DRP or DX is easily decomposed or oxidized and inactivated. At the same time, the highly acidic environment (pH 1-3) in the stomachs of dogs and cats may also significantly reduce their bioavailability after ingestion.

[0004] If the three core challenges of stability, palatability, and industrialization feasibility of DRP or DX can be solved simultaneously, then DX, a highly promising active substance, can be transformed into a commercial product that can benefit a wide range of pets. Summary of the Invention

[0005] To address the core technical challenges of the active substance Deinoxanthin in *D. radiophila* postbiotics (DRP), including its easy degradation, low bioavailability, and inability to stably exert its antioxidant and gut microbiota-regulating functions during pet food processing and digestion, this invention aims to provide a systematic technology employing a "liquid + solid-state staged fermentation + microencapsulation" approach. This technology optimizes the complex substrate fermentation system to enhance DRP yield and activity, and utilizes a multi-layered synergistic encapsulation system of "chitosan-low-ester pectin-rosemary" to simultaneously improve DRP retention in the acidic gastric environment and enhance its antioxidant stability during storage.

[0006] The technical solution for achieving the objective of this invention is as follows: A method for preparing a postbiotic from *Radiata spp.* includes the following steps: (1) Preparation of liquid seed: Deinococcus radiodurans was inoculated into liquid seed TGY medium and cultured with shaking at a temperature of 28-32℃ and a rotation speed of 200-240 rpm / min until the cells reached the logarithmic growth phase to obtain liquid seed liquid; (2) Solid-state fermentation: The liquid seed liquid obtained in step (1) is inoculated onto a solid fermentation medium at 5–8% (v / w). The solid fermentation medium is composed of 50–60% soybean meal, 30–40% sunflower seeds, 4–12% flaxseed meal, and 2–8% palm fruit pomace by mass percentage. Before inoculation, the medium is sterilized at 121°C for 15 minutes, and the moisture content is controlled at 40–45%. The medium is then fermented at a constant temperature of 28–32°C for 48–60 hours. (3) Post-processing: After fermentation, the fermented material is dried at a low temperature of 55-65℃ until the moisture content is ≤5% to obtain radiation-resistant cytokine post-biotic (DRP) powder.

[0007] The solid fermentation medium described in step (2) consists of 55% soybean meal, 35% sunflower seeds, 6% flaxseed meal, and 4% palm fruit residue.

[0008] A method for microencapsulation of radiation-resistant *Gastrodinium spp.* metabiotics, comprising: (1) Preparation of wall material solution: Mix 0.5–1% (w / v) of modified chitosan, 1.5–2.5% (w / v) of low esterification pectin, and 0.1–0.5% (accounting for the total solids of the wall material) of rosemary extract and dissolve them in water to form a homogeneous aqueous phase; (2) Core material dispersion: The DRP powder is dispersed in the aqueous phase at a core-to-wall ratio of 1:2–1:4 and homogenized to form a suspension; (3) Microcapsule formation: The suspension is sprayed into a 0.1 M calcium chloride solution through a spray drying device, so that the low-esterification degree pectin and calcium ions form an "egg box" structure cross-linking to form microcapsules; (4) Post-processing: Collect the microcapsules, rinse with deionized water, and dry in a fluidized bed to obtain microcapsule powder containing DRP.

[0009] The modified chitosan is N-trimethyl chitosan or acetylated chitosan, with a degree of deacetylation ≥85%.

[0010] A functional pet food comprising an effective amount of the microencapsulated postbiotic, wherein the food is cat food, dog food, or rodent food, and the amount of DRP added is 0.5–2.0% (w / w).

[0011] The amount of DRP added is 1.5% (w / w).

[0012] The feed contains the following ingredients by weight: 60 parts frozen fresh chicken meat, 10 parts frozen fresh duck breast meat, 10 parts bone-in chicken meat, 5 parts enzymatically hydrolyzed chicken liver, 4.5 parts fresh chicken heart, 3 parts chicken oil, 3 parts tapioca starch, 0.5 parts egg yolk powder, 0.5 parts krill powder, and 1.5 parts pet compound additive; the DRP is uniformly sprayed onto the surface of the feed in microencapsulated form.

[0013] The pet compound additive contains taurine, vitamin C, brewer's yeast, light calcium carbonate, citric acid, yeast selenium, vitamin A acetate, vitamin D3, dl-α-tocopherol acetate, thiamine nitrate, vitamin B2, vitamin B6, vitamin B12, D-calcium pantothenate, nicotinamide, folic acid, D-biotin, choline chloride, zinc methionine chelate, iron glycine chelate, and copper glycine chelate.

[0014] The method is used in the preparation of functional pet food for regulating the weight of dogs and cats.

[0015] The beneficial effects of this invention are: This invention focuses on DRP (dihydrotestosterone) fermentation products from *Trichophyton radiata*, forming a complete solution encompassing process innovation, improved product stability, safety and efficacy verification, and the development of functional food formulations for dogs and cats. The traditional single-soybean meal-based fermentation medium has been optimized by innovatively employing a solid multi-layer composite medium, making it more suitable for pet food applications and enabling more efficient preparation of DRP with various biological activities. Based on fruit-based edible coating technology, customized edible functional coatings suitable for dogs and cats have been developed, allowing DRP to better exert its effects in feed (rodent food, cat food), such as anti-oxidation, regulating intestinal flora, and regulating weight. Furthermore, its safety has been scientifically verified, providing strong evidence for its application in pet food and other fields, thereby enhancing the overall value and application scope of the product.

