A method for directional isolation of b. longum from human intestinal samples

Abstract

This invention provides a method for the targeted isolation of *Bifidobacterium longum* from human intestinal samples. The method includes: mixing a human intestinal sample with an anaerobic preservative solution to obtain a sample suspension; performing species-level target detection on a portion of the sample suspension to obtain the target abundance level; determining the enrichment inoculum size, enrichment culture time, and separation dilution factor based on the target abundance level; inoculating another portion of the sample suspension into a biased enrichment culture system for anaerobic enrichment to obtain an enriched solution; inoculating the enriched solution into a selective separation medium to form isolated colonies; performing micro-sampling and species-level determination on the isolated colonies, retaining and labeling positive original isolated colonies; and then performing recovery culture, streak purification, and species-level verification to obtain a pure culture of *Bifidobacterium longum*. This invention can adjust the isolation conditions according to the abundance of the target bacteria, reduce the ineffective amplification and identification of non-target suspected colonies, and improve the efficiency of targeted isolation of *Bifidobacterium longum*.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation and culture technology, and in particular to a method for the directional isolation of Bifidobacterium longum from human intestinal samples. Background Technology

[0002] Bifidobacterium longum is one of the most common Bifidobacteria in the human gut and has high research value in intestinal microecology research, screening of probiotic candidate strains, and construction of human-derived strain resource banks. Human intestinal samples, especially fecal samples, intestinal contents samples, or intestinal mucosa-related samples, have a complex microbial composition, including various Bifidobacteria, as well as lactic acid bacteria, Enterobacteriaceae, Bacteroides, and other anaerobic or facultative anaerobic microorganisms. Bifidobacterium longum has specific requirements for its culture environment, oxygen exposure conditions, and nutrient substrates. Improper handling conditions during sample collection, transportation, pretreatment, and isolation culture can easily lead to a decrease in the activity of the target bacteria or its masking by rapidly growing other bacteria.

[0003] Existing methods for isolating Bifidobacteria typically involve steps such as sample dilution, anaerobic enrichment, selective culture plate isolation, picking of suspected colonies, and molecular identification. While these methods can improve the yield of Bifidobacterium strains to some extent, they often target the entire genus Bifidobacterium. During isolation, species-level pre-identification of *Bifidobacterium longum* in the sample is usually not performed before culture, nor are adjustments made to the inoculum size, enrichment culture time, isolation dilution gradient, and picking strategy based on the abundance differences of the target bacteria in the sample.

[0004] In actual isolation processes, the content of *Bifidobacterium longum* varies significantly among different human intestinal samples. If fixed enrichment conditions and dilution plating methods are used, when the abundance of the target bacteria in the sample is low, it may be difficult for the target bacteria to form a sufficient number of pickable colonies; when the proportion of non-target bacteria or other bifidobacteria in the sample is high, a large number of suspected colonies may form on the culture plate, significantly increasing the workload for subsequent identification. Especially when relying solely on colony morphology, Gram staining, or ordinary bifidobacterium selection media for initial screening, it is difficult to accurately distinguish *Bifidobacterium longum* from other closely related bifidobacteria before picking, resulting in a high proportion of invalid picks.

[0005] Furthermore, existing methods often place molecular identification after colony picking and amplification. That is, a large number of suspected colonies are picked first, and then nucleic acid amplification, sequencing or species-level identification are performed one by one. Although this method can confirm the identity of the target bacteria, the identification step is at the end of the isolation process and cannot effectively guide the enrichment, dilution and picking process at the front end. When the proportion of target bacteria in the sample is low or the interference of other bacteria is strong, problems such as prolonged screening cycle, increased culture consumables and unstable efficiency of target bacteria acquisition are likely to occur.

[0006] Therefore, it is necessary to provide a method for the targeted isolation of Bifidobacterium longum that can pre-identify the target bacteria before isolation and combine the pre-identification results with the enrichment, isolation, determination and purification steps. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the targeted isolation of Bifidobacterium longum from human intestinal samples, in order to solve the problems of existing Bifidobacterium isolation methods, which mostly use Bifidobacterium genus as the whole screening object, lacking pre-culture target prediction and isolation parameter adjustment for Bifidobacterium longum, resulting in unstable target positive colony ratio, high invalid bacterial selection ratio, and large workload for subsequent identification and purification.

