Intestinal flora metabolite extract in periplaneta americana excrement as well as preparation method and application of intestinal flora metabolite extract

Abstract

The invention relates to an intestinal flora metabolite extract in periplaneta americana excrement and a preparation method and application thereof, and relates to the technical field of coprophilous fungi, and the preparation method comprises the following steps: S1, collecting excrement, pretreating, and preserving the excrement by using an excrement preserving fluid; s2, putting the stored periplaneta americana excrement suspension into a freeze dryer, performing vacuum drying, crushing and sieving, and then performing ethanol reflux extraction; S3, performing reduced pressure concentration on filtrate at 70 DEG C to obtain extract, recovering ethanol, and stirring to prepare dispersion liquid; s4, sequentially extracting the dispersion liquid by using petroleum ether, ethyl acetate and n-butyl alcohol to obtain a powdery metabolite extract; integrity and metabolic activity of intestinal flora preservation are improved through excrement preservation liquid, and then an extract with a high anti-inflammatory effect is obtained through extraction.

Description

Technical Field

[0001] The present invention relates to the technical field of fecal microbes, and in particular to an extract of intestinal microbiota metabolites in American cockroach feces, a preparation method and an application thereof. Background Art

[0002] American cockroach ( Periplaneta americana ) is the main source of the traditional Chinese medicine "Cockroach", and its extracts have a broad research base in tissue repair, anti-inflammation, and immune regulation. In recent years, studies have found that the parasitic flora in the intestines of American cockroaches have unique metabolic capabilities, and their metabolites may be involved in regulating the host's inflammatory response, antimicrobial defense, and regeneration and repair physiological processes. However, there is currently a lack of methods to systematically preserve the flora in feces and extract their metabolites. Although the metabolic mechanisms of intestinal flora in insects and mammals are different, many core metabolic pathways (such as short-chain fatty acid and aromatic compound metabolism) are highly conserved.

[0003] For example, Chinese patent application number 202210435558.7 discloses a method for preparing a fecal preservation solution and a fecal bacterial suspension. The fecal preservation solution comprises the following components in parts by weight: 20-40 parts of glycerol; 60-80 parts of water; 1-5 parts of disodium ethylenediaminetetraacetic acid; 1-3 parts of sodium chloride; 1-3 parts of sodium citrate; and 1-3 parts of an extract. The method for preparing the fecal bacterial suspension comprises the following steps: taking feces, then adding the fecal preservation solution, and mixing evenly to obtain the fecal bacterial suspension. The fecal preservation solution can effectively improve the stability of microbial DNA in feces and significantly extend the storage time of feces at room temperature; thereby enabling feces to be stored for a long time at room temperature without the need for liquid nitrogen freezing. Furthermore, using the fecal preservation solution to store feces at low temperatures has a longer storage time and can maintain the stability and activity of the bacterial flora for a long time.

[0004] However, since the intestinal flora of American cockroaches is mainly composed of high-temperature-resistant bacteria and extreme environment bacteria (such as Bacillus), which is significantly different from the human intestinal flora (mainly Bacteroidetes and Firmicutes), the American cockroach flora may have thicker cell walls (such as spores), and glycerol is difficult to penetrate into the cells to inhibit metabolism, resulting in insufficient inhibition of DNAase. The intestine of American cockroaches contains more chitinase and heat-resistant DNAase, and the chelation effect of EDTA may not be sufficient to completely inhibit their activity.

[0005] For example, Chinese patent application number CN202010640421.6 discloses an extract of metabolites from the intestinal flora of the American cockroach, a preparation method thereof, and its use in the preparation of anti-inflammatory or anti-ulcer products. This extract, made from American cockroach feces, is extracted through ethanol reflux, filtered, and concentrated to form an extract, which is then dispersed with water and extracted with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate extract and the n-butanol extract are then collected. The extract has anti-inflammatory and anti-ulcer effects, and its efficacy is superior to that of an extract derived from the body of the American cockroach.

[0006] However, if the feces are not processed promptly after collection or are stored under inappropriate conditions (such as high temperature and humidity), the active ingredients may be oxidized, degraded or contaminated by microorganisms, resulting in insufficient concentration of active ingredients in the extract or impurities, which significantly reduces the anti-inflammatory and anti-ulcer effects. Summary of the Invention

[0007] The present invention aims to solve the technical problems existing in the prior art and provides an extract of intestinal flora metabolites in American cockroach feces, a preparation method and an application thereof.