[0016] (1) Improved fermentation efficiency: The composite substrate enabled the viable count of DRP to reach 4.5 × 10⁻⁶. 9CFU / g, 40.6% higher than traditional soybean meal-based; (2) Improved stability: After encapsulation, the retention rate of DX in simulated gastric fluid reached 1529.9 ng / g, which was 375249.3% higher than that of the unencapsulated group and 35.1% higher than that of the sodium alginate system; (3) Functional validation (animal feeding experiment): In the mouse obesity model, 1.5% DRP significantly increased the abundance of Clostridium pluvialis (7.3-fold), butyrate production (35%), intestinal barrier score (37%) and improved body shape score (p<0.05); In the canine and feline obesity models, 1.5% DRP increased the abundance of Clostridium pluvialis (25%) and butyrate production (36%). (4) Good palatability: DRP-containing cat food has a consumption rate of >98% and no refusal to eat. Attached Figure Description

[0017] Figure 1 This is a flowchart of the liquid + solid fermentation and microencapsulated DR bacteria cat food preparation process.

[0018] Figure 2 It is the DPPH free radical scavenging rate (%).

[0019] Figure 3 This describes the effect of different doses of DRP on the body weight of mice.

[0020] Figure 4a The effect of different doses of DRP intervention on the blood biochemistry of mice (serum total cholesterol (TC)).

[0021] Figure 4b The effect of different doses of DRP intervention on blood biochemistry (low-density lipoprotein (LDL)) in mice.

[0022] Figure 4c The effect of different doses of DRP intervention on blood biochemistry (triglycerides (TG)) in mice.

[0023] Figure 5a The effects of different doses of DRP intervention on mouse liver (liver morphology). Figure 5b This describes the effect of different doses of DRP intervention on mouse livers (liver weight).

[0024] Figure 5c This describes the effects of different doses of DRP intervention on mouse liver (liver fat deposition).

[0025] Figure 5d The effect of different doses of DRP intervention on mouse liver (percentage of liver Oil Red O staining).

[0026] Figure 6 This describes the effect of different doses of DRP intervention on fat in mice.

[0027] Figure 7a shows the effect of different doses of DRP intervention on the overall gut microbiota (gut microbiota balance index) in mice.

[0028] Figure 7b This refers to the effect of different doses of DRP intervention on the overall gut microbiota (microbial diversity, MD) in mice.

[0029] Figure 7c This refers to the effect of different doses of DRP intervention on the overall gut microbiota (number of species, NS) in mice.

[0030] Figure 8a This study investigated the effects of different doses of DRP intervention on the phylum of the mouse gut microbiota (Firmicutes).

[0031] Figure 8b This study investigated the effects of different doses of DRP intervention on the phylum of the intestinal flora in mice (Clostridium).

[0032] Figure 8c The effect of different doses of DRP intervention on the phylum of the intestinal flora in mice (Clostridium prausnitzii).

[0033] Figure 8d This study investigated the effects of different doses of DRP intervention on the metabolism of intestinal flora in mice (butyrate).

[0034] Figure 8e This study investigated the effects of different doses of DRP intervention on the metabolism of the intestinal microbiota in mice (Intestinal Barrier Index, IBI).

[0035] Figure 9 This is a top-down view of the cats participating in the feeding experiment.

[0036] Figure 10a It refers to the effect of DRP-containing cat food on the gut microbiota phylum of cats (Gut Microbiota Balance (GMB)).

[0037] Figure 10b This refers to the effect of DRP-containing cat food on the phylum of the cat's gut microbiota (microbial diversity, MD).

[0038] Figure 10cThis refers to the overall effect of feeding cats with DRP-containing cat food on the gut microbiota (number of species, NS).

[0039] Figure 10d It is the effect of DRP-containing cat food on the phylum of the cat's gut microbiota (Clostridium).

[0040] Figure 10e It is the effect of DRP-containing cat food on the phylum of the cat's gut microbiota (F. prausnitzii).

[0041] Figure 10f The effect of DRP-containing cat food on the metabolism of cat gut microbiota ( Figure 10f Butyrate. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0043] Example 1: Fermentation process of radiation-resistant *Gastrodinium radiata* metabiotic (DRP) Step 1: Liquid fermentation and plate preparation Culture medium formulation (TGY): Tryptone 5 g / L, Yeast Extract 3 g / L, Glucose 1 g / L, Agar 15 g / L, pH 7.0 ± 0.2.

[0044] Inoculate DR bacteria onto TGY solid medium plates using an inoculation loop. Culture parameters: Invert the streaked plate in a 28°C incubator and incubate in the dark for 36 hours. After incubation, observe the plate. Plump, smooth, red or pink single colonies should form. Select morphologically typical single colonies with neat edges for subsequent experiments to ensure strain purity and viability. Liquid seed culture: In a clean bench, use a sterile inoculation loop to pick the above single colonies and inoculate them into a sterilized Erlenmeyer flask containing liquid TGY medium. Culture parameters: Temperature: 30°C, shaker. Rotation speed: 220 rpm / min. Culture time: Approximately 12-16 hours, monitored periodically by OD. 600 (Absorbance) is used for precise judgment. When OD 600 When the value reaches 0.8, the bacterial cell activity is at its highest, and this is used as the seed culture.

[0045] Step 2: Fermentation in a multi-layered composite solid culture medium Fermentation substrate formula 1: 60% soybean meal (providing high-quality protein and nitrogen source), 30% sunflower seeds (providing oil, protein and cellulose), 6% flaxseed meal (rich in polysaccharides and residual protein) and 4% palm fruit pomace (providing fiber structure and fermentable sugars). All of the above fermentation substrate raw materials are listed in the "Feed Raw Material Catalog". All raw materials need to be crushed to a suitable particle size (40 mesh) to increase the specific surface area, which is conducive to the attachment and growth of microorganisms.

[0046] Fermentation substrate formula 2: 90% soybean meal (providing high-quality protein and nitrogen source) and 10% corn flour (providing protein and carbon source). All of the above fermentation substrate raw materials are listed in the "Feed Raw Material Catalog". All raw materials need to be crushed to a suitable particle size (40 mesh) to increase the specific surface area, which is conducive to bacterial attachment and growth.

[0047] Liquid seed culture in the logarithmic growth phase was evenly sprayed onto the two sterilized fermentation substrates (121℃, 15 minutes) at a predetermined inoculation rate (5% v / w). Fermentation parameters: temperature controlled at 30℃, moisture content of fermentation material controlled at 42%, constant temperature fermentation for 48 hours. After fermentation, three replicates were taken for each formulation, and the effects of the two fermentation substrates were evaluated using the following parameters (Table 1): ① Microbial growth: endpoint viable count (CFU / g), specific growth rate; ② Matrix transformation: acid-soluble protein / total protein, degradation rate of anti-nutritional factors, and degradation rate of crude fiber; ③ Metabolic activity: pH value variation, total organic acid content; After fermentation, the material is immediately transferred to a 60℃ circulating air oven or fluidized bed dryer for drying. Drying continues until the moisture content of the material is below 5%, and the particle size distribution of the final product is controlled to be 75μm-200μm (about 80 mesh).

[0048]

[0049] The test results are as follows: Regarding microbial growth, Formula 1 provides a superior environment for DR cell growth, specifically reflected in higher cell yield and growth rate. Formula 1 achieved a 40.6% higher DR cell yield, with a endpoint viable count reaching 4.5 × 10⁻⁶. 9 The CFU / g was significantly higher than that of Formula 2 (3.2 × 10⁻⁶). 9 CFU / g (P<0.01). This indicates that the fermentation substrate of Formula 1 has a more comprehensive nutrient composition, supporting a higher cell density. Formula 1 exhibits a faster growth rate, with a specific growth rate of 0.35 h⁻¹, significantly faster than the 0.25 h⁻¹ of Formula 2 (P<0.05). These results demonstrate that in Formula 1, the microbial community can enter the rapid proliferation phase more quickly, shortening the fermentation cycle and improving production efficiency.

[0050] Regarding fermentation substrate conversion, Formula 1 showed better protein quality optimization, with an acid-soluble protein / total protein ratio as high as 28.2%, significantly higher than Formula 2's 20.1% (P<0.001). The results suggest that Formula 1 produced more small peptides and free amino acids after fermentation, greatly improving protein digestibility and absorption, which is crucial for its use as feed. Formula 1 also demonstrated stronger fiber degradation capabilities: the crude fiber degradation rate of Formula 1 was 17.9%, significantly higher than Formula 2's 11.0% (P<0.0001). This indicates that the complex substrate in Formula 1 (such as palm fruit pomace containing soluble sugars) effectively stimulated the cellulase activity of microorganisms, better disrupting plant cell walls. Regarding the degradation of anti-nutritional factors: the degradation rate of Formula 1 (65%) was numerically higher than that of Formula 2 (61%), but there was no statistically significant difference (P>0.05). This indicates that both formulas were similarly effective in degrading such substances, with degradation rates exceeding 60%, meeting the requirements for use as cat food ingredients.

[0051] Regarding metabolic activity: Formula 1 exhibited stronger acid-producing capacity, with an organic acid content of 45.6 mg / g after fermentation, significantly higher than Formula 2's 32.5 mg / g (P<0.001). Organic acids are important prebiotics and can effectively inhibit harmful microorganisms. Formula 1 showed a more significant pH decrease: the pH decrease of Formula 1 (Δ-1.8) was significantly greater than that of Formula 2 (Δ-1.4) (P<0.05). This further confirms that Formula 1 produced more acidic metabolites during fermentation, corroborating the higher organic acid content.