[0008] To achieve the above objectives, the present invention provides a method for the targeted isolation of Bifidobacterium longum from human intestinal samples, comprising the following steps: S1. Mix the human intestinal sample with the anaerobic preservation solution to obtain a sample suspension; S2. Take a portion of the sample suspension for species-level target detection of Bifidobacterium longum to obtain the target abundance level of Bifidobacterium longum in the human intestinal sample; S3. Based on the target abundance level of Bifidobacterium longum, determine the enrichment inoculum amount, enrichment culture time, and isolation dilution factor according to the preset correspondence. S4. Inoculate another portion of the sample suspension into the Bifidobacterium longum biased enrichment culture system according to the determined enrichment inoculation amount, and carry out enrichment culture under anaerobic conditions for the determined enrichment culture time to obtain the enriched solution; the Bifidobacterium longum biased enrichment culture system contains oligosaccharide carbon source, slow-release carbon source, nitrogen source and reducing component; S5. The enriched solution is inoculated into a selective separation medium according to the determined separation dilution ratio, and cultured under anaerobic conditions to form isolated colonies that can be picked up individually. S6. Take a small sample of the isolated colonies and determine the species level of Bifidobacterium longum from the sample. Retain the original isolated colonies that were sampled and mark the original isolated colonies that were positive in the species level determination. S7. Select the original isolated colonies that are positive at the species level for recovery culture, streak purification and species level verification to obtain a pure culture of Bifidobacterium longum.

[0009] Furthermore, the human intestinal sample is a fecal sample, intestinal contents sample, or intestinal mucosal swab sample; the time from collection to mixing with the anaerobic preservation solution does not exceed 4 hours. By performing anaerobic preservation treatment promptly after sample collection, the impact of oxygen exposure on the survival status of Bifidobacterium longum is reduced, providing a sample basis for subsequent targeted enrichment and isolation.

[0010] Furthermore, the anaerobic protective solution contains a phosphate buffer component, cysteine ​​salt, ascorbate, and glycerol; the mass-to-volume ratio of the human intestinal sample to the anaerobic protective solution is 1 g: (5-20) mL.

[0011] By combining buffering, reducing and protecting components, the sample suspension is kept in a low-oxygen protective environment suitable for the survival of Bifidobacterium longum during the pretreatment stage.

[0012] Furthermore, in step S2, the species-level target detection of Bifidobacterium longum adopts species-specific nucleic acid detection of Bifidobacterium longum, and the detection target is selected from one of the 16S rRNA gene, hsp60 gene, groEL gene, and tuf gene.

[0013] By detecting the target species, the abundance information of the target bacteria can be obtained before culture and isolation, so that the subsequent enrichment inoculum amount, enrichment culture time and isolation dilution factor can be matched according to the target bacteria.

[0014] Furthermore, the target abundance levels of *Bifidobacterium longum* include high abundance, medium abundance, and low abundance levels; when the target abundance level of *Bifidobacterium longum* is high abundance, the inoculum size is 1%–3% of the enrichment culture system volume, the enrichment culture time is 6–10 hours, and the isolation dilution factor is 10. -5 ~10 -7 A 10-fold dilution was used; when the target abundance level of *Bifidobacterium longum* was medium, the inoculum size was 3%–6% of the enrichment culture system volume, the enrichment culture time was 10–16 hours, and the separation dilution factor was 10. -4 ~10 -6 The dilution factor was 10:1; when the target abundance level of Bifidobacterium longum was low, the inoculum size was 6%–10% of the enrichment culture system volume, the enrichment culture time was 16–24 hours, and the separation dilution factor was 10:1. -2 ~10 -4 Diluted solution.

[0015] Through the above correspondence, samples with lower target bacterial abundance can obtain more sufficient enrichment conditions, while samples with higher target bacterial abundance can avoid over-enrichment and excessive colony density, thereby increasing the probability of forming target bacterial colonies that can be picked on the isolation plate.

[0016] Furthermore, the oligosaccharide carbon source is selected from at least one of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and inulin; the slow-release carbon source is selected from at least one of resistant starch, soluble dietary fiber, and pectin hydrolysate.

[0017] By combining oligosaccharide carbon sources with slow-release carbon sources, competition from other bacteria caused by the rapid utilization of simple sugars is reduced, making the enrichment process more suitable for the targeted proliferation of Bifidobacterium longum.

[0018] Furthermore, in the *Bifidobacterium longum* biased enrichment culture system, the mass concentration of oligosaccharide carbon source is 5–20 g / L, the mass concentration of slow-release carbon source is 1–8 g / L, the mass concentration of monosaccharide carbon source does not exceed 2 g / L, and the mass concentration of reducing component is 0.2–1.5 g / L. This culture system, by limiting monosaccharide carbon source and configuring oligosaccharide carbon source, slow-release carbon source, and reducing component, ensures that the enrichment stage balances the growth of target bacteria with the control of competition from other bacteria.

[0019] Furthermore, the selective isolation culture medium contains basic nutrient components for Bifidobacteria, blood components, reducing components, and impurity-inhibiting components; the impurity-inhibiting components include at least two of cephalosporin antibacterial agents, lithium salts, and propionates.

[0020] By using nutrient support and inhibitory components in selective separation media, interference from non-target bacteria on the isolation plates is reduced, allowing isolated colonies to form a state that can be picked up individually.

[0021] Further, in step S6, the micro-sampling involves sampling from the edge of the isolated colony, and the original isolated colony is retained on the selective isolation medium after sampling. When the *Bifidobacterium longum* species determination result of the micro-sample is positive, the corresponding original isolated colony is marked and picked within 24 hours. By performing micro-sampling determination before picking and retaining the original isolated colony, the amplification operation of non-target colonies can be reduced, and the accuracy of the picked target can be improved.

[0022] Further, in step S7, the recovery culture is carried out using a low-selectivity pressure recovery medium, which does not contain the inhibitory component of the selective isolation medium in step S5, or the concentration of the inhibitory component in the low-selectivity pressure recovery medium is less than 50% of the concentration of the inhibitory component in the selective isolation medium; after the single colony obtained after streak purification is confirmed to be positive by the Bifidobacterium longum species-level verification, a pure culture of Bifidobacterium longum is prepared.

[0023] By integrating recovery culture, streak purification, and seed-level verification, positive colonies can be restored to growth under reduced selection pressure, and pure cultures of Bifidobacterium longum can be obtained.

[0024] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: I. This invention performs species-level target detection of Bifidobacterium longum in human intestinal samples before isolation and culture, and determines the enrichment inoculum amount, enrichment culture time and isolation dilution factor according to the target abundance level. This allows the isolation process to be adjusted according to the difference in the abundance of target bacteria in the sample, avoiding blind screening based solely on fixed conditions, thereby increasing the probability of Bifidobacterium longum forming pickable colonies.

[0025] II. This invention employs a combination of a Bifidobacterium longum biased enrichment culture system and a selective separation culture medium. During the enrichment stage, oligosaccharide carbon sources, slow-release carbon sources, and reducing components provide suitable growth conditions for the target bacteria. During the separation stage, impurity-suppressing components reduce interference from non-target bacteria, thereby increasing the proportion of Bifidobacterium longum positive colonies in the isolated colonies and reducing ineffective bacterial picking.

[0026] Third, this invention performs micro-sampling and species-level determination of isolated colonies before picking and expanding the culture, and retains the original isolated colonies. Only positive original isolated colonies are subjected to recovery culture, streak purification, and species-level verification. This method moves the species-level determination step forward to before expansion culture, reduces the workload of repeated expansion culture and identification of non-target suspected colonies, and helps to shorten the screening process and obtain pure cultures of Bifidobacterium longum.

[0027] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a flowchart of a method for the targeted isolation of Bifidobacterium longum from human intestinal samples according to the present invention; Figure 2 This is a schematic diagram illustrating the matching relationship between the target abundance level and the separation parameters of this invention; Figure 3 This is a flowchart illustrating the process of micro-sampling, labeling, and purifying isolated bacterial colonies according to the present invention. Detailed Implementation

[0030] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0031] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0032] like Figure 1As shown, this invention provides a method for the targeted isolation of *Bifidobacterium longum* from human intestinal samples. The human intestinal sample is divided into a target detection portion and an isolation and culture portion. The target detection portion is used to obtain the target abundance level of *Bifidobacterium longum* in the sample before culture. The isolation and culture portion determines the enrichment inoculum amount, enrichment culture time, and isolation dilution factor based on the target abundance level. Then, through biased enrichment, selective isolation, micro-sampling judgment, and re-screening purification, a pure culture of *Bifidobacterium longum* is obtained. This method differs from conventional isolation methods that only use *Bifidobacterium* genus as the screening target. Its key point is to move the species-level target detection result forward and use it to adjust subsequent isolation conditions, so that human intestinal samples with different target bacterial abundances can be isolated and processed according to the corresponding conditions.

[0033] I. Sample processing and anaerobic preservation solution The human intestinal sample of this invention can be a fecal sample, an intestinal contents sample, or an intestinal mucosal swab sample. In practice, the sample should be transferred to the anaerobic preservation treatment step as soon as possible after collection to reduce the adverse effects of exposure to air on the survival of Bifidobacterium longum; the time from collection to mixing of the human intestinal sample with the anaerobic preservation solution should be controlled to not exceed 4 hours.

[0034] In one embodiment, the anaerobic preservation solution contains a phosphate buffer, cysteine, ascorbate, and glycerol. The phosphate buffer maintains a buffered environment for the sample suspension; the cysteine ​​and ascorbate provide a reducing environment; and the glycerol provides protection during sample suspension preparation and subsequent short-term storage.

[0035] Specifically, the anaerobic preservation solution can be prepared as follows: using sterile water as the solvent, each liter of anaerobic preservation solution contains 8.0g of phosphate buffer, 5.0g of sodium chloride, 0.5g of cysteine, 0.5g of ascorbate, and 100mL of glycerol. The pH is adjusted to 6.8-7.2, and the solution is sterilized before use.

[0036] The above formulation is one possible implementation method. As long as the anaerobic protection solution contains phosphate buffer components, cysteine ​​salt, ascorbate and glycerol, and can form an environment suitable for sample suspension and hypoxia protection, it can be used in this invention.

[0037] When preparing the sample suspension, human intestinal samples are mixed with anaerobic protective solution at a mass-volume ratio of 1g:(5-20)mL; for fecal samples, 1g of sample can be added to 5-20mL of anaerobic protective solution, and the sample can be fully dispersed by shaking, vortexing or homogenization to obtain the sample suspension.

[0038] For intestinal mucosal swab samples, the swabs can be placed in 5-10 mL of anaerobic preservative solution and eluted by shaking to obtain a sample suspension. The prepared sample suspension is divided into two parts: one part is used for the detection of Bifidobacterium longum at the species level, and the other part is used for enrichment culture.

[0039] II. Species-level target detection and abundance classification of Bifidobacterium longum Before culturing and isolating, this invention first performs species-level target detection of Bifidobacterium longum on the sample suspension to obtain the target abundance level of Bifidobacterium longum. The purpose of this step is to provide a basis for determining the subsequent enrichment inoculum amount, enrichment culture time, and isolation dilution factor, rather than passively identifying the colony identity only after the isolation is completed.

[0040] Species-level target detection of Bifidobacterium longum can be performed using species-specific nucleic acid detection of Bifidobacterium longum; the detection target can be selected from one of the 16S rRNA gene, hsp60 gene, groEL gene, and tuf gene; the detection method can be conventional PCR, real-time quantitative PCR, or other nucleic acid detection methods that can obtain species-level target detection results.

[0041] In one embodiment, 1 mL of sample suspension is taken, and after centrifugation to collect bacterial cells, total DNA is extracted. Real-time quantitative PCR is then performed using the hsp60 gene or groEL gene as the detection target. The target abundance level of *Bifidobacterium longum* is obtained based on the amplification signal. Simultaneously, the universal 16S rRNA gene can be detected as a reference signal for total bacterial count. The target abundance level is then determined based on the relative relationship between the target detection signal and the total bacterial reference signal, facilitating comparison between different samples.

[0042] When using real-time quantitative PCR detection, abundance levels can be classified as follows: when the Ct value for Bifidobacterium longum is no greater than 25, it is classified as high abundance; when the Ct value is greater than 25 but no greater than 30, it is classified as medium abundance; and when the Ct value is greater than 30 but no greater than 35, it is classified as low abundance. For different detection platforms or different targets, specific thresholds can be calibrated using standard samples, but the high abundance, medium abundance, and low abundance levels should correspond to three sample states with the highest to lowest target bacterial content, respectively.

[0043] III. Correspondence between abundance levels and separation parameters like Figure 2 As shown, after obtaining the target abundance level of Bifidobacterium longum, the enrichment inoculum size, enrichment culture time, and isolation dilution factor are determined based on the target abundance level; the technical logic of this correspondence is as follows: Samples with high abundance of target bacteria do not need to be over-enriched, otherwise it may lead to overly dense colonies on the plate; samples with low abundance of target bacteria need to increase the inoculum size and extend the enrichment time to increase the chance of the target bacteria forming pickable colonies.

[0044] In this invention, the target abundance level and isolation parameters of Bifidobacterium longum can be determined according to Table 1. Table 1. Correspondence between target abundance levels and separation parameters High abundance level Enrichment culture system volume 1%–3% 6 to 10 hours <![CDATA[10 -5 ~10 -7 [Dilution] Medium abundance level Enrichment culture system volume 3%–6% 10-16 hours <![CDATA[10 -4 ~10 -6 [Dilution] Low abundance level Enrichment culture system volume 6%–10% 16-24 hours <![CDATA[10 -2 ~10 -4 [Dilution] The above correspondence allows samples with different target bacterial abundances to be placed under matching enrichment and isolation conditions. For high abundance samples, using a lower inoculum size, shorter enrichment culture time, and higher isolation dilution factor helps avoid overly dense colonies on the plate. For low abundance samples, using a higher inoculum size, longer enrichment culture time, and lower isolation dilution factor increases the chance of Bifidobacterium longum forming individually pickable colonies on the isolation plate.

[0045] IV. Bifidobacterium longum biased enrichment culture The present invention provides a biased enrichment culture system for Bifidobacterium longum containing oligosaccharide carbon sources, slow-release carbon sources, nitrogen sources, and reducing components. This culture system is used to enrich sample suspensions under anaerobic conditions, making the enriched solution more suitable as a source of bacteria for subsequent selective isolation.

[0046] The carbon source of oligosaccharides can be selected from at least one of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and inulin.

[0047] The slow-release carbon source can be selected from at least one of resistant starch, soluble dietary fiber, and pectin hydrolysate.

[0048] The nitrogen source can be one or more of peptone, tryptone, or yeast extract.

[0049] The reducing component can be cysteine ​​salt, ascorbate or thioglycolate.

[0050] In one embodiment, the Bifidobacterium longum biased enrichment culture system contains, per liter of culture medium, 5-20 g of oligosaccharide carbon source, 1-8 g of slow-release carbon source, 8-12 g of peptone, 3-8 g of yeast extract, 1-3 g of dipotassium hydrogen phosphate, 0.5-2 g of potassium dihydrogen phosphate, 0.1-0.5 g of magnesium sulfate, 0.02-0.1 g of manganese sulfate, 0.5-1.5 mL of Tween 80, and 0.2-1.5 g of reducing components; wherein the mass concentration of monosaccharide carbon source does not exceed 2 g / L. After sterilization, the culture system is equilibrated in an anaerobic environment before use.

[0051] The above culture system provides a relatively suitable nutritional and hypoxic environment for the enrichment of target bacteria through the combination of oligosaccharide carbon sources, slow-release carbon sources, and reducing components; at the same time, by limiting the content of monosaccharide carbon sources, it reduces the competitive interference caused by the rapid amplification of some non-target bacteria relying on simple sugars.

[0052] It should be noted that the purpose of this enrichment culture system is to improve the adaptability of the isolation process to Bifidobacterium longum, not to make the culture system only allow the growth of Bifidobacterium longum.

[0053] V. Selective Separation Culture Media After enrichment culture is completed, the enriched solution is inoculated into selective isolation medium according to the determined separation dilution factor, and cultured under anaerobic conditions to form isolated colonies that can be picked up individually; the selective isolation medium contains basic nutrients for Bifidobacteria, blood components, reducing components and impurity inhibitors.

[0054] The basic nutrient components for Bifidobacteria may include peptone, yeast extract, salts, and agar. Blood components may be sterile defibrinated sheep blood. Reducing components may be cysteine ​​salts, ascorbate, or thioglycolate. Inhibiting components include at least two of cephalosporin antibiotics, lithium salts, and propionates.

[0055] In one embodiment, the selective separation medium, per liter of medium, contains 10g peptone, 5g yeast extract, 5g soluble starch, 5g sodium chloride, 2g dipotassium hydrogen phosphate, 15g agar, 0.5g cysteine, 50mL sterile defibrinated sheep blood, and a contaminant; the contaminant contains at least two of cefepime, lithium chloride, and sodium propionate. After sterilization and cooling to a suitable temperature, the blood component and the heat-sensitive contaminant are added to the medium, mixed thoroughly, and then poured into trays for use.

[0056] The purpose of selective isolation medium is to reduce the interference of some non-target bacteria on the plate, so that the enriched solution can form isolated colonies that can be picked up individually after dilution and inoculation. This medium does not necessarily have to completely exclude all non-target bacteria. As long as it can reduce the interference of non-target bacteria and form isolated colonies that can be used for micro-sampling and subsequent determination, it can meet the isolation purpose of this invention.

[0057] VI. Micro-sampling, species-level determination, and original colony marking like Figure 3 As shown, after the isolated colonies are formed on the selective isolation medium, the present invention does not directly pick all suspected colonies for expansion, but first performs micro-sampling on the isolated colonies and determines the species level of Bifidobacterium longum from the micro-samples; this step is an important part of the present invention that distinguishes it from the conventional process of "picking and expanding bacteria first, and then identifying species level".

[0058] In practice, select isolated colonies that match the morphological characteristics of suspected Bifidobacterium colonies and can be picked up individually. Use a sterile inoculation needle or sterile pipette tip to gently touch and sample from the edge of the colony. The sample size should be sufficient to complete the species determination. After sampling, the original isolated colonies should still be retained on the selective isolation medium. Add a small amount of sample to sterile water or lysis buffer, and perform rapid lysis to determine the species level of Bifidobacterium longum.

[0059] When the species determination result of the trace sample is positive for Bifidobacterium longum, the corresponding original isolated colony is marked and picked within 24 hours. The location marking can be done by numbering the back of the plate, using coordinate marking, or recording the colony image. In this way, the species determination information can be obtained first without destroying the original isolated colony, and then the positive original isolated colony can be picked at a specific point, reducing the amplification and repeated identification of non-target suspected colonies.

[0060] VII. Recovery culture, streak purification and seed-level verification After selecting the original isolates that were determined to be positive at the seed level, they were inoculated into low-selectivity pressure recovery medium for recovery culture. The low-selectivity pressure recovery medium did not contain the inhibitory components of the selective isolation medium, or the concentration of the inhibitory components in the low-selectivity pressure recovery medium was less than 50% of the concentration of the inhibitory components in the selective isolation medium.

[0061] In one embodiment, the low-selectivity pressure recovery medium can be a liquid medium containing peptone, yeast extract, oligosaccharide carbon source, slow-release carbon source, and reducing components, without the addition of cephalosporin antibiotics, lithium salts, and propionates, or with only a low concentration of inhibitory components. Recovery culture is carried out under anaerobic conditions at a temperature of 35–38°C for 12–24 hours.

[0062] After recovery culture, the culture medium is streaked onto a solid medium without strong selective pressure for purification culture. After single colonies form, a single colony is picked for further testing for *Bifidobacterium longum*. If the test result is positive, the single colony is expanded to obtain a pure culture of *Bifidobacterium longum*. The obtained pure culture can be preserved using glycerol cryopreservation, for example, by mixing the bacterial culture with sterile glycerol cryopreservation solution to a final glycerol concentration of 15%–25%, and then storing it at low temperature.

[0063] Example 1: Targeted Separation of High Abundance Samples In this embodiment, human fecal samples were used as human intestinal samples. Samples were processed within 2 hours of collection. 1g of fecal sample was added to 10mL of anaerobic preservative solution and vortexed until the sample was evenly dispersed, resulting in a sample suspension.

[0064] Total DNA was extracted from 1 mL of the sample suspension, and the *Bifidobacterium longum* strain was targeted for detection. The results showed a Ct value of 23.8, indicating that the sample was of high abundance.

[0065] Based on the high abundance level correspondence, 2% of the sample suspension from the enrichment culture system was inoculated into the Bifidobacterium longum biased enrichment culture system and enriched under anaerobic conditions at 37℃ for 8 hours. In the biased enrichment culture system, the oligosaccharide carbon source was fructooligosaccharide at a concentration of 10 g / L; the slow-release carbon source was resistant starch at a concentration of 3 g / L; the monosaccharide carbon source was 1 g / L; and the reducing component was cysteine ​​at a concentration of 0.5 g / L.

[0066] After the enrichment culture was completed, the enriched solution was serially diluted, and 10 was selected. -6 The diluted solution was inoculated into selective isolation medium and cultured under anaerobic conditions at 37°C for 48 hours to form isolated colonies that could be picked up individually.

[0067] Microsamples were taken from suspected colonies, and the corresponding original isolated colonies were retained. The microsamples were subjected to *Bifidobacterium longum* species-level determination. Positive original isolated colonies were marked and picked within 24 hours. The picked colonies were inoculated into low-selectivity recovery medium and incubated anaerobically at 37°C for 18 hours, followed by streak purification. Single colonies were picked after purification for species-level verification. If the verification confirmed *Bifidobacterium longum* positivity, a pure culture of *Bifidobacterium longum* was obtained.

[0068] Example 2: Directed Separation of Abundance Grade Samples In this embodiment, human fecal samples were used as human intestinal samples. 1g of sample was added to 10mL of anaerobic preservation solution and mixed thoroughly to obtain a sample suspension.

[0069] A portion of the sample suspension was used for species-level detection of Bifidobacterium longum. The target gene was groEL, and the result showed a Ct value of 28.1, indicating that the sample was of medium abundance.

[0070] Based on the correspondence between abundance levels, 5% of the sample suspension from the enrichment culture system was inoculated into the Bifidobacterium longum biased enrichment culture system and enriched under anaerobic conditions at 37℃ for 14 hours. In the biased enrichment culture system, the oligosaccharide carbon source was galactooligosaccharide and inulin, with a combined mass concentration of 15 g / L; the slow-release carbon source was soluble dietary fiber, with a mass concentration of 5 g / L; the monosaccharide carbon source had a mass concentration of 1.5 g / L; and the reducing components were cysteine ​​salt and ascorbate, with a combined mass concentration of 0.8 g / L.

[0071] After enrichment culture, the enrichment solution was serially diluted, and 10⁻⁶ ppm was selected. -5The diluted solution was inoculated into selective isolation medium and cultured anaerobically at 37°C for 48 hours. The selective isolation medium contained basic nutrients for Bifidobacteria, sterile defibrinated sheep blood, reducing agents, and inhibitors. The inhibitors were cefepime, lithium chloride, and sodium propionate.

[0072] After isolated colonies have formed and can be picked individually, a small sample is taken from the edge of the isolated colony, and the original isolated colony is preserved. The small sample is then subjected to species-level determination of *Bifidobacterium longum*, and positive original isolated colonies are marked and picked. The picked original isolated colonies are then transferred to low-selectivity recovery medium for re-culturing, followed by streak purification and species-level verification to obtain a pure culture of *Bifidobacterium longum*.

[0073] Example 3: Targeted Separation of Low Abundance Grade Samples In this embodiment, human intestinal mucosal swab samples were used as human intestinal samples. The swabs were placed in 8 mL of anaerobic preservative solution and eluted by shaking to obtain a sample suspension.

[0074] A portion of the sample suspension was used for species-level detection of Bifidobacterium longum. The target gene was tuf, and the result showed a Ct value of 32.6, indicating that the sample was classified as low abundance.

[0075] Based on the correspondence between low abundance levels, 8% of the sample suspension from the enrichment culture system was inoculated into the Bifidobacterium longum biased enrichment culture system and enriched under anaerobic conditions at 37℃ for 20 hours. In the biased enrichment culture system, the oligosaccharide carbon source was xylooligosaccharide and fructooligosaccharide, with a combined mass concentration of 18 g / L; the slow-release carbon source was pectin hydrolysate, with a mass concentration of 6 g / L; the monosaccharide carbon source mass concentration did not exceed 2 g / L; and the reducing component was cysteine ​​salt, with a mass concentration of 1.0 g / L.

[0076] After enrichment culture, 10 were selected -3 The diluted solution was inoculated into selective isolation medium and incubated anaerobically at 37°C for 60 hours to form isolates that could be picked individually. Micro-sampling and species-level determination were performed on the isolates, and positive original isolates were marked and picked promptly. After recovery culture, streak purification, and species-level verification, pure cultures of *Bifidobacterium longum* were obtained.

[0077] In this embodiment, by using a higher enrichment inoculum amount, a longer enrichment culture time, and a lower separation dilution factor for low abundance level samples, the target bacteria in the samples are more likely to enter the separable state, which is beneficial for obtaining positive colonies that can be picked individually in the future.

[0078] Comparative Example 1: Separation method without abundance level parameter matching This comparative example used human intestinal samples from the same source as in Examples 1-3, but did not match the target abundance level of Bifidobacterium longum with the isolation parameters. All samples were treated under fixed conditions: the inoculum size was 5% of the enrichment culture volume, the enrichment culture time was 12 hours, and the isolation dilution factor was uniformly set to 10-1. -5 Diluted solution.

[0079] Other sample processing, biased enrichment culture systems, selective separation media, micro-sampling determination, and re-screening purification conditions are the same as in the examples. This comparative example is used to compare the differences between fixed separation parameters and the separation parameters of this invention based on target abundance levels.

[0080] Comparative Example 2: Separation method without micro-sampling judgment This comparative example uses the same human intestinal sample processing method, target abundance level determination method, enrichment inoculum amount, enrichment culture time, separation dilution factor, biased enrichment culture system and selective separation culture medium as Example 2.

[0081] The difference lies in that, after the isolated colonies formed, this comparative example did not perform micro-sampling or pre-selection species-level determination. Instead, it directly selected suspected colonies based on colony morphology for amplification, and then performed Bifidobacterium longum species-level identification after amplification. This comparative example was used to compare the impact of moving the species-level determination to before amplification in this invention on invalid selection and repeated identification.

[0082] Test Example 1: Test for the proportion of Bifidobacterium longum positive colonies The methods of Examples 1-3, Comparative Examples 1 and 2 were used for isolation. For each group, the number of suspected colonies that could be picked up individually, the number of colonies that entered subsequent processing, and the number of colonies that were finally confirmed as positive for Bifidobacