Intestinal flora excrement sample preserving fluid and preparation method thereof

Abstract

The invention relates to the technical field of biomedicine and microorganisms, and provides an intestinal flora excrement sample preserving fluid which comprises a buffer system, a chelating agent, a reductive anaerobic protective agent, a cell lysis-bacteriostatic agent and a biomacromolecular stabilizer, wherein the buffer system is a combination of a Tris-HCl buffer solution and sodium chloride, the chelating agent is ethylenediamine tetraacetic acid disodium salt, the reductive anaerobic protective agent is L-cysteine hydrochloride, the cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, and the biomacromolecular stabilizer is a combination of trehalose and glycerin. The invention also provides a preparation method of the preserving fluid. According to the invention, the microbial DNA and community structure in the excrement can be stably preserved at room temperature for a long time without a cold chain; meanwhile, the preservation solution can effectively protect DNA of strict anaerobic bacteria from oxidative degradation, flora composition is prevented from shifting, and the reliability of subsequent molecular detection and analysis is finally ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical and microbiological technology, and particularly relates to a fecal sample preservative for intestinal flora and a preparation method thereof. BACKGROUND

[0002] In the study of human intestinal microbiome, how to effectively preserve the microbial community structure and nucleic acid integrity of the fecal sample after sampling is a core link to ensure the accuracy of subsequent analysis. Ideally, fresh fecal samples should be analyzed as soon as possible or immediately preserved at-20°C or-80°C for low-temperature freezing to maintain the original state of the sample to the greatest extent. However, in actual application scenarios, immediate low-temperature preservation is often difficult to achieve, especially for large-scale cohort studies, home sampling or sample collection in remote areas.

[0003] Under room temperature conditions, the DNA in the feces will rapidly degrade, and the metabolic activity of the microorganisms will cause changes in the community structure. However, the existing chemical preservation schemes have many limitations and cannot meet the three core requirements of "long-term room temperature preservation", "unbiased protection of the microbial community" and "compatibility with downstream molecular analysis". The specific problems are as follows: Limitations of direct freezing method: As the "gold standard" for microbial community preservation, direct freezing (such as-80°C long-term preservation) can well maintain the original state of the microbial community, but it has very high requirements for the cold chain conditions and cannot adapt to the transportation requirements of remote sampling or home sampling. In addition, repeated freezing and thawing will cause DNA degradation and changes in the community structure, further limiting its application range.

[0004] Defects of traditional chemical preservatives: Commonly used traditional chemical preservatives, such as RNAlater solution, 95% ethanol and Tris-EDTA (TE) buffer, all have obvious shortcomings. Among them, RNAlater has been widely used in microbiome research, but subsequent studies have shown that its preservation effect is poor and the microbial community structure is significantly different from that of fresh samples. Although 95% ethanol is low in cost and can preserve samples at room temperature for several weeks, and the microbial community after preservation has a higher correlation with the frozen sample, ethanol is flammable and its transportation is limited. For low-biomass samples (such as skin swabs), ethanol will also reduce the abundance of microbial cells and disturb the community composition. The protective effect of TE buffer on fecal samples is weak, and some studies have shown that its preservation effect is similar to that of samples without any addition, and it is difficult to effectively prevent changes in the community.

[0005] Limitations of commercialized special-purpose preservative reagents: In recent years, commercialized preservative kits such as OMNIgene•GUT, DNA / RNA Shield, PrimeStore MTM have appeared. Although most of them are composite formulations and can stabilize samples at room temperature for a short period of time (several days to two weeks) (such as OMNIgene•GUT, which claims to be stable at room temperature for 14-21 days), they have three major problems: first, they are expensive and not conducive to large-scale popularization and application; second, different reagents have different preferences and biases in preserving the flora, and some reagents (such as Norgen preservative solution, which has better effect, but RNAlater, FTA and simple TE buffer have poorer effect) cannot protect the flora comprehensively; third, some solutions containing denaturing reagents (such as guanidine salt) can inactivate pathogens and stabilize nucleic acids, but may have potential interference on downstream analysis.

[0006] Limitations of new preservation schemes: To solve the defects of existing schemes, some researchers have explored new material preservation technologies. For example, the BGI team proposed the N-octylpyridinium bromide (NOPB) preservation method, which can preserve samples at room temperature for at least 14 days. Hao et al. studied an ionic liquid-based preservative, which can improve the medium-term preservation stability. However, these schemes still have shortcomings: the room temperature preservation time is generally not more than 4 weeks, and most of them have only verified the effectiveness for 1-2 weeks; some reagents (such as NOPB) have residual toxicity, which may inhibit downstream sequencing; and a few companies claim long-term preservation schemes (such as Norgen preservation tube, which claims to preserve DNA for more than 2 years at 15-25°C), which rely on high-concentration chemical reagents or drying technology, have high cost and the formula is not disclosed, making it difficult for researchers to optimize and improve.

[0007] In summary, the existing fecal preservation technology still has obvious shortcomings in long-term preservation (≥4 weeks) at room temperature, comprehensive protection of anaerobic bacterial DNA, and maintaining the neutrality of the flora structure. The composition of the flora changes significantly after 48 hours of room temperature storage of samples without preservation treatment, and there is often excessive proliferation of facultative anaerobes and attenuation of sensitive species. Even with some preservatives, due to the limitations of the composition design, it is easy to favor the preservation of certain flora or introduce new biases. Therefore, it is urgent to develop a new formula of intestinal flora fecal sample preservative, break through the limitations of traditional materials, achieve efficient preservation at room temperature for 4 weeks or more, effectively protect the overall microbial community structure and DNA integrity including strict anaerobes, and meet the subsequent molecular detection needs. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a new formula of intestinal flora fecal sample preservation liquid and a preparation method thereof, which can stably store microbial DNA and community structure in feces at room temperature for a long time (at least 4 weeks) without cold chain; at the same time, the preservation liquid can effectively protect the DNA of strict anaerobes from oxidative degradation, prevent the shift of flora composition due to oxygen exposure and microbial metabolism, and ultimately ensure the reliability of subsequent molecular detection analysis, and make up for the short board of the prior art in the three core needs of "long-term room temperature storage", "unbiased protection of flora", and "compatible downstream molecular analysis".

[0009] The present application solves the above technical problems by adopting the following technical solutions: The intestinal flora fecal sample preservation liquid comprises the following components: a buffer system, a chelating agent, a reducing anaerobic protective agent, a cell lysis-bacteriostatic agent, and a biological macromolecule stabilizer. The buffer system is a combination of Tris-HCl buffer and sodium chloride, wherein the pH value of the Tris-HCl buffer is 7.5-8.0, the final concentration in the preservation liquid is 40-60 mM, and the final concentration of sodium chloride is 10-50 mM. The chelating agent is disodium ethylenediaminetetraacetate (EDTA), and the final concentration in the preservation liquid is 10-100 mM. The reducing anaerobic protective agent is L-cysteine hydrochloride, and the final concentration in the preservation liquid is 0.3-1% w / v. The cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, and the final concentration in the preservation liquid is 1-2% w / v. The biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the preservation liquid is 8-12% w / v, and the final concentration of glycerol is 12-18% w / v.

[0010] As one of the preferred modes of the present application, the pH value of the Tris-HCl buffer is 8.0, and the final concentration in the preservation liquid is 50 mM; the final concentration of sodium chloride in the preservation liquid is 20 mM.

[0011] As one of the preferred modes of the present application, the final concentration of disodium ethylenediaminetetraacetate in the preservation liquid is 50 mM.

[0012] As one of the preferred modes of the present application, the final concentration of L-cysteine hydrochloride in the preservation liquid is 0.5% w / v.

[0013] As one of the preferred modes of the present application, the final concentration of sodium lauroyl sarcosinate in the preservation liquid is 1% w / v.

[0014] As one of the preferred modes of the present application, the final concentration of trehalose in the preservation solution is 10% w / v, and the final concentration of glycerol in the preservation solution is 15% w / v.

[0015] As one of the preferred modes of the present application, an antioxidant and a bacteriostatic agent are further included.

[0016] As one of the preferred modes of the present application, the antioxidant is tea polyphenol (such as epigallocatechin gallate EGCG) or rosemary extract, and the final concentration in the preservation solution is 0.1% w / v; the bacteriostatic agent is lauric acid ester (such as acetylated lauroyl arginine LAE), and the final concentration in the preservation solution is 0.05% w / v.

[0017] A preparation method of the above-mentioned preservation solution, comprising the following steps: S1, adding Tris base to deionized water, stirring to dissolve, then adjusting the pH value to 7.5-8.0 with hydrochloric acid, and then adding sodium chloride and stirring to dissolve, followed by adding disodium ethylenediaminetetraacetate and stirring to dissolve, to obtain a buffer base solution; S2, adding glycerol and trehalose to the buffer base solution obtained in step S1, and stirring until completely dissolved to obtain a mixed solution; S3, after the mixed solution obtained in step S2 is cooled to room temperature, L-cysteine hydrochloride is added and stirred until dissolved, and then sodium lauroyl sarcosinate is added and stirred until dissolved; then, the mixed solution is used in the subsequent step S4, or after further adding an antioxidant and a bacteriostatic agent, it is used in step S4; S4, adding deionized water to the solution obtained in step S3 to make up the volume, and then sterilizing by filtration to obtain a preservation solution for intestinal flora fecal samples.

[0018] As one of the preferred modes of the present application, in step S3, the addition of cysteine slightly reduces the pH, and if the pH value of the mixed solution deviates from 7.5-8.0, the pH value is adjusted to 7.5-8.0 with a base solution.

[0019] As one of the preferred modes of the present application, in step S4, the sterilization by filtration uses a 0.22 μm filter membrane.

[0020] Core component efficacy: Buffer system: the pH is stabilized at 7.5-8.0 with a weak alkaline buffer, and the osmotic pressure is maintained with 10-50 mM sodium chloride; among them, the weak alkaline environment is beneficial to the stability of DNA and reduces the risk of acid hydrolysis.

[0021] Chelating agent: the addition of EDTA (disodium ethylenediaminetetraacetate) with a final concentration of 10-50 mM can effectively complex divalent metal ions (such as Mg 2+ , Ca 2+), inhibit DNase and other nuclease activity; wherein, EDTA can also destroy the stability of the outer membrane of gram-negative bacteria, and synergize with subsequent components to enhance the bacteriostatic and DNA-protecting effects.

[0022] Reducing anaerobic protective agent: adding reducing natural compounds (L-cysteine hydrochloride) to scavenge dissolved oxygen and simulate an anaerobic environment, which can consume dissolved oxygen, generate dithiols to reduce the oxidation-reduction potential, and protect the structure of anaerobic microorganisms from direct damage by oxygen.

[0023] Cell lysis-bacteriostatic agent: introducing a natural fatty acid-derived anionic surfactant (such as sodium lauroyl sarcosinate), which can destroy microbial cell membranes at a concentration of 1-2% w / v, killing most bacteria (including aerobic and anaerobic bacteria), and instantaneously "freezing" the original community structure, and denaturing gently to avoid excessive shearing damage to DNA, ensuring DNA length and downstream analysis quality.

[0024] Biological macromolecule stabilizer: adding natural trehalose and polyols (glycerol), preferably 10% trehalose + 15% glycerol (w / v); wherein, trehalose stabilizes nucleic acid structure through hydrogen bonding, and glycerol increases solution viscosity and reduces water activity, both forming a "bioglass" microenvironment to slow down DNA degradation, breaking through the limitations of traditional preservation solutions.

[0025] Antioxidants and bacteriostatic agents: to further prevent DNA oxidation and microbial degradation, low concentrations of natural phenolic antioxidants or broad-spectrum bacteriostatic agents can be added; for example, natural antioxidants such as tea polyphenols (such as epigallocatechin EGCG) or rosemary extract can provide additional free radical scavenging effects to protect nucleic acids from oxidative damage; in addition, trace amounts of food-grade bacteriostatic agents such as lauric acid esters (such as acetylated lauroyl arginine, LAE) can be added to synergistically inhibit the reproduction of residual microorganisms; these adjuvants are of natural origin or derivatives, safe and non-toxic, and in this invention, they are only used to synergistically enhance, and are not necessary.

[0026] The synergistic effect of the above core components achieves long-term preservation of fecal microbial samples through a comprehensive strategy of "alkaline buffer + enzyme inhibition by chelating agent + deoxygenation by reducing agent + broad-spectrum sterilization by surfactant + DNA protection by stabilizer", meeting the three core requirements of "long-term room temperature preservation", "unbiased protection of microbial community", and "compatibility with downstream molecular analysis".

[0027] The advantages of the present invention over the prior art are: 1. Breakthrough in long-term room temperature preservation, without the need for cold chain This invention utilizes the synergistic effect of a "Tris-HCl buffer system + L-cysteine ​​hydrochloride + trehalose-glycerol composite stabilizer" to stably preserve fecal samples at room temperature (15~25°C) for more than 4 weeks, with experimental verification showing a maximum preservation period of up to 8 weeks. During the preservation period, the DNA yield and quality of the samples do not decrease significantly, fully meeting the needs of downstream molecular analysis.

[0028] 2. Provides comprehensive protection for the DNA of strictly anaerobic bacteria, preventing oxidative damage to the bacterial community. This invention innovatively introduces L-cysteine ​​hydrochloride as a reducing anaerobic protectant, which can create a localized anaerobic, low-oxidative-stress environment in the sample, effectively preventing the growth of strictly anaerobic bacteria (such as Bacteroides). Bacteroides Butyric acid-producing bacteria Faecalibacterium The DNA of anaerobic bacteria (such as the proportion of large DNA fragments) degrades rapidly upon exposure to air; experimental results show that after 4 weeks of treatment with this preservation solution, the integrity indicators of anaerobic bacterial DNA (such as the proportion of large DNA fragments) are almost no different from those of fresh samples.

[0029] 3. Protect the fungal community structure and avoid deviations and interference. This invention utilizes a cell lysis-antibacterial agent (sodium lauroyl sarcosinate) to rapidly kill and fix various bacteria in samples, maximizing the preservation of the original fecal microbial community composition. Compared to immediate freezing as a baseline, the α-diversity (Shannon index) and β-diversity (community differences) of the samples after 4 weeks of preservation showed no significant changes, and the community structure was highly correlated (Bray-Curtis dissimilarity was extremely low, see experimental data described later). This means that the preservation solution of this invention does not selectively enrich or lose specific bacterial groups, avoiding structural deviations caused by the proliferation or death of certain bacteria in traditional preservation methods.

[0030] 4. Compatible with downstream molecular analysis, with no residual interference. The formulation of this invention does not contain any components that strongly inhibit PCR or sequencing. Samples treated with this preservation solution can be directly processed into the conventional nucleic acid extraction process. The surfactants and other components in the formulation (such as sodium lauroyl sarcosinate) are easily removed or washed away during the extraction process, leaving no residual interference to the enzyme reaction. The DNA purity (A260 / 280 ratio) and integrity of the samples treated with the preservation solution meet the requirements of 16S amplicon sequencing and metagenomic sequencing, and the species profile of the sequencing results is consistent with that of fresh samples.

[0031] 5. Safe, environmentally friendly, and cost-controllable, making it easy to promote on a large scale. The core components (Tris-HCl, sodium chloride, L-cysteine hydrochloride, trehalose, glycerol, etc.) of the application are natural or biocompatible materials, non-toxic, non-irritating, safe for operators and the environment; tea polyphenols, acetylated lauroyl arginine (LAE) and other auxiliary agents are food grade or natural sources, further improving safety; at the same time, all raw materials are easy to obtain, and the preparation process only needs common steps such as dissolution, constant volume, sterile filtration, etc., the cost is much lower than that of commercialized patent reagent kit, especially suitable for large-scale cohort study, primary medical sampling and other scenes, and has wide scientific research and industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the comparison of fecal sample flora diversity under different preservation conditions (in the figure, A is the alpha diversity Shannon index, and B is the Bray-Curtis distance relative to the fresh baseline sample, that is, the beta diversity difference; it can be seen that the diversity and community composition of the preservation liquid group are highly close to the baseline, which is obviously better than ethanol, RNAlater and the unsaved group; the error bar represents the standard deviation of three biological repeats); Figure 2 is the composition of the main bacterial phylum under different preservation conditions (in the figure, each column represents a preservation condition LN, PB, EtOH, RNA or None, and the stacking of each column shows Firmicutes , Bacteroidetes , Proteobacteria , Others The proportion of the four bacterial phyla can be directly seen: the structure of LN and PB groups is almost the same; the EtOH and RNA groups are slightly offset; the None group appears significant structural imbalance, Proteobacteria Significant increase); Figure 3 is the relative abundance of key genera under different preservation conditions of the application (in the figure, each column represents the stacking distribution of five key genera Faecalibacterium , Bacteroides , Prevotella , Megamonas , Escherichia / Shigella And among them: Faecalibacterium and Bacteroides are stable in LN and PB groups; in the None group Escherichia / Shigella obvious proliferation (accounting for more than 10%), indicating that a large number of facultative bacteria proliferate in the sample without preservation liquid; the RNA group also shows that some anaerobic genera decrease).

[0033] Figure 4 is a PCoA dimensionality reduction analysis chart (in the figure, different colors represent different preservation conditions, and the dotted ellipse represents the clustering range of the samples in the group. The coordinate axes PCoA1 and PCoA2 are marked with the explained variation proportion, reflecting the overall difference and similarity of the fecal microbial community structure under different preservation conditions); Figure 5 The sequencing data quality comparison results (in the figure, the blue column represents the average sequence length bp, and the red line represents the GC content %; the LN and PB groups are almost the same, showing high-quality long reads and stable GC content; the None group is significantly reduced, indicating that the DNA of the sample without the preservative solution is broken and the GC content is severely deviated). DETAILED DESCRIPTION

[0034] The following detailed description of the embodiments of the present application is based on the technical solutions of the present application, and detailed implementation methods and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0035] Example 1 The fecal sample preservative solution of the enteric flora of the present embodiment comprises the following components: a buffer system, a chelating agent, a reducing anaerobic protective agent, a cell lysis-bacteriostatic agent, and a biological macromolecule stabilizer.

[0036] In the present embodiment, the buffer system is a combination of Tris-HCl buffer (pH 8.0) and sodium chloride, wherein the final concentration of Tris-HCl buffer in the preservative solution is 50 mM, and the final concentration of sodium chloride is 20 mM; the chelating agent is EDTA, and the final concentration in the preservative solution is 50 mM; the reducing anaerobic protective agent is L-cysteine hydrochloride, and the final concentration in the preservative solution is 0.5% w / v; the cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, and the final concentration in the preservative solution is 1% w / v; and the biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the preservative solution is 10% w / v, and the final concentration of glycerol is 15% w / v.

[0037] Preparation method: S1, add Tris base to deionized water, stir to dissolve, adjust the pH value to 8.0 with hydrochloric acid, then add sodium chloride and stir to dissolve, then add EDTA and stir to dissolve, to obtain a buffer base solution.

[0038] S2, add glycerol and trehalose to the buffer base solution obtained in step S1, stir until completely dissolved, to obtain a mixed solution. If dissolution is difficult, appropriate warming (≤40°C) can be used to promote dissolution. The obtained solution should be a clear colorless viscous liquid.

[0039] S3, cool the mixed solution obtained in step S2 to room temperature, add L-cysteine hydrochloride and stir until dissolved, then add sodium lauroyl sarcosinate and stir until dissolved, and then adjust the pH value to 8.0 with 1M NaOH.

[0040] S4, add deionized water to the solution obtained in step S3 to make each component meet the final concentration requirements of each component described above; then, filter through a 0.22 μm filter membrane to obtain the intestinal flora fecal sample preservation solution.

[0041] The preservation solution of this example is a clear slightly viscous colorless solution, which can be stored at room temperature in the dark for at least 12 months without precipitation or activity decline.

[0042] Example 2 The intestinal flora fecal sample preservation solution of this example comprises the following components: buffer system, chelating agent, reducing anaerobic protective agent, cell lysis-bacteriostatic agent, biological macromolecule stabilizer, antioxidant, bacteriostatic agent.

[0043] In this example, the buffer system is a combination of Tris-HCl buffer (pH 8.0) and sodium chloride, wherein the final concentration of Tris-HCl buffer in the preservation solution is 50 mM, and the final concentration of sodium chloride is 20 mM; the chelating agent is EDTA, with a final concentration of 50 mM in the preservation solution; the reducing anaerobic protective agent is L-cysteine hydrochloride, with a final concentration of 0.5% w / v in the preservation solution; the cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, with a final concentration of 1% w / v in the preservation solution; the biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the preservation solution is 10% w / v, and the final concentration of glycerol is 15% w / v; the antioxidant is epicatechin, with a final concentration of 0.1% w / v in the preservation solution; and the bacteriostatic agent is acetylated lauroyl arginine, with a final concentration of 0.05% w / v in the preservation solution.

[0044] Preparation method: S1, add Tris base to deionized water, stir to dissolve, then adjust the pH to 8.0 with hydrochloric acid, then add sodium chloride and stir to dissolve, then add EDTA and stir to dissolve to obtain a buffer base solution.

[0045] S2, add glycerol and trehalose to the buffer base solution obtained in step S1, stir until completely dissolved to obtain a mixture. If dissolution is difficult, appropriate warming (≤40°C) can be used to promote dissolution. The resulting solution should be a clear colorless viscous liquid.

[0046] S3, cool the mixture obtained in step S2 to room temperature, add L-cysteine hydrochloride and stir until dissolved, then add sodium lauroyl sarcosinate and stir until dissolved, adjust the pH to 8.0 with 1M NaOH; then, continue to add epicatechin and acetylated lauroyl arginine and stir until dissolved.

[0047] S4, add deionized water to the solution obtained in step S3 to make each component meet the final concentration requirements of each component described above; then, filter through a 0.22 μm filter membrane to obtain the intestinal flora fecal sample preservative.

[0048] The preservative solution of this example is a clear slightly sticky colorless solution, which can be stored at room temperature in the dark for at least 12 months without precipitation or activity decrease.

[0049] Example 3 The intestinal flora fecal sample preservative of this example is basically the same as that of Example 2, the main difference being that the final concentration of L-cysteine hydrochloride in the preservative solution of this example is adjusted to 1% to enhance the deoxygenation ability of the solution and further protect the DNA of strict anaerobes, which is suitable for the preservation of samples collected in extreme anaerobic environments.

[0050] Example 4 The intestinal flora fecal sample preservative of this example is basically the same as that of Example 2, the main difference being that the final concentration of EDTA in the preservative solution of this example is adjusted to 100 mM to strengthen the complexing effect on divalent metal ions and more completely inhibit nuclease activity, which is suitable for the preservation of samples that may contain high nuclease.

[0051] Example 5 The intestinal flora fecal sample preservative of this example comprises the following components: buffer system, chelating agent, reducing anaerobic protective agent, cell lysis-bacteriostatic agent, biological macromolecule stabilizer, antioxidant, bacteriostatic agent.

[0052] In this example, the buffer system is a combination of Tris-HCl buffer (pH 7.5) and sodium chloride, wherein the final concentration of Tris-HCl buffer in the preservative solution is 40 mM and the final concentration of sodium chloride is 10 mM; the chelating agent is EDTA, with a final concentration of 10 mM in the preservative solution; the reducing anaerobic protective agent is L-cysteine hydrochloride, with a final concentration of 0.3% w / v in the preservative solution; the cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, with a final concentration of 1.5% w / v in the preservative solution; the biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the preservative solution is 8% w / v and the final concentration of glycerol is 12% w / v; the antioxidant is epicatechin, with a final concentration of 0.1% w / v in the preservative solution; and the bacteriostatic agent is acetylated lauroyl arginine, with a final concentration of 0.05% w / v in the preservative solution.

[0053] Preparation method: S1, add Tris base to deionized water, stir to dissolve, adjust the pH to 7.5 with hydrochloric acid, then add sodium chloride and stir to dissolve, then add EDTA and stir to dissolve to obtain a buffer base solution.

[0054] S2, glycerol and trehalose were added to the buffer base solution obtained in step S1, and stirred until completely dissolved to obtain a mixed solution. If dissolution is difficult, appropriate warming (≤40°C) can be used to promote dissolution. The obtained solution should be a clear colorless viscous liquid.

[0055] S3, after the mixed solution obtained in step S2 was cooled to room temperature, L-cysteine hydrochloride was added and stirred until dissolved, then sodium lauroyl sarcosinate was added and stirred until dissolved, and the pH value was adjusted to 7.5 with 1M NaOH; subsequently, epicatechin and acetylated lauroyl arginine were continuously added and stirred until dissolved.

[0056] S4, deionized water was added to the solution obtained in step S3 to make up the volume, so that each component meets the final concentration requirement of each component described above; subsequently, it was filtered through a 0.22μm filter membrane to obtain an intestinal flora fecal sample preservation solution.

[0057] The preservation solution of this example is a clear slightly viscous colorless solution, which can be stored at room temperature in the dark for at least 12 months without precipitation or activity decline.

[0058] Example 6 An intestinal flora fecal sample preservation solution of this example includes the following components: buffer system, chelating agent, reducing anaerobic protective agent, cell lysis-bacteriostatic agent, biological macromolecule stabilizer, antioxidant, bacteriostatic agent.

[0059] In this example, the buffer system is a combination of Tris-HCl buffer (pH 7.8) and sodium chloride, wherein the final concentration of Tris-HCl buffer in the preservation solution is 60mM, and the final concentration of sodium chloride is 50mM; the chelating agent is EDTA, with a final concentration of 100mM in the preservation solution; the reducing anaerobic protective agent is L-cysteine hydrochloride, with a final concentration of 1% w / v in the preservation solution; the cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, with a final concentration of 2% w / v in the preservation solution; the biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the preservation solution is 12% w / v, and the final concentration of glycerol is 18% w / v; the antioxidant is epicatechin, with a final concentration of 0.1% w / v in the preservation solution; the bacteriostatic agent is acetylated lauroyl arginine, with a final concentration of 0.05% w / v in the preservation solution.

[0060] Preparation method: S1, Tris base was added to deionized water, stirred and dissolved, then the pH value was adjusted to 7.8 with hydrochloric acid, sodium chloride was added and stirred until dissolved, and then EDTA was added and stirred until dissolved to obtain a buffer base solution.

[0061] S2, glycerol and trehalose were added into the buffer solution obtained from step S1, and stirred until completely dissolved to obtain a mixed solution. If dissolution is difficult, appropriate warming (≤40°C) can be used to promote dissolution. The obtained solution should be a clear colorless viscous liquid.

[0062] S3, the mixed solution obtained from step S2 was cooled to room temperature, L-cysteine hydrochloride was added and stirred until dissolved, then sodium lauroyl sarcosinate was added and stirred until dissolved, and the pH value was adjusted to 7.8 with 1M NaOH; subsequently, epicatechin and acetylated lauroyl arginine were continuously added and stirred until dissolved.

[0063] S4, deionized water was added to the solution obtained from step S3 to make up the volume, so that each component meets the final concentration requirement of each component described above; subsequently, filtration was performed through a 0.22 μm filter membrane to obtain a fecal sample preservation solution for intestinal flora.

[0064] The preservation solution of the present embodiment is a clear slightly viscous colorless solution, which can be stably stored at room temperature away from light for at least 12 months without precipitation or activity decline.

[0065] Example 7 The storage and use method of the above preservation solution of the present embodiment.

[0066] 1, Sampling tool preparation A randomly selected fresh human fecal sample was used to simulate the actual household sampling process. Before sampling, the preservation solution preloaded tube (containing 5 mL of the preservation solution of the above embodiment in each tube) of the present invention and a plastic fecal spoon or swab were prepared. The preservation solution preloaded tube should be confirmed to be well sealed and room temperature balanced before use.

[0067] 2, Sample collection The subject collected the feces of pea size (2-3 g) by himself, and tried to take it from the middle part of the feces which had not been in contact with air for a long time. The sample was placed into the preservation solution tube using the fecal spoon; if a swab was used, it was inserted into the preservation solution after being fully dipped in the feces. The tube cap was immediately tightened and vigorously shaken for 15 s to fully mix and contact the fecal sample with the preservation solution. Note: since the preservation solution contains lysis components, shaking will cause the sample to rapidly disintegrate to form a suspension, which is a normal phenomenon.

[0068] 3, Storage conditions The mixed sample preservation solution suspension was allowed to stand for 1 min to complete the cell lysis and protection reaction. Subsequently, it can be stored for a long time at room temperature. In the present embodiment, the sample tube was directly placed in a 25°C room at room temperature for light-free storage.

[0069] 4, Subsequent processing After storage at different time points, the sample was taken for DNA extraction.

[0070] A portion of the preserved samples was taken out at 0 days (immediate treatment), 7 days, 14 days, 30 days, and 60 days for subsequent analysis. At the time of extraction, each time, the sample suspension was first gently shaken to restore the mixed state, and then an equal volume of sample suspension (containing 200 mg of original feces) was taken for standard fecal DNA extraction (using a commercial column extraction kit or the Phenol-Chloroform method). The extraction process can appropriately add proteinase K digestion and denaturation buffer to ensure that the proteins / bacteriostatic agents that may remain in the preservation solution of the application are fully removed. The DNA solution obtained by extraction is redissolved with TE buffer, and then concentration, purity determination, and downstream sequencing library construction are performed.

[0071] To verify the effectiveness of the preservation solution of the application, the following experiment was designed to evaluate the DNA quality and microbial community composition changes of fecal samples under different preservation conditions.

[0072] Example 1, verification of the effectiveness of the preservation solution of the application: I. Experimental design (core goal: systematic comparison of the effects of different preservation conditions) 1. Experimental objects and grouping Sample source: fresh fecal samples from 3 healthy volunteers (referred to as A, B, and C), which were mixed and equally divided immediately after laboratory on-site collection (collection method as in Example 7). Each group had 3 technical repeats, for a total of 3 (volunteers) x 5 (main conditions) x 3 (repeats) = 45 samples.

[0073] The preservation conditions are shown in Table 1.

[0074] Table 1, preservation condition grouping

[0075] 2. Experimental procedures and detection indicators (1) Sample processing: all samples were taken out after 4 weeks of preservation, and DNA was extracted using the same batch of commercial DNA extraction kit (to eliminate extraction bias), and redissolved with TE buffer.

[0076] (2) Core detection indicators: DNA yield and quality detection: DNA concentration and A260 / 280 purity ratio were determined using a NanoDrop spectrophotometer and fluorescence quantification method.

[0077] 16S rRNA gene sequencing analysis: Amplicon library was constructed for the V3-V4 region of 16S gene of each DNA sample, and high-throughput sequencing was performed using the Illumina sequencing platform. At least 30,000 valid sequences were obtained for each sample. QIIME2 software was used for quality control and OTU / ASV classification annotation, and the alpha diversity index (Shannon index, species richness, etc.) of each sample and the Bray-Curtis distance beta diversity index between samples were calculated. The immediate freezing group (LN) was used as the baseline reference, and the differences between other condition groups and the baseline were compared.

[0078] Microbial community composition comparison: The relative abundance of species at the phylum and genus levels of each sample was extracted, and the abundance changes of major anaerobic bacterial groups (such as Clostridia and Bacteroidales) were focused on. At the same time, it was detected whether there was overgrowth of specific flora (such as the proliferation of Enterobacteriaceae under poor storage conditions). The significant differences in flora diversity and composition between different storage conditions were evaluated.

[0079] Through the above experimental design, we can systematically evaluate the performance of the preservation solution of the application in terms of DNA preservation effect and flora structure preservation, and intuitively compare it with the traditional scheme.

[0080] II. Experimental results and analysis The experimental results show that the preservation solution of the application performs excellently in various indicators, successfully realizing the long-term room temperature preservation of intestinal flora fecal samples. The specific analysis is as follows: 1. DNA yield and purity: The DNA yield and quality extracted from samples under different storage conditions are shown in Table 2. It can be seen that the DNA extraction rate of the immediate freezing sample is about 200 ng / mg per gram of feces, and the preservation solution of the application has almost no loss, about 190 ng / mg, and the purity A260 / 280≈1.85, which is comparable to the freezing control. The DNA of the 95% ethanol group is slightly reduced (about 180 ng / mg, A260 / 280≈1.80), and the RNAlater group is reduced more obviously and the purity is lower (only ~170 ng / mg, A260 / 280≈1.70, indicating the presence of impurities). The non-preservation treatment group decreased the most, with a yield of less than half of the control group (about 100 ng / mg), and the purity was significantly lower (about 1.50), reflecting a large amount of impurities and partial DNA degradation. Therefore, the preservation solution of the application can effectively protect the sample DNA from degradation and loss, maintain high purity, and is obviously superior to the non-preservation or traditional preservation conditions.

[0081] Table 2. DNA yield and purity extracted from fecal samples under different storage conditions

[0082] Note: The data are the average value ± standard deviation of 3 subjects.

[0083] The above results show that the preservation solution can maintain high DNA recovery rate and good purity after 4 weeks of room temperature preservation, with little difference from frozen preservation, while the DNA in the no preservation treatment group is severely lost, and the traditional liquid such as RNAlater also has a certain loss. This verifies the effectiveness of the measures such as EDTA chelation, preservative enzyme inhibition added in the formula.

[0084] 2. Microbial community diversity: Calculate the 16S sequencing data of the fecal samples under each condition to evaluate the alpha and beta diversity of the flora. In terms of alpha diversity, take the Shannon diversity index as an example, the results are shown in Figure 1 A. The Shannon index of the preservation solution group of the application is almost the same as the immediate freezing baseline (average 4.9 vs 5.0, no statistically significant difference, p>0.05), indicating that the richness and evenness are basically unaffected. The 95% ethanol group decreased slightly (about 4.7), the RNAlater group decreased significantly (about 4.3), and the no preservation group decreased significantly (only about 3.5, p<0.01, significantly decreased compared with the baseline). It can be seen that the preservation solution of the application effectively maintains the species diversity of the sample, avoids the loss of sensitive flora, and has better performance than conventional preservation solutions and no preservation.

[0085] In terms of beta diversity, the Bray-Curtis distance is used to measure the difference in flora composition between each group and the baseline sample, and the results are shown in Figure 1 B. The smaller the distance, the more similar the community composition. It can be seen that the Bray-Curtis distance of the preservation solution group of the application and the baseline is only about 0.10, which is the smallest among all conditions, indicating that the flora structure after 4 weeks of preservation is highly consistent with the fresh sample. The 95% ethanol group is second, with a distance of about 0.15. The deviation of the RNAlater group increases, with a distance of about 0.25. The no preservation group has the largest difference from the baseline, with a Bray-Curtis distance of 0.50, showing significant community changes. Statistical analysis shows that the effects of different preservation methods on flora composition are significantly different (Kruskal-Wallis test, p<0.001). Compared with the baseline, only the preservation solution group of the application has no statistically significant difference (p=0.34, Wilcoxon paired test), while the ethanol, RNAlater and no preservation groups all have significant differences from the baseline (p<0.01). This proves that the preservation solution has the best effect in maintaining the overall structure of the flora, and the technical bias introduced by preservation can be almost ignored.

[0086] Further community structure analysis ( Figure 2 and Figure 3 ) also supports the above conclusion.

[0087] Take the relative abundance of the main bacterial phylum as an example (Table 3, Figure 2), the dominant phylum in the frozen baseline sample was Firmicutes ( Firmicutes , ~55%), Bacteroidetes ( Bacteroidetes , ~40%), and a small amount of Proteobacteria ( Proteobacteria , <5%). After 4 weeks of storage, the phylum distribution of the present preservative group was almost identical to the baseline ( Firmicutes , ~54%, Bacteroidetes 39%, Proteobacteria 5%), and no significant change in the proportion of any phylum was observed (p>0.5). In contrast, the group without preservative showed significant dysbiosis: the proportion of Bacteroidetes dropped to only 10%, while Proteobacteria (mainly Enterobacteriaceae and other facultative bacteria) soared to 30%. This indicates that under the unprotected room temperature condition, some facultative bacteria proliferate greatly, while many anaerobes decrease significantly in number. The RNAlater group also observed a decrease in the relative abundance of Bacteroidetes (~25%), suggesting that it is not sufficient to protect anaerobes. The present preservative successfully prevents the secondary growth of facultative bacteria and the death of strict anaerobes by rapid inactivation and anti-oxidation measures, thereby maintaining the original composition of the flora.

[0088] In addition to the overall composition, we also investigated the stability of several key genus-level taxonomic units under different conditions (Table 4, Figure 3 ). For example Faecalibacterium (Clostridia, important probiotics in Firmicutes, anaerobic): about 8.5% of the total sequences in the baseline sample, 8.0% after 4 weeks of storage with the present preservative, no significant change; while the group without preservation is almost undetectable (reduced to 0.5%), and the RNAlater group is also reduced to 3%. For example Escherichia / Shigella (E. coli, etc., facultative bacteria, oxygen-tolerant): 0.8% in the baseline, still 0.7% in the present preservative group, no abnormal proliferation; but the unsaved group increased to more than 10%, showing obvious over-proliferation. The above comparison clearly proves that the present preservative can simultaneously avoid the loss of anaerobic probiotics and the false proliferation of potential pathogenic bacteria, thereby ensuring the authenticity of the flora structure.

[0089] Table 3, Relative abundance (%) of main phylum under different storage conditions

[0090] Note: Each value is the relative abundance percentage, calculated based on the high-throughput 16S rNA gene sequencing results of three subjects (n=3) fecal samples, taking the mean value; compared with the LN group, the PB group has no significant change in the abundance of each phylum (p>0.05), the EtOH and RNA groups have partial deviation, and the None group has obvious dysbiosis ( Bacteroidetes significant decrease, Proteobacteria significant increase).

[0091] Table 4. Relative abundance (%) of some key genera under different storage conditions

[0092] Note: Each value is the relative abundance percentage, calculated based on high-throughput 16S rRNA gene sequencing results of fecal samples from three subjects (n = 3), and the mean value is taken. The selected genera include Butyricimonas (Firmicutes, important probiotics), Bacteroides (Bacteroidetes), Prevotella (Firmicutes), Megamonas (Bacteroidetes), Enterococcus (Firmicutes), Escherichia / Shigella (Proteobacteria, facultative bacteria). The abundance of each key genus in the PB group is close to that in the LN group (p > 0.05), and the samples without storage Faecalibacterium significantly decrease while Bacteroides significantly proliferate, and a decrease is also observed in the RNA group, indicating that it is insufficient to protect anaerobic probiotics. Prevotella Megamonas Escherichia / Shigella Faecalibacterium Escherichia / Shigella Faecalibacterium Figure 4. PCoA dimensionality reduction analysis plot (PCoA1 vs. PCoA2) intuitively shows the distribution difference of triple-replicate fecal samples in two-dimensional principal component space under different storage conditions (LN: frozen baseline, PB: the present storage solution, EtOH: 95% ethanol, RNA: RNAlater, None: no storage solution). Different colors represent different storage conditions, and the dashed oval defines the clustering range of samples within the group. The coordinate axes (PCoA1 and PCoA2) are labeled with the explained variation proportion, reflecting the overall difference and similarity of microbial community structure under different storage conditions. The analysis results show that the community structure distance between the samples in the present storage solution group and the frozen baseline group is the smallest, and the clustering is highly coincident, indicating that the present storage solution can effectively maintain the original structure of the fecal flora at room temperature, which is comparable to the effect of long-term cryopreservation, thereby providing a feasible and stable technical solution for the room temperature storage of clinical and scientific research samples.

[0093] Figure 4

[0094] Based on the comprehensive data, the samples stored using the present storage solution are close to the ideal state of immediate cryopreservation in various key indicators. For example, the Shannon diversity retention rate is more than 98%, the Bray-Curtis similarity to the baseline is more than 90%, and the relative abundance change of the main flora is less than 5%. However, the samples stored at ordinary room temperature (without storage solution) often deteriorate by more than 50% in these indicators.

[0095] ​​​​​​​The application also simultaneously made a 4°C refrigeration / OMNIgene•GUT control group. Even compared with the current better preservation solution (such as ethanol and OMNIgene-GUT), the application scheme also shows longer effective preservation time and smaller deviation. It is especially valuable that the protection of DNA and abundance of strict anaerobes is significantly better than the control scheme. This fully verifies the correctness and effectiveness of the design idea of the application formula.

[0096] In addition, it is worth mentioning that the DNA samples treated by the preservation solution are also suitable for metagenomic shotgun sequencing. The metagenomic library of some samples was constructed for sequencing, and the results also showed that the species and functional gene profiles of the application preservation solution group and the frozen group were highly consistent, while the unsaved group was significantly degraded in sequencing data quality (sequence length, GC content distribution) and result deviation. Figure 5 This further indicates that the protection of the preservation solution to nucleic acids and communities is comprehensive, and is not limited to 16S analysis.

[0097] 3. Statistical analysis: For all comparisons, strict statistical tests were performed. For example, the effect of different preservation methods on alpha diversity, repeated measurement ANOVA analysis showed that the method main effect was significant (p<0.01), and in the post hoc comparison, the application preservation solution and the frozen group had no difference (p=0.78), and the RNAlater and unsaved groups were significantly lower than the frozen group (p<0.01). The difference in community composition was also proved by PERMANOVA analysis that the application preservation solution group and the frozen group had the smallest and non-significant difference, while the other groups had significant difference with the frozen group. All these statistical results support that the application scheme is excellent and stable in performance.

[0098] III. Summary of experimental results and analysis of technical innovation The innovative intestinal flora fecal sample preservation solution of the application is novel and non-obvious in formula and performance, which is embodied in the following aspects: 1. The originality of the formula combination: The preservative solution formula of the present application is a breakthrough innovation to the prior art. The combination of reductive anaerobic protective agent + natural stabilizer + mild surfactant for fecal sample preservation has not been reported in the disclosed literature and existing patents. The formula introduces a variety of new or natural components such as L-cysteine, trehalose, sodium lauroyl sarcosinate, etc., forming a synergistic effect, which fundamentally distinguishes it from the traditional preservative solution that mainly relies on high-concentration salt or organic solvent. For example, the main components of the existing RNAlater preservative solution are high-concentration ammonium sulfate and EDTA solution, which uses high salt to inhibit enzyme activity, but high salt does not actively protect anaerobic bacteria and may cause some bacterial groups to lose water and die. For another example, the NOPB method developed by BGI uses the cationic surfactant N-octylpyridinium bromide to kill bacteria, while the present application selects a less toxic and naturally derived anionic surfactant, supplemented by an antioxidant system, which provides more comprehensive protection for anaerobic bacterial DNA. These formula differences make the overall scheme of the present application unique.

[0099] 2. The prominence of functional effect: The preservative solution of the present application has achieved effects that the prior art cannot achieve in realizing the technical target. On the one hand, the room temperature storage time reaches 4 weeks or even longer, far exceeding the 1-2 week stabilization period reported by most existing schemes. Even though some commercial products claim to be able to be stored for a long time (such as Norgen for 2 years), our formula does not rely on highly specialized drying media, but achieves long-term preservation in liquid state through chemical combination, which belongs to a different innovative path. On the other hand, the present application pays special attention to the protection of anaerobic bacteria and the unbiased fidelity of the community, which is a weak link in many traditional schemes. Experimental data show that the community after preservation by the preservative solution of the present application is highly consistent with the fresh state, especially with almost zero loss of oxygen-sensitive bacterial groups, while schemes such as RNAlater and FTA card introduce significant bias. This combination of long-term and faithful performance is not only superior to the existing technology that improves a single indicator.

[0100] 3. Non-obviousness: In the opinion of those skilled in the art, it is not easy to make breakthroughs in both long-term room temperature storage and maintenance of diversity. The present application successfully solves this problem by boldly using a variety of new materials and optimizing the ratio. The proposal of this innovative scheme is not a trivial logical extension, but a creative idea after considering the principles of microbial ecology, biochemistry, and molecular biology. For example, it is difficult to predict that the introduction of sugars (trehalose) and amino acids (cysteine) into the preservative solution will have such a significant positive impact on DNA stability; again, traditional views may tend to use more powerful denaturants for sterilization (such as guanidine salt), but we unexpectedly found that mild surfactants are sufficient to inhibit microorganisms in the presence of chelating agents and antioxidants, and are more friendly to DNA.

[0101] 4. Differences from existing schemes: Compared with the 95% ethanol preservation method, the preservation solution of the present application does not contain high-concentration organic solvents, avoiding the shortcomings of ethanol such as flammability and excessive dehydration. The present application stops microbial activity by chemical inhibition rather than complete dehydration, thus causing less damage to certain cell structures and having higher fidelity. At the same time, the preservation solution of the present application can also provide antioxidant protection to DNA, while ethanol does not have the function of preventing oxidative damage.

[0102] Compared with RNAlater and other high-salt preservation solutions, the present application does not rely on an extremely high-salt environment, but rather uses a variety of protective agents to achieve preservation under mild conditions. RNAlater has been shown to have significant bias in bacterial community preservation; by adding an anaerobic protective agent, the present application avoids such bias, representing a qualitative difference.

[0103] Compared with the formula of commercial products such as OMNIgene•GUT, the present application fully discloses the composition and formula principle. According to literature reports, OMNIgene•GUT performs well in preserving bacterial communities, but its specific formula is not disclosed, and it is speculated that it may contain some buffer salts and enzyme inhibitors. The present application uses natural components (sugars, polyols, etc.) and new materials (surfactants, etc.) in a different way, and the experiments have proved that the effect is better and more durable. Therefore, we provide a completely new technical solution, rather than a copy or simple improvement of existing commercial formulas.

[0104] Compared with the NOPB scheme of BGI, the present application has obvious differences in material selection and mechanism. NOPB is a quaternary ammonium salt surfactant, which mainly achieves short-term (14 days) preservation through strong membrane disruption disinfection; the present application emphasizes multi-factor synergy, including antioxidant, chelation, and enzyme inhibition, to comprehensively improve the preservation time and effect. In addition, the lauryl amino acid salt used by the present application is derived from natural amino acid derivatives, which has lower toxicity and is more environmentally friendly than NOPB, embodying a unique innovative idea.

[0105] In summary, the preservation solution of the present application can maintain the DNA integrity and composition stability of fecal microbial communities at room temperature for a long time, and its effect is comparable to or even better than the best commercial solution. This will provide strong technical support for large-scale intestinal flora research and the collection and preservation of related medical samples.

[0106] The above only describes the preferred embodiments of the present application and should not be used to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A fecal sample preservative for intestinal flora, characterized by comprising: It comprises the following components: a buffer system, a chelating agent, a reducing anaerobic protective agent, a cell lysis-bacteriostatic agent, a biological macromolecule stabilizer; The buffer system is a combination of Tris-HCl buffer and sodium chloride, wherein the pH value of the Tris-HCl buffer is 7.5-8.0, the final concentration in the storage solution is 40-60 mM, and the final concentration of sodium chloride is 10-50 mM; The chelating agent is ethylenediaminetetraacetic acid disodium salt, and the final concentration in the storage solution is 10-100 mM; The reducing anaerobic protective agent is L-cysteine hydrochloride, and the final concentration in the storage solution is 0.3-1% w / v; The cell lysis-bacteriostatic agent is sodium lauroyl sarcosinate, and the final concentration in the storage solution is 1-2% w / v; The biological macromolecule stabilizer is a combination of trehalose and glycerol, wherein the final concentration of trehalose in the storage solution is 8-12% w / v, and the final concentration of glycerol is 12-18% w / v.

2. The preservative solution according to claim 1, wherein The pH value of the Tris-HCl buffer is 8.0, and the final concentration in the storage solution is 50 mM; the final concentration of sodium chloride in the storage solution is 20 mM.

3. The preservative solution of claim 1, wherein The final concentration of ethylenediaminetetraacetic acid disodium salt in the storage solution is 50 mM.

4. The preservative solution of claim 1, wherein The final concentration of L-cysteine hydrochloride in the storage solution is 0.5% w / v.

5. The preservative solution of claim 1, wherein The final concentration of sodium lauroyl sarcosinate in the storage solution is 1% w / v.

6. The preservative solution of claim 1, wherein The final concentration of trehalose in the storage solution is 10% w / v, and the final concentration of glycerol in the storage solution is 15% w / v.

7. The preservative solution according to any one of claims 1 to 6, wherein It also includes an antioxidant and a bacteriostatic agent; the antioxidant is tea polyphenol or rosemary extract, and the final concentration in the storage solution is 0.1% w / v; the bacteriostatic agent is lauric acid ester, and the final concentration in the storage solution is 0.05% w / v.

8. A method for preparing the preservative solution according to any one of claims 1 to 7, characterized by, It comprises the following steps: S1, add Tris base to deionized water, stir to dissolve, adjust the pH value to 7.5-8.0 with hydrochloric acid, then add sodium chloride and stir to dissolve, then add ethylenediaminetetraacetic acid disodium salt and stir to dissolve, to obtain a buffer base solution; S2, add glycerol and trehalose to the buffer base solution obtained in step S1, stir until completely dissolved, to obtain a mixed solution; S3, cool the mixed solution obtained in step S2 to room temperature, add L-cysteine hydrochloride and stir until dissolved, then add sodium lauroyl sarcosinate and stir until dissolved; then, the mixed solution is used in subsequent step S4, or after adding an antioxidant and a bacteriostatic agent, it is used in step S4; S4, add deionized water to the solution obtained in step S3 to constant volume, then sterile filter to obtain an intestinal flora fecal sample storage solution.

9. The production method according to claim 8, characterized by, In step S3, if the pH value of the mixed solution deviates from 7.5-8.0, add a step of adjusting the pH value to 7.5-8.0 with a base solution.

10. The preparation method according to claim 8, characterized in that, In step S4, sterile filtration uses a 0.22 μm filter membrane.

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