[0052] Example 2: Edible microencapsulation technology for protecting DRP I. Wall Material Selection and Addition Ratio Formula 1: Modified chitosan (0.5%-1%, w / v): a cationic polysaccharide used as the wall material matrix. Its amino groups are protonated under acidic conditions, resisting gastric acid and dissolving and releasing contents in the neutral environment of the intestine; Low-esterification pectin (1.5%-2.5%, w / v) anionic polysaccharide (LMP): forms a polyelectrolyte complex with chitosan through electrostatic interactions and hydrogen bonds, constituting the main framework of the microcapsules. Its low esterification characteristic allows it to undergo ionic cross-linking with Ca²⁺, forming an "egg-box" structure, further enhancing gel strength; Rosemary extract (0.1%-0.5%, accounting for wall material solids): a natural antioxidant and antibacterial agent. Its main active ingredients, such as rosmarinic acid and salicylic acid, can effectively scavenge free radicals and prevent the lipids and active ingredients in the core material from being oxidized during storage; Calcium chloride (0.1 M, post-treatment solution): a cross-linking agent. By crosslinking the carboxyl ions of LMP, the surface of the microcapsule is solidified to form a dense network structure, thereby improving the mechanical strength and sustained-release performance of the microcapsule.

[0053] Formula 2: Sodium alginate 2.0% (w / v): a natural polysaccharide whose molecular chain is composed of G units (α-L-guluronic acid) and M units (β-D-mannuronic acid). The G units are key to gel formation.

[0054] Each G unit has a negatively charged carboxyl group (-COO⁻). When it encounters a positively charged divalent cation (such as Ca²⁺), the carboxyl groups on the two G units will chelate with one Ca²⁺ cation, forming a bridge; 0.1 M CaCl₂ crosslinking: same as formula one.

[0055] II. Experimental Procedure Step 1: Aqueous phase preparation The modified chitosan was dissolved in dilute acid, and LMP and rosemary extract were added and stirred until homogeneous.

[0056] Step 2: Core material dispersion DRP (core material) is slowly added to the above wall material solution at a core-to-wall ratio of 1:3, and homogenized to form a uniform suspension.

[0057] Step 3: Microcapsule Formation Using a spray dryer / electrospinning device, the suspension is atomized and sprayed into a 0.1 M calcium chloride solution, where Ca²⁺ is instantaneously cross-linked to form microcapsules.

[0058] Step 4: Post-treatment and drying The microcapsules were collected, rinsed lightly with deionized water, and finally dried into a powder by fluidized bed drying or freeze drying.

[0059] The core antioxidant component of DRP is Deinoxanthin (DX). DX is chemically classified as: lipids and their analogues → terpenoid lipids → tetraterpenoids → carotenoids → lutein. Therefore, lutein content was used as the metric to evaluate the effectiveness of the two encapsulation materials, with three replicates per group.

[0060] The test results are as follows: In terms of storage stability analysis (based on lutein content), Formula 1 showed the highest lutein content at 30 and 60 days (1930.2 ng / g and 1895.2 ng / g, respectively), which was approximately 10.9% and 14.6% higher than Formula 2 (1740.9 ng / g and 1652.9 ng / g), indicating that Formula 2 had the best protective effect under long-term storage. With prolonged storage, the lutein content decreased in all three groups, but the decrease was smaller in the encapsulated group, indicating that the microcapsules effectively delayed the degradation of the active ingredient (results shown in Table 2).

[0061] Regarding simulated gastric acid tolerance, in a simulated gastric juice environment, Formula 1 exhibited the highest lutein retention (1529.9 ng / g), approximately 35.1% higher than Formula 2 (1132.3 ng / g), indicating that its wall material structure more effectively resists gastric acid erosion. In contrast, the unencapsulated group showed the most severe loss in gastric juice, suggesting that DX is easily destroyed by gastric acid without protection (results are shown in Table 2).

[0062]

[0063] In terms of antioxidant activity, Formulation 1 exhibited the highest DPPH free radical scavenging rate (95.1%), approximately 11.1% higher than Formulation 2 (85.6%), indicating that it more effectively protects the antioxidant capacity of DX (results are shown in Figure 1). Figure 2 (As shown).

[0064] Formula 1 (modified chitosan + LMP + rosemary extract) performed best in all evaluation indicators, especially in gastric acid tolerance (35.1% higher than formula 2) and antioxidant retention rate (11.1% higher than formula 2), showing significant differences compared with the control group.

[0065] Example 3: Safety and dose-effect assessment of DRP in a high-fat diet-induced mouse model Animal selection: Healthy adult mice (strain C57BL / 6, half male and half female, 8 weeks old) were selected.

[0066] Grouping Design: The animals are divided into 4 groups, with 8 animals in each group. The specific feeding plan is as follows: ①Grouping and rodent food formulation The control group's rat diet consisted of: 47.98% corn starch, 18.96% casein, 11.85% maltodextrin, 6.52% sucrose, 4.74% cellulose, 2.37% soybean oil, 1.90% lard, 1.56% potassium citrate monohydrate, 1.23% dicalcium phosphate, 0.95% mineral premix, 0.95% vitamin premix, 0.52% calcium carbonate, 0.28% cystine, 0.19% choline, 0.0038% yellow dye, and 0.0009% blue dye.

[0067] High-fat (HFD) rat food formula: 25.85% casein, 31.66% lard, 16.16% maltodextrin, 8.89% sucrose, 6.46% cellulose, 3.23% soybean oil, 2.13% potassium citrate monohydrate, 1.68% dicalcium phosphate, 1.29% mineral premix, 1.29% vitamin premix, 0.71% calcium carbonate, 0.39% cystine, 0.26% choline, 0.006% blue dye.

[0068] HFD+0.5%DRP formulation: 25.35% casein, 31.66% lard, 16.16% maltodextrin, 8.89% sucrose, 6.46% cellulose, 3.23% soybean oil, 2.13% potassium citrate monohydrate, 1.68% dicalcium phosphate, 1.29% mineral premix, 1.29% vitamin premix, 0.71% calcium carbonate, 0.5% radiation-resistant probiotic (DRP), 0.39% cystine, 0.26% choline, 0.006% blue dye.

[0069] HFD+1.5%DRP formulation: 24.35% casein, 31.66% lard, 16.16% maltodextrin, 8.89% sucrose, 6.46% cellulose, 3.23% soybean oil, 2.13% potassium citrate monohydrate, 1.68% dicalcium phosphate, 1.29% mineral premix, 1.29% vitamin premix, 0.71% calcium carbonate, 1.5% radiation-resistant probiotic (DRP), 0.39% cystine, 0.26% choline, 0.006% blue dye.

[0070] ② Feeding method

[0071] ③ Sample collection 1. Weight and body shape data Data collection method: The mice were weighed daily using an electronic balance; photos were taken weekly to record changes in body shape and appearance.

[0072] 2. Blood sample Collection method: Whole blood is collected by enucleation. The blood is collected in anticoagulant tubes (such as EDTA tubes) or procoagulant tubes, centrifuged, and then separated to obtain plasma or serum for biochemical index detection.

[0073] 3. Tissue Samples Liver: The liver was completely removed, photographed, and weighed to record the wet weight. One portion was immediately rinsed with physiological saline and fixed in paraformaldehyde for paraffin embedding and sectioning (histological analysis); the other portion was rapidly frozen at -80°C for subsequent molecular biological testing.

[0074] Adipose tissue: A specific fat pad was collected from the subcutaneous fat of the groin and weighed. A portion was fixed for sectioning, and the other portion was frozen.

[0075] Feces: Collect colon contents or fresh feces and immediately freeze them at -80°C for intestinal flora DNA extraction and sequencing analysis.

[0076] ④Indicator Testing Blood: 34 biochemical tests (serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C)).

[0077] Tissue: Routine liver pathology, Oil Red O; Tissue sample: Subcutaneous fat (groin fat).

[0078] Fecal intestinal flora: flora index, phylum level, genus level, etc.

[0079] The test results are as follows: Regarding weight, the HFD group gained 10.1% more weight than the Con group, indicating that the high-fat diet successfully induced obesity. After 8 weeks of intervention, the 0.5% DRP group had an average weight loss of 1.47g (4.0%) less than the HFD group, showing a weaker effect. After 8 weeks of intervention, the 1.5% DRP group had an average weight loss of 2.25g (6.2%) less than the HFD group, showing a more significant weight loss effect, indicating a dose-dependent effect (results are shown in Figure 1). Figure 3 (As shown).

[0080] In terms of blood biochemistry, DRP significantly reduced serum total cholesterol (TC) levels, with a 1.5% dose showing better efficacy (possibly due to dose dependence). Adding 1.5% DRP significantly reduced low-density lipoprotein (LDL-), with a slightly better effect at the 1.5% dose. Adding 1.5% DRPE improved triglycerides (TG), while adding 0.5% DRP showed no significant improvement. The HFD group showed significantly elevated TC and LDL levels, consistent with the characteristics of metabolic syndrome induced by a high-fat diet (see results). Figure 4a Serum total cholesterol (TC) Figure 4b Low-density lipoprotein (LDL). Figure 4c Triglycerides (TG)

[0081] Regarding liver indicators, after 20 weeks of feeding (4 weeks of intervention feeding), the livers of the CON group exhibited typical normal physiological conditions, showing a uniform dark red color and good tissue luster, indicating intact liver lobule structure and adequate blood supply. The HFD group showed significant pathological changes: the liver tissue color was noticeably lighter, the liver became enlarged, and the surface luster decreased, confirming the successful modeling of the high-fat diet. The livers of the HFD+1.5%DRP group were slightly lighter in color, with relatively intact liver lobule structure, and surface luster was better than the HFD+0.5%DRP group and the HFD group, approaching that of the CON group. The HFD+0.5%DRP group showed some improvement, but the effect was not as good as the high-dose group (results are shown in the figure). Figure 5a (As shown). After intervention with different doses of DRP, the liver weight of mice decreased slightly. The average weight of the high-dose group was slightly lighter than that of the HFD group, but still higher than that of the CON group (as shown). Figure 5b(As shown). After 20 weeks of feeding, the CON group showed sparse red staining and clearly visible hepatocytes, while the HFD group showed abundant red lipid droplets, blue collagen deposition, and increased lipid droplet size, forming macrovesicular steatosis. Feeding a high-fat diet containing 1.5% DRP for 4 weeks significantly improved liver steatosis, reducing lipid droplet size, and the effect was superior to feeding a high-fat diet containing 0.5% DRP (as shown). Figure 5c (As shown). A high-fat diet significantly increased lipid deposition, and the positive rate in the HFD group was significantly higher than that in the CON group. Compared with the HFD group, the positive rate in the HFD + 1.5% DRP group was significantly lower than that in the HFD group, while the change in the HFD + 0.5% DRP group was not significant, and even the positive rate increased at week 20, indicating that 1.5% DRP has an inhibitory effect on fatty degeneration (e.g., Figure 5d (As shown).

[0082] Regarding fat: Feeding with a high-fat diet containing HFD + 1.5% DRP significantly improved fat hypertrophy, showing better results than the HFD + 0.5% DRE group. This indicates that adding DRP to the high-fat diet improved adipocyte hypertrophy, exhibiting a dose-dependent effect (e.g., ...). Figure 6 (As shown).

[0083] Regarding gut microbiota: A high-fat diet (HFD) reduces gut microbiota balance and diversity but increases the number of bacterial species. Adding DRP, especially at a concentration of 1.5%, effectively reversed the negative effects of HFD, and significantly increased gut microbiota balance, diversity, and quantity compared to the control group, demonstrating its strong potential for regulating the gut microbiota (e.g., Figure 7a , 7b (As shown in 7c).

[0084] At the phylum level, a high-fat diet (HFD) significantly affects the gut microbiota structure, while DRP supplementation exhibits different regulatory effects at different doses, with some indicators showing dose-dependent effects. In the Firmicutes phylum, HFD reduces levels, low-dose DRP further reduces them, while high-dose DRP restores them, approaching the levels of the CON group (e.g., ...). Figure 8a (As shown). Firmicutes are the dominant phylum in the gut, and their abundance changes are often associated with energy metabolism and obesity. HFD reduces their abundance, while low-dose DRP exacerbates this effect, and high-dose DRP shows a certain "restorative" or "antagonistic" effect. This may mean that there is a "U-shaped" or "inverted U-shaped" dose-response effect of DRP on Firmicutes, that is, low doses inhibit, and high doses partially restore.

[0085] Clostridium: HFD caused its level to rise, low-dose DRP could bring it back to normal, while high-dose DRP caused it to rise again. The HFD + 1.5% DRP group (5.829) was 2.606 higher than the CON group (3.223). Figure 8b (As shown). Clostridium is a large genus containing a variety of pathogens and probiotics. HFD increases its abundance, which may be related to intestinal microecological imbalance. Low-dose DRP can restore its abundance to near basal levels, showing a good regulatory effect; however, high-dose DRP leads to a re-increase in its abundance, so further analysis of the changes in the number of its subclasses within the Clostridium genus is needed. Analysis of changes in Clostridium subclasses revealed that, compared with the CON group, high-dose DRP significantly increased the level of *F. prausnitzii* (p < 0.01), from 0.006 to 0.044, an increase of 7.3 times (e.g., as shown). Figure 8c As shown). *Clostridium plasminogen lysate* is a key anti-inflammatory bacterium in the gut, primarily producing short-chain fatty acid butyrate to suppress intestinal inflammation. Further studies found that butyrate levels significantly decreased after high-fat feeding (p < 0.05). After high-dose DRP treatment, the butyrate production capacity of the gut microbiota recovered to some extent, significantly increasing from HFD (15) to 1.5% DRP (25), which was somewhat different from the CON group (37), but not significantly different. This result also proves that increased *Clostridium plasminogen lysate* levels can improve the overall butyrate production capacity of the gut microbiota. In contrast, low-dose DRP further reduces the butyrate production capacity of the gut microbiota (e.g., ...). Figure 8d (As shown). Regarding intestinal barrier function, compared to the CON group, the intestinal barrier score of the high-dose DRP group significantly increased from 38.7 to 53.0 (p < 0.05). Increased butyrate secretion levels helped improve intestinal barrier function, indicating that high-dose DRP can also improve overall intestinal barrier function to reduce intestinal inflammation (e.g., Figure 8e (As shown). The mechanism by which DRP improves gut microbiota imbalance caused by a high-fat diet: A high-fat diet disrupts gut microbiota balance, damages the intestinal barrier, and leads to a decrease in butyrate secretion levels. It precisely regulates the internal structure of Clostridium → by increasing the level of Clostridium praosporum → restores butyrate synthesis capacity → butyrate mediates the strengthening of the intestinal barrier, forming a positive feedback loop of "microbe-metabolism-barrier", optimizing gut microbiota structure, and improving gut microbiota balance, diversity, and quantity.

[0086] Example 4: Preparation of DRP-containing functional food and feeding experiment with pet cats. According to the Body Condition Score (BCS) system for cats, overweight domestic cats with a score of 6 or higher (meaning indistinct ribs, significant fat accumulation, a slightly flat waist, and a slightly protruding abdomen) are considered. Ten overweight, spayed cats of similar age and weight (BCS > 6) were selected and fed cat food containing 1.5% DRP (preparation process as follows) during their rearing period. Figure 1 As shown in the image, the basic formula of the cat food remained the same before and after feeding, the difference being whether it contained 1.5% DRP. Approximately 50g per day (adjusted according to body weight), with the 1.5% addition based on the dosage used in mouse feeding experiments, and a feeding period of 28 days. Fecal samples were collected on days 0 and 28 for gut microbiota testing, denoted as D0 and D28. Body weight, waist circumference, BCS, and muscle condition scores were recorded on days 0, 7, 14, and 28, denoted as D0, D7, D14, and D28. Fecal scores, feed intake rate, food intake, and cat food and water were changed daily.