[0008] The technical solution of the present invention to solve the above technical problems is as follows: A method for preparing an extract of intestinal flora metabolites in American cockroach feces comprises the following steps: S1. After collecting feces, pre-treat them and then preserve them in fecal preservation solution. Mix the feces with the preservation solution at a ratio of 1g:5ml to prepare a fecal bacterial suspension, which is then stored at 5°C. The fecal preservation solution consisted of 5 mM EDTA-Mg, 15% glycerol, 2% mealworm chitin hydrolyzate, 1% symbiotic bacterial metabolite extract, 0.1% glutathione, and the rest was solvent water; S2, the American cockroach feces suspension of preservation was placed in a freeze dryer, -50 ℃ of pre-freeze, then vacuum dried, pulverized through a 20 mesh sieve, to obtain a coarse powder, which was then subjected to ethanol reflux extraction, and extracted 3 times, each time for 2 hours. Every 1kg feces coarse powder was added with 10L ethanol solution, and each extraction used different ethanol concentrations. From the first to the third time, 20% aqueous ethanol solution, 50% aqueous ethanol solution, and 90% aqueous ethanol solution were used sequentially, and the filtrate was combined to remove the residue. S3. The filtrate is concentrated under reduced pressure at 70°C to an extract, the ethanol is recovered, and water is added to the extract at a mass ratio of 1:5 and stirred to form a dispersion; S4. Extract the dispersion with petroleum ether, ethyl acetate, and n-butanol in sequence, extract with each solvent 5 times, and combine the extracts with the same solvent; then concentrate the ethyl acetate and n-butanol extracts under reduced pressure, recover the solvents, and then freeze-dry to obtain a powdered metabolite extract.

[0009] Furthermore, it is characterized in that the steps of feces pretreatment are: after liquid nitrogen freezing and grinding, adding 1% TritonX-100, mixing evenly, pretreatment at 37°C for 10 minutes, ultrasonic fragmentation pulse mode, lasting for 5 minutes, the fragmentation efficiency is improved by 30%, using recombinant chitinase Chit42 at a concentration of 2 U / mg feces, and adding 0.5 mM MgCl2, incubating at 37°C for 1.5 hours.

[0010] Furthermore, it is characterized in that glutathione is encapsulated by liposomes, and the preparation method is as follows: the liposome-encapsulated glutathione is composed of dipalmitoylphosphatidylcholine, cholesterol, and phospholipid-polyethylene glycol, with dipalmitoylphosphatidylcholine: cholesterol: phospholipid-polyethylene glycol = 6:3:1, the lipid mixture is dissolved in chloroform, rotary evaporation is performed to form a film, vacuum drying is carried out overnight, the lipid film is hydrated with PBS containing 5 mM glutathione, and liposomes with a particle size of 100±20 nm are obtained by high-pressure homogenization, and free GSH is removed by ultrafiltration and centrifugation.

[0011] Furthermore, the method is characterized in that the yellow mealworm chitin hydrolyzate is prepared by grinding the dried yellow mealworm shells through a 100-mesh sieve, mixing the dried shells with 6 M HCl at a ratio of 1:10, reacting the mixture under reflux at 110° C. for 4 hours, cooling the mixture and centrifuging the mixture, taking the supernatant, neutralizing the mixture with NaOH to pH 7.0, concentrating the mixture to 1 / 5 of the original volume by rotary evaporation, and freeze-drying the mixture to obtain a light yellow hydrolyzate powder.

[0012] Furthermore, it is characterized in that the symbiotic bacteria metabolite extract uses human intestinal symbiotic bacteria (Bacteroides thetaiotaomicron), which is inoculated into GAM culture medium, and the bacteria are removed by centrifugation. The supernatant is filtered through a 0.45 μm filter membrane, and the filtrate is filtered through a 3 kDa ultrafiltration membrane to intercept small molecule metabolites, and the brown extract is obtained by freeze-drying.

[0013] Furthermore, the invention is characterized in that glutathione is loaded on core-shell structure particles, and the specific preparation method is: ZnFe2O4 magnetic nanoparticles are synthesized by hydrothermal method, loaded with Zn(NO3)2 and FeCl3, coated with mesoporous silica by sol-gel method, then modified with chitin-binding peptide and polyethylene glycol, and finally loaded with glutathione.

[0014] Furthermore, it is characterized in that, during the pretreatment process, 5 mM CaCl2 is added while adding 0.5 mM MgCl2, and incubated at 37°C for 1.5 hours under the guidance of a 0.5 T magnetic field.

[0015] Furthermore, the core-shell structured MSNs include core-shell structured MSNs with three different core structures, including high magnetic particles with Fe3O4@Co as the core, medium magnetic particles with MnFe2O4 as the core, and low magnetic particles with γ-Fe2O3 as the core, with a mass ratio of 0.5:1:1.5. The high magnetic particles are loaded with 0.2 mM zinc ions, the medium magnetic particles are loaded with 10 wt% GSH and 0.1 mM iron ions, and the geomagnetic particles are loaded with 20 wt% trehalose.

[0016] The application of the extract of intestinal flora metabolites in the feces of American cockroaches is used to treat inflammation.

[0017] The beneficial effects of the present invention are: First, the integrity and metabolic activity of intestinal flora are increased through fecal preservation fluid, laying a high-quality sample foundation for subsequent metabolite extraction.

[0018] Second, metal ion compensation (Zn 2+ / Fe 3+ ) is combined with mesoporous silica nanoparticles (MSN) to encapsulate glutathione (GSH), and the utilization rate of glutathione and the ·OH clearance rate are improved through three-level synergy: magnetic targeting (atomic level) → controlled release (molecular level) → enzyme / DNA protection (submicroscopic), laying the foundation for the efficient extraction of intestinal flora metabolites.

[0019] Third, Co doping makes the saturation magnetization reach 85 emu / g (pure Fe3O4 is only 60 emu / g), and the moving speed is improved under 0.5 T magnetic field. 2+ Through the controlled release of the PVP layer (release rate ≈ 60% in 72h), it binds to the ZnuABC transporter in the bacterial membrane and promotes the expression of DNA repair protein (RecA); Mn 2+ / Fe 3+ The alternating valence state generates heat under an alternating magnetic field (42±1℃), and local temperature rise promotes the release of GSH. The weak magnetism (25 emu / g) reduces particle collisions and prevents trehalose leakage. Trehalose dehydrates on the surface of the biofilm to form an amorphous glass layer, inhibiting ice crystal damage. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of this application.

[0021] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the described features. In the description of this application, "plurality" means two or more, unless otherwise specifically specified.

[0022] In the description of this application, the term "for example" is used to mean "used as an example, illustration or explanation". Any embodiment described as "for example" in this application is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is given to enable any person skilled in the art to implement and use the present invention. In the following description, details are listed for the purpose of explanation. It should be understood that a person of ordinary skill in the art will recognize that the present invention can be implemented without using these specific details. In other examples, well-known structures and processes will not be elaborated in detail to avoid obscuring the description of the present invention with unnecessary details. Therefore, the present invention is not intended to be limited to the embodiments shown, but is consistent with the widest scope consistent with the principles and features disclosed in this application.

[0023] Example 1: A method for preparing an extract of intestinal flora metabolites from American cockroach feces, comprising the following steps: S1. After collecting feces, pre-treat them and then preserve them in fecal preservation solution. Mix the feces with the preservation solution at a ratio of 1g:5ml to prepare a fecal bacterial suspension, which is then stored at 5°C. Fecal pretreatment steps were as follows: after freezing and grinding in liquid nitrogen, 1% Triton X-100 was added, mixed evenly, pretreated at 37°C for 10 minutes, and ultrasonically disrupted (20 kHz, 150 W) in pulse mode (5 s on / 5 s off) for 5 minutes to increase the disruption efficiency by 30%. Recombinant chitinase Chit42 (EDTA-Mg resistant and uninhibited) was used at a concentration of 2 U / mg feces, and 0.5 mM MgCl2 was added, and incubated at 37°C for 1.5 hours. Fecal preservation solution includes 5 mM EDTA-Mg (ethylenediaminetetraacetic acid magnesium disodium salt), 15% (v / v) glycerol, 2% (w / v) mealworm chitin hydrolyzate, 1% (w / v) symbiotic bacterial metabolite extract, and 0.1% (w / v) liposome-encapsulated glutathione; The chitin hydrolyzate of Tenebrio molitor was prepared by grinding the dried shells of Tenebrio molitor (chitin content ≥ 30%) through a 100-mesh sieve and mixing them with 6M HCl at a ratio of 1:10 (w / v). The mixture was refluxed at 110°C for 4 hours, cooled, and centrifuged (8000×g, 15 minutes). The supernatant was neutralized with NaOH to pH 7.0, concentrated to 1 / 5 of the original volume by rotary evaporation, and lyophilized to obtain a light yellow hydrolyzate powder. Symbiotic metabolite extracts were prepared using human intestinal symbiotic bacteria (Bacteroides thetaiotaomicron) inoculated into GAM medium (anaerobic culture at 37°C for 48 h). The bacteria were removed by centrifugation (10,000 × g, 20 min). The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was filtered through a 3 kDa ultrafiltration membrane to retain small molecular metabolites. The brown extract was freeze-dried and dissolved in preservation solution at a 1% mass concentration (10 g / L). Liposome-encapsulated glutathione consists of dipalmitoylphosphatidylcholine, cholesterol, and phospholipid-polyethylene glycol (DSPE-PEG2000) at a molar ratio of DPPC:cholesterol:DSPE-PEG2000 = 6:3:1. The lipid mixture was dissolved in chloroform, rotary evaporated to form a film, and vacuum-dried overnight. The lipid film was hydrated with PBS (pH 6.5) containing 5 mM glutathione (GSH) (ultrasonication at 60°C for 30 minutes). Liposomes with a particle size of 100 ± 20 nm were obtained by high-pressure homogenization (15,000 psi, 3 cycles). Free GSH was removed by ultrafiltration and centrifugation (100 kDa), and preservation solution was added at a concentration of 0.1% (1 g / L). S2. The preserved American cockroach feces suspension was placed in a freeze dryer and pre-frozen at -50°C for 4 to 6 hours, followed by sublimation drying under vacuum conditions for 24 to 48 hours to obtain a dry solid, which was crushed through a 20-mesh sieve to obtain a coarse powder, and then subjected to ethanol reflux extraction, the extraction being performed 3 times, each time for 2 hours, with 10 L of ethanol solution added to every 1 kg of fecal coarse powder, and the ethanol concentration used for each extraction being different, using 20% ​​(v / v) ethanol aqueous solution, 50% (v / v) ethanol aqueous solution, and 90% (v / v) ethanol aqueous solution from the first to the third extraction, the filtrates were combined, and the residue was removed; S3. The filtrate is concentrated under reduced pressure at 70°C to an extract, the ethanol is recovered, and water is added to the extract at a mass ratio of 1:5 and stirred to form a dispersion; S4. Extract the dispersion with petroleum ether (80°C), ethyl acetate, and n-butanol in sequence, extracting five times with each solvent, and combine the extracts with the same solvent; then, concentrate the ethyl acetate and n-butanol extracts under reduced pressure, recover the solvents, and then freeze-dry to obtain powdered metabolite extracts.

[0024] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The integrity and metabolic activity of intestinal flora are increased by fecal preservation fluid, laying a foundation for high-quality samples for subsequent metabolite extraction.

[0025] Tenebrio molitor chitin hydrolysate includes N-acetylglucosamine (GlcNAc) oligomers and short-chain chitin fragments. GlcNAc, as a carbon and nitrogen source, is taken up by intestinal flora through ABC transporters (such as NagP), promoting bacterial metabolism. Chitin fragments activate Toll-like receptors (such as TLR2 / 4) or NOD-like receptors (NLRs), inducing the flora to produce stress-resistant proteins (heat shock protein Hsp20). The positively charged amino groups in the hydrolysate bind to negatively charged phospholipids (such as phosphatidylglycerol) on the surface of the bacterial membrane, enhancing the stability of the membrane. The positively charged amino groups in the chitin hydrolysate and the negatively charged surface of the liposome (DSPE-PEG) form a protective layer through electrostatic interaction, reducing liposome aggregation. Symbiotic bacterial metabolite extracts include short-chain fatty acids (SCFAs, such as acetate and propionate), polysaccharide-degrading enzymes (such as glycosidases), and vitamins (B group). SCFAs enter the bacteria through diffusion or MCT transporters, inhibiting histone deacetylases (HDACs) and upregulating the expression of DNA repair genes (RecA). Glycosidases hydrolyze host polysaccharides (such as mucins) to release monosaccharides (such as fucose), which serve as cross-feeding substrates and maintain the bacterial interaction network. Vitamin B acts as a coenzyme in methionine synthesis, reducing oxidative stress damage to the bacteria. Liposomes are 100 nm nanoparticles composed of DPPC (rigid lipid membrane) and DSPE-PEG (hydrophilic coating). The sulfhydryl group (-SH) of GSH directly scavenges ROS (such as ·OH) and protects bacterial DNA (preventing guanine from being oxidized to 8-oxo-dG). Liposomes enter the bacteria through membrane fusion or endocytosis, releasing GSH into the cell and improving its bioavailability (free GSH has low utilization due to poor membrane permeability). The hydrophobic core of the liposome can load fat-soluble antioxidants and can absorb small amounts of vitamin E contained in other components to form a multi-stage antioxidant system. GlcNAc increases NADPH production (required for the regeneration of reduced glutathione) through the hexosamine pathway, synergistically maintaining intracellular redox homeostasis with liposomal GSH.

[0026] The mechanism of the chitin hydrolyzate of mealworms and liposome-encapsulated glutathione was verified. In the experiment of this example, the verification dosage of the chitin hydrolyzate of mealworms was 1%-3%, and the verification dosage of the liposome-encapsulated glutathione was 0.05%-0.2%. The other components and conditions were the same as the dosage experiment in Example 1; the fecal bacteria suspension stored in the preservation solution for 7 days and 14 days was used for experimental verification. The specific experiments included bacterial activity detection with reference to GB 4789.2-2022, using plate count agar medium for plate count detection; short-chain fatty acids (SCFAs) were detected by high performance liquid chromatography, with reference to GB / T 32465-2015, using a C18 reverse phase column (particle size 5 μm, inner diameter 4.6 mm), and a wavelength of 210 nm; DNA integrity was detected by agarose gel electrophoresis + qPCR, and the DNA was detected by agarose gel electrophoresis (0.8% gel, 1×TAE buffer, 100 V, 45 minutes), and then fluorescent quantitative PCR targeting long fragment genes (16S rRNA gene>1.5 kb), the proportion of long fragment DNA is the integrity result; the DPPH free radical scavenging rate is detected by spectrophotometry. The fecal bacteria suspension is mixed with 0.1 mM DPPH ethanol solution at a volume of 4:1, and the mixture is kept in the dark for 30 minutes, and then the absorbance is measured at 517 nm. The control was fresh feces without preservation. The degree of DNA oxidative damage was verified by detecting the marker (8-OHdG) using LC-MS / MS. The chromatographic column was C18 column (2.1×100 mm, 1.7 μm), and the ion source was electrospray positive ion (ESI + ) Quantitative ion pair: 8-OHdG m / z 284→168. The fecal bacterial suspension was centrifuged (10,000 × g, 10 min) and filtered through a 0.22 μm filter membrane. After solid phase extraction (SPE) purification, it was used as a sample. The test results are shown in Tables 1 and 2. Table 1 7-day storage experimental verification results in Example 1

[0027] Table 2 Experimental verification results of 14-day storage in Example 1

[0028] An experiment was designed to verify the extraction effect of intestinal flora metabolite extracts from feces after using the preservation solution. Short-chain fatty acids including acetic acid, propionic acid, and butyric acid were detected by GC-MS. Glutathione (GSH) and polyphenols (calculated as gallic acid) were detected by HPLC (UV detection at 280 nm, reverse C18 column). Total polysaccharides were determined by the phenol-sulfuric acid method. Vitamins (vitamin B12, wavelength 361 nm, C18 column) were detected using an amino acid analyzer (Hitachi L-8900) and HPLC-UV. The results are shown in Table 3. The experimental group was extracted after 7 days of preservation in Experiment 2. The control group was extracted after 7 days of preservation at 5°C without using the fecal preservation solution. The fresh group was directly extracted without preservation. Table 3 Validation of the extract from Example 1

[0029] The anti-inflammatory effect of each group was tested at the cellular level. RAW264.7 mouse macrophages were stimulated with LPS (1 μg / mL) for 24 hours to induce an inflammatory response. Then, the experimental group, control group, fresh group, and positive control group (dexamethasone) were set up respectively. After incubation for 24 hours, IL-6 and TNF-α levels were detected by ELISA, ROS clearance rate in cell lysate was detected by DCFH-DA fluorescent probe, and NF-κB p65 inhibition rate was detected by Western Blot. The results are shown in Table 4. Table 4 Verification of anti-inflammatory level

[0030] SCFAs (such as butyrate) in the fresh group were easily oxidized and degraded, while the preservation solution of the experimental group protected the active ingredients through liposome GSH and EDTA-Mg. The mealworm hydrolysate in the experimental group promoted the secretion of polysaccharide-degrading enzymes by the bacterial community, released fucosylated oligosaccharides, and synergistically inhibited TLR4 / NF-κB signaling with SCFAs, ultimately resulting in a certain disadvantage in the anti-inflammatory effect of the fresh group.

[0031] Example 2. In Example 1, a chemically stable environment was constructed to inhibit the degradation of microbial DNA by using a combination of mealworm chitin hydrolyzate and liposome-encapsulated glutathione with glycerol and EDTA. However, the presence of glycerol and EDTA-Mg destroyed the liposome bilayer membrane structure, resulting in a decrease in the encapsulation efficiency, which in turn caused free glutathione to form a GSH-Mg-EDTA ternary complex with EDTA-Mg. The efficiency of scavenging hydroxyl radicals (·OH) still needed to be improved, and it was found that the effective concentration of GSH was reduced to 31% of the original concentration after 72 hours. In this regard, further improvements were made based on Example 1.

[0032] The liposome-encapsulated glutathione is changed to a core-shell structure MSN loaded with glutathione; the preparation method of the core-shell structure MSN loaded with glutathione is as follows: Zn(NO3)2·6H2O (0.1 M) and FeCl3·6H2O (0.2 M) were dissolved in 50 mL of ethylene glycol with a molar ratio of Zn:Fe = 1:2. Sodium citrate (0.5 g) was added as a morphology modifier and magnetically stirred for 30 min. The mixture was then transferred to a 100 mL polytetrafluoroethylene autoclave and reacted at 200°C for 12 h. After cooling to room temperature, the precipitate was collected by centrifugation (12000 rpm, 10 min), washed three times with ethanol and deionized water, and dried at 60°C in a vacuum oven for 6 h to obtain ZnFe2O4 nanoparticles (diameter 50±5 nm). The nanoparticles were then immersed in a mixture of Zn(NO3)2 (0.1 mM) and FeCl3 (0.05 mM) and stirred at 50°C for 24 h to immobilize the metal ions by ion exchange. The particles were then centrifuged to remove free ions and dried at 60°C. ZnFe2O4 nanoparticles (100 mg) were dispersed in 100 mL of ethanol / water (4:1) mixture and ultrasonicated for 30 minutes. CTAB (0.5 g, template) and ammonia (1 mL, 28%) were added in sequence and stirred for 30 minutes. Tetraethyl orthosilicate (TEOS, 1 mL) was slowly added dropwise and reacted at 40°C for 6 hours to form CTAB@SiO2 / ZnFe2O4. The product was collected by centrifugation and refluxed with ethanol / hydrochloric acid (0.1 M) mixture for 12 hours to remove the CTAB template, resulting in core-shell structured MSNs containing a mesoporous SiO2 shell (pore size 3±0.5 nm). MSN was dispersed in toluene, and APTES (3-aminopropyltriethoxysilane, 1 mL) was added and refluxed at 80°C for 4 hours to introduce amino groups (-NH2) and activate CHBP (KLVFFAK): the carboxyl groups were activated with EDC / NHS (molar ratio 1:1.2) in PBS (pH 6.0) for 30 minutes. The activated peptide solution was mixed with the amino-modified MSN and shaken at room temperature for 12 hours to complete the grafting through amide bonds. The unreacted peptide was removed by centrifugation and the CHBP-MSN was lyophilized for storage. mPEG-SCM (molecular weight 5000 Da, 0.1 g) was reacted with CHBP-MSN in PBS (pH 7.4) for 4 hours to complete PEG modification through amino-carboxyl coupling. The CHBP-PEG-MSN was purified by centrifugation and lyophilized to obtain CHBP-PEG-MSN. CHBP-PEG-MSN was dispersed in a pH 3.0 hydrochloric acid solution and ultrasonicated for 30 minutes to open the mesoporous channels. GSH (10 wt%) was added and the pH was adjusted to 7.4. GSH was captured in the mesopores due to the change in isoelectric point (pI 5.9). TEOS (0.1 mL) was added and reacted at room temperature for 2 hours to seal the pores to prevent GSH leakage, thus obtaining a core-shell structured MSN loaded with glutathione.

[0033] During the fecal pretreatment, 0.5 mM MgCl2 was added together with 5 mM CaCl2, and the feces were incubated at 37°C for 1.5 hours under the guidance of a 0.5 T magnetic field.

[0034] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The metal ion compensation (Zn 2+ / Fe 3+ ) is combined with mesoporous silica nanoparticles (MSN) to encapsulate glutathione (GSH), and the utilization rate of glutathione and the ·OH clearance rate are improved through three-level synergy: magnetic targeting (atomic level) → controlled release (molecular level) → enzyme / DNA protection (submicroscopic), laying the foundation for the efficient extraction of intestinal flora metabolites.

[0035] Zn 2+ / Fe 3+ In ethylene glycol solvent, a spinel structure (ZnFe2O4) is formed, and its crystal space group is Fd3m, Fe 3+ Occupies octahedral sites, Zn 2+ Occupying tetrahedral sites, this arrangement produces superparamagnetism (saturation magnetization ~65 emu / g), which drives the nanoparticles to move to the surface of the biofilm under a 0.5 T magnetic field and fixes Zn on the surface through ion exchange. 2+ / Fe 3+ Finally, these metal ions act as Lewis acid sites, capturing bacterial membrane phospholipids (such as negatively charged phosphatidylserine) through electrostatic interaction, thereby enhancing the adsorption capacity of the nanocarriers to the bacterial community. The mesoporous silica shell precisely matches the molecular size of glutathione (GSH) (kinetic diameter ~0.8 nm) and is controlled by pH response: under acidic conditions (pH 3.0), GSH is positively charged (isoelectric point pI=5.9) and is electrostatically captured by the amino-modified pores (-NH2); under neutral conditions (pH 7.4), GSH becomes negatively charged and confined within the pores. TEOS hydrolysis then forms a Si-O-Si covalent network to seal the pore opening, reducing the GSH leakage rate. The β-pleated conformation of the CHBP peptide (KLVFFAK) specifically binds to the hydrophobic pocket of the Gram-negative bacterial outer membrane protein OmpA, increasing the enrichment efficiency of MSN on the bacterial membrane surface by 3 times; the long-chain polyethylene glycol of mPEG5000 forms a steric hindrance, inhibiting the adsorption of fecal proteins on the MSN surface (reducing the "protein corona" effect) and maintaining the dispersion stability of the nanoparticles.

[0036] In traditional liposomes, free GSH easily forms [GSH-Mg-EDTA] with EDTA-Mg. 3- Complex (complex constant K=10 5 M- 1 ), reducing the OH removal efficiency, while MSN encapsulation completely isolates this side reaction, and GSH reduces Fe 2+ , , Fe 2+ Triggering a Fenton-like reaction, , and then the generated ·OH is removed by the adjacent GSH to form , while regenerating Fe 3+ Entering the next cycle, 1 molecule of GSH can remove 4 molecules of OH (the original liposome is only 1:1), Zn 2+ Activate the synthesis of metallothionein (MT), cooperate with GSH to remove hydroxyl radicals (·OH), and the MSN pores limit Zn 2+ / Fe 3+ Contact with other components in the preservation solution (such as EDTA-Mg) to prevent them from being chelated and inactivated; and the additional addition of Ca 2+ Stabilize the β-barrel structure of the enzyme protein, increase the enzyme activity retention rate, Mg 2+ It chelates the bacterial DNA phosphate backbone, blocks the cleavage site of DNase I, and increases DNA integrity.

[0037] The preservation solution of Example 2 was verified using the same test method as in Example 1, with the only difference being that the amount of glutathione loaded into the core-shell MSN used in Example 2 was also verified, with 0.1% (w / v) liposome-encapsulated glutathione serving as the control group. The results are shown in Tables 5 and 6. Table 5 7-day storage experimental verification results in Example 2

[0038] Table 6 Experimental verification results of 14-day storage in Example 2

[0039] Example 3: Example 2 is further improved on the basis of Example 2 by using three-level synergy to improve the utilization rate of glutathione, thereby increasing the extraction activity of the bacterial flora and simplifying the production process.

[0040] The core-shell MSNs include core-shell MSNs with three different core structures: high magnetic particles with Fe3O4@Co as the core, medium magnetic particles with MnFe2O4 as the core, and low magnetic particles with γ-Fe2O3 as the core. The mass ratio is 0.5:1:1.5. The high magnetic particles are loaded with 0.2 mM zinc ions, the medium magnetic particles are loaded with 10 wt% GSH and 0.1 mM iron ions, and the geomagnetic particles are loaded with 20 wt% trehalose. Fe3O4@Co was prepared by dispersing ferroferric oxide in ethylene glycol, then adding CoCl2·6H2O and polyvinylpyrrolidone (PVP), passing nitrogen gas to remove oxygen, heating to 180°C, and slowly adding NaBH4 solution dropwise. After reacting for 2 hours, Fe3O4@Co particles were collected by centrifugation. MnFe2O4 was prepared by mixing Mn(NO3)2 and Fe(NO3)3 in a 1:2 molar ratio, adding NaOH solution (pH=12) dropwise, stirring at 80℃ for 2 hours, centrifugal washing, and high temperature annealing (600℃, 2 hours, air atmosphere).

[0041] γ-Fe2O3 is prepared by heating Fe3O4 nanoparticles to 200-300℃ in an air atmosphere and oxidizing them for 2 hours.

[0042] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Co doping makes the saturation magnetization reach 85 emu / g (pure Fe3O4 is only 60 emu / g), and the moving speed is improved under 0.5 T magnetic field. 2+ Through the controlled release of the PVP layer (release rate ≈ 60% in 72h), it binds to the ZnuABC transporter in the bacterial membrane and promotes the expression of DNA repair protein (RecA); Mn 2+ / Fe 3+ The alternating valence state generates heat under an alternating magnetic field (42±1℃), and local temperature rise promotes the release of GSH. The weak magnetism (25 emu / g) reduces particle collisions and prevents trehalose leakage. Trehalose dehydrates on the surface of the biofilm to form an amorphous glass layer, inhibiting ice crystal damage.

[0043] Fe3O4@Co is distributed on the surface of the biofilm and quickly delivers Zn 2+DNA is stabilized, MnFe2O4 is distributed in the peribacterial space, GSH is released to eliminate extracellular ROS, and γ-Fe2O3 is distributed inside the bacterial biofilm, where trehalose penetrates and protects intracellular proteins.

[0044] Verify the performance comparison after saving for 7 days. The results are shown in Table 7. Table 7 Magnetic gradient matching verification of Example 3

[0045] The recovery rate of Fe3O4@Co is 98%, and it can be reused more than 50 times. The recovery rate of MnFe2O4 is 85%, and it can be reused more than 30 times. The recovery rate of γ-Fe2O3 is 92%, and it can be reused more than 100 times.

[0046] Example 4: When the electromagnetic array does not dissipate enough heat, the magnetic field intensity fluctuates, and the particles are layered and mixed, it is easy to cause cross-contamination of high / low magnetic particles and disorder of the repair-protection sequence. Further improvement is made by utilizing the fluctuation.

[0047] The magnetic field includes a steady-state magnetic field and a chaotic magnetic field. The steady-state magnetic field is 0.5T, and the chaotic magnetic field has a change rate of 0.1-1.5T / s. The steady-state magnetic field and the chaotic magnetic field are used alternately and are changed every 5 minutes.

[0048] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: By actively using a chaotic magnetic field, the layered structure is disrupted and forced to mix, resulting in global coverage of disordered diffusion. This allows particles to accumulate (highly magnetic particles aggregate) at the edges or corners of the container, leading to the re-dispersion of particles in heat dissipation dead zones or unevenly distributed reactants. Random pulses generate a magnetic vortex effect, which draws accumulated particles into the flow zone, lowering the local temperature peak and forcing the contact area between the fungi and other substances to increase metabolic efficiency by 22%. Through magnetic turbulence, different magnetic particles are deeply interwoven, forming a three-dimensional network structure, increasing the specific surface area, generating a microscale cavitation effect in the liquid phase, and breaking the diffusion boundary layer. Through the use of a chaotic magnetic field, the bacterial activity can reach 8.7 CFU / g, ×10 8 , which greatly improves the activity of the bacterial flora and lays a good foundation for the subsequent extraction of metabolites.

[0049] It should be noted that, in the above embodiments, the description of each embodiment has its own focus. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0050] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0051] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an extract of intestinal flora metabolites from American cockroach feces, characterized in that: The following steps are involved: S1. After collecting feces, pre-treat them and then preserve them in fecal preservation solution. Mix the feces with the preservation solution at a ratio of 1g:5ml to prepare a fecal bacterial suspension, which is then stored at 5°C. The fecal preservation solution consisted of 5 mM EDTA-Mg, 15% glycerol, 2% mealworm chitin hydrolyzate, 1% symbiotic bacterial metabolite extract, 0.1% glutathione, and the rest was solvent water; S2, the American cockroach feces suspension of preservation was placed in a freeze dryer, -50 ℃ of pre-freeze, then vacuum dried, pulverized through a 20 mesh sieve, to obtain a coarse powder, which was then subjected to ethanol reflux extraction, and extracted 3 times, each time for 2 hours. Every 1kg feces coarse powder was added with 10L ethanol solution, and each extraction used different ethanol concentrations. From the first to the third time, 20% aqueous ethanol solution, 50% aqueous ethanol solution, and 90% aqueous ethanol solution were used sequentially, and the filtrate was combined to remove the residue. S3. The filtrate is concentrated under reduced pressure at 70°C to an extract, the ethanol is recovered, and water is added to the extract at a mass ratio of 1:5 and stirred to form a dispersion; S4. Extract the dispersion with petroleum ether, ethyl acetate, and n-butanol in sequence, extract with each solvent 5 times, and combine the extracts with the same solvent; then concentrate the ethyl acetate and n-butanol extracts under reduced pressure, recover the solvents, and then freeze-dry to obtain a powdered metabolite extract.

2. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 1, characterized in that: The feces pretreatment steps are as follows: after freezing and grinding in liquid nitrogen, add 1% Triton X-100, mix evenly, pretreat at 37°C for 10 minutes, ultrasonic fragmentation pulse mode for 5 minutes, which increases the fragmentation efficiency by 30%, use recombinant chitinase Chit42 at a concentration of 2 U / mg feces, add 0.5 mM MgCl2, and incubate at 37°C for 1.5 hours.

3. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 1, characterized in that: Glutathione is encapsulated by liposomes, and the preparation method is as follows: liposome-encapsulated glutathione is composed of dipalmitoylphosphatidylcholine, cholesterol, and phospholipid-polyethylene glycol, with dipalmitoylphosphatidylcholine: cholesterol: phospholipid-polyethylene glycol = 6:3:

1. The lipid mixture is dissolved in chloroform, rotary evaporated to form a film, and vacuum dried overnight. The lipid film is hydrated with PBS containing 5 mM glutathione, and liposomes with a particle size of 100±20 nm are obtained by high-pressure homogenization. Free GSH is removed by ultrafiltration and centrifugation.

4. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 1, characterized in that: The chitin hydrolyzate of mealworm was prepared by grinding the dried mealworm shells through a 100-mesh sieve and mixing them with 6 M HCl at a ratio of 1:

10. The mixture was refluxed at 110°C for 4 hours, cooled and centrifuged, and the supernatant was neutralized with NaOH to pH 7.

0. The supernatant was concentrated to 1 / 5 of the original volume by rotary evaporation and freeze-dried to obtain a light yellow hydrolyzate powder.

5. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 1, characterized in that: The symbiotic bacteria metabolite extract was prepared by inoculating human intestinal symbiotic bacteria into culture medium, removing the bacteria by centrifugation, filtering the supernatant through a 0.45 μm filter membrane, and filtering the filtrate through a 3 kDa ultrafiltration membrane to retain small molecule metabolites. The extract was then freeze-dried to obtain a brown extract.

6. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 1, characterized in that: Glutathione is loaded on core-shell structure particles. The specific preparation method is: ZnFe2O4 magnetic nanoparticles are synthesized by hydrothermal method, loaded with Zn(NO3)2 and FeCl3, coated with mesoporous silica by sol-gel method, and then modified with chitin-binding peptide and polyethylene glycol, and finally loaded with glutathione.

7. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 2, characterized in that: During the pretreatment process, 0.5 mM MgCl2 was added together with 5 mM CaCl2, and the cells were incubated at 37°C for 1.5 hours under the guidance of a 0.5 T magnetic field.

8. The method for preparing an extract of intestinal flora metabolites from American cockroach feces according to claim 6, characterized in that: The core-shell structure MSNs include core-shell structure MSNs with three different core structures, including high magnetic particles with Fe3O4@Co as the core, medium magnetic particles with MnFe2O4 as the core, and low magnetic particles with γ-Fe2O3 as the core, with a mass ratio of 0.5:1:1.

5. The high magnetic particles are loaded with 0.2 mM zinc ions, the medium magnetic particles are loaded with 10 wt% glutathione and 0.1 mM iron ions, and the geomagnetic particles are loaded with 20 wt% trehalose.

9. An extract of intestinal flora metabolites from American cockroach feces, characterized in that: Prepared by any one of the preparation methods of claims 1-9.

10. A use of the extract of intestinal flora metabolites from American cockroach feces as claimed in claim 9, characterized in that: Used to treat inflammation.

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