[0087] DRP-containing cat food formula (compliant with GB / T 31217-2014 Complete Pet Food for Cats): 60 portions of frozen fresh chicken, 10 portions of frozen fresh duck breast, 10 portions of bone-in chicken, 5 portions of enzymatically hydrolyzed chicken liver, 4.5 portions of chilled chicken hearts, 3 portions of chicken fat, 3 portions of tapioca starch, 1.5 portions of DRP, 0.5 portions of egg yolk powder, 0.5 portions of krill powder, and 1.5 portions of pet compound additive (composition: taurine, vitamin C, brewer's yeast, light calcium carbonate, citric acid, yeast selenium, vitamin A acetate, vitamin D3, dl-α-tocopherol acetate, thiamine nitrate, vitamin B2, vitamin B6, vitamin B12, D-calcium pantothenate, nicotinamide, folic acid, D-biotin, choline chloride, zinc methionine chelate, iron glycine chelate, and copper glycine chelate), totaling 100 portions of staple food formula.

[0088] The feeding results are as follows: Regarding growth performance: There was no significant change in average food intake before and after feeding DRP cat food, and the feed intake rate was consistently high (>98%) with no significant change. The average fecal score was around 2.5, which is considered normal fecal condition (as shown in Table 4).

[0089]

[0090] The results of the cat body shape indicators are as follows: After 28 days of feeding, the average weight of the 10 cats decreased by 0.18 kg, showing an overall downward trend. Body shape scores also tended towards the ideal body shape range (BCS score 4-6). Muscle scores showed significant differences (p < 0.05), but overall scores were all above 3, falling within the ideal normal range. Waist circumference also decreased slightly, with no significant change (as shown in Table 5). In terms of overall sensory evaluation, the body shape showed a trend towards becoming leaner. At the beginning of feeding, the waistline of the 10 cats was not obvious, but after 28 days, 3 cats showed a defined waistline, with a noticeably thinner waist and abdomen (e.g., ...). Figure 9 (As shown). Feeding cats with DRP-containing food improved their overall body shape, suggesting that high-fiber DRP may enhance satiety, prolong digestion time, and reduce energy density.

[0091]

[0092] Regarding the gut microbiota results, the gut microbiota balance index increased from 72.1 at D0 (before the experiment) to 79.4 at D28 (after the experiment), indicating better microbiota balance. The gut microbiota diversity index also improved to some extent after feeding (from 48.1 at D0 to 65.2 at D28), and the total number of microbiota increased significantly, from 352.8 at D0 to 458.1. p <0.05), indicating that DRP may act as a prebiotic, providing more fermentable substrates for gut microbiota, thereby promoting the overall proliferation of the gut microbiota (e.g., Figure 10a (As shown in 10b and 10c). Overall, the significant increase in the total number of bacteria indicates that DRP, as a prebiotic, provides more fermentable substrates for the gut microbiota, thereby promoting the overall proliferation of the gut microbiota.

[0093] Regarding gut bacterial abundance, the abundance of Clostridium species was approximately 1.1 at day 0 and increased to approximately 1.3 at day 28. Clostridium is a large taxonomic unit containing many important butyric acid-producing bacteria, and its increased abundance is generally positively correlated with gut health, particularly with the ability to produce short-chain fatty acids (e.g., ...). Figure 10d As shown). The abundance of Clostridium praosporum, a subfamily of Clostridium, was approximately 0.8 at D0, and significantly increased to approximately 1.0 at D28 (as shown). p <0.05), Clostridium plasminogen lysate (one of the most important butyric acid-producing bacteria in the animal gut. It not only produces butyric acid efficiently, but also has strong anti-inflammatory properties (such as...). Figure 10e (As shown). Simultaneously, the detection of butyrate levels, a key intestinal metabolite, also confirmed the increased butyrate production capacity in the intestine; the butyrate index was approximately 14 at D0 and rose to approximately 19 at D28 (as shown). Figure 10f(As shown). Butyrate is the primary energy source for intestinal epithelial cells and can regulate immunity, suppress inflammation, and affect satiety and energy metabolism. In overweight cats, increased butyrate levels help reduce "leaky gut" and decrease systemic inflammation caused by endotoxins entering the bloodstream. Butyrate can also help control weight by regulating energy metabolism and insulin sensitivity, and it also has the effect of regulating appetite and affecting intestinal hormone secretion, which may help reduce overeating.

[0094] Overall analysis of the gut microbiota revealed a positive regulatory effect of 1.5% DRP in cat food on the gut microbiota of overweight, spayed, and neutered cats. The mechanism of action can be summarized as follows: DRP acts as a prebiotic → promotes the proliferation of beneficial bacteria such as Clostridium perfringens → increases the production of short-chain fatty acids such as butyrate → improves the intestinal barrier, reduces inflammation, and regulates metabolism → ultimately helps improve obesity. The trends in the DRP-fed cat food results were largely consistent with those in mice, indicating that, in both mice and cats, DRP, as a prebiotic, can be efficiently utilized by these key butyrate-producing bacteria, thus initiating a positive cycle of "anti-inflammatory-repair-metabolic regulation." These two feeding experiments provided important data on the safety, dosage, and mechanism of DRP, offering strong evidence for its application as a functional ingredient in pet food, and in particular, providing a scientific basis for developing functional cat foods specifically for managing pet obesity.

[0095] The above examples are merely some specific embodiments of the present invention. It should be noted that the present invention is not limited to the above embodiments and can have many other modifications, including changes to the industrial production parameters of *Bacillus radiodurans* post-biotics (without affecting the core process), parameters for the microencapsulation and embedding of *Bacillus radiodurans* post-biotics, application of *Bacillus radiodurans* post-biotics in other animal models (normal-sized dogs and cats, obese dogs and cats, etc.), and changes in the addition ratio of *Bacillus radiodurans* post-biotics. The above content is merely illustrative and explanatory of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing a postbiotic from *Radiata spp.*, characterized in that, Includes the following steps: (1) Preparation of liquid seed: Deinococcus radiodurans was inoculated into liquid seed TGY medium and cultured with shaking at a temperature of 28-32℃ and a rotation speed of 200-240 rpm / min until the cells reached the logarithmic growth phase to obtain liquid seed liquid; (2) Solid-state fermentation: The liquid seed liquid obtained in step (1) is inoculated onto a solid fermentation medium at 5–8% (v / w). The solid fermentation medium is composed of 50–60% soybean meal, 30–40% sunflower seeds, 4–12% flaxseed meal, and 2–8% palm fruit pomace by mass percentage. Before inoculation, the medium is sterilized at 121°C for 15 minutes, and the moisture content is controlled at 40–45%. The medium is then fermented at a constant temperature of 28–32°C for 48–60 hours. (3) Post-processing: After fermentation, the fermented material is dried at a low temperature of 55-65℃ until the moisture content is ≤5% to obtain radiation-resistant cytokine post-biotic (DRP) powder.

2. The method according to claim 1, characterized in that, The solid fermentation medium described in step (2) consists of 55% soybean meal, 35% sunflower seeds, 6% flaxseed meal, and 4% palm fruit residue.

3. A method for microencapsulation and embedding of *Radiata susceptibility-resistant* metagenin, characterized in that, include: (1) Preparation of wall material solution: Mix 0.5–1% (w / v) of modified chitosan, 1.5–2.5% (w / v) of low esterification pectin, and 0.1–0.5% (accounting for the total solids of the wall material) of rosemary extract and dissolve them in water to form a homogeneous aqueous phase; (2) Core material dispersion: The DRP powder according to claim 1 or 2 is dispersed in the aqueous phase at a core-to-wall ratio of 1:2–1:4 and homogenized to form a suspension; (3) Microcapsule formation: The suspension is sprayed into a 0.1 M calcium chloride solution through a spray drying device, so that the low-esterification degree pectin and calcium ions form an "egg box" structure cross-linking to form microcapsules; (4) Post-processing: Collect the microcapsules, rinse with deionized water, and dry in a fluidized bed to obtain microcapsule powder containing DRP.

4. The microencapsulation embedding method according to claim 3, characterized in that, The modified chitosan is N-trimethyl chitosan or acetylated chitosan, with a degree of deacetylation ≥85%.

5. A functional pet food, characterized in that, The feed contains an effective amount of the microencapsulated biogener of claim 3 or 4, wherein the feed is cat food, dog food, or rodent food, and the amount of DRP added is 0.5–2.0% (w / w).

6. The functional pet food according to claim 5, characterized in that, The amount of DRP added is 1.5% (w / w).

7. The functional pet food according to claim 5 or 6, characterized in that, The feed contains the following ingredients by weight: 60 parts frozen fresh chicken meat, 10 parts frozen fresh duck breast meat, 10 parts bone-in chicken meat, 5 parts enzymatically hydrolyzed chicken liver, 4.5 parts fresh chicken heart, 3 parts chicken oil, 3 parts tapioca starch, 0.5 parts egg yolk powder, 0.5 parts krill powder, and 1.5 parts pet compound additive; the DRP is uniformly sprayed onto the surface of the feed in microencapsulated form.

8. The functional pet food according to claim 7, characterized in that, The pet compound additive contains taurine, vitamin C, brewer's yeast, light calcium carbonate, citric acid, yeast selenium, vitamin A acetate, vitamin D3, dl-α-tocopherol acetate, thiamine nitrate, vitamin B2, vitamin B6, vitamin B12, D-calcium pantothenate, nicotinamide, folic acid, D-biotin, choline chloride, zinc methionine chelate, iron glycine chelate, and copper glycine chelate.

9. Use of the method of any one of claims 1–4 in the preparation of functional pet food for regulating the weight of dogs and cats.

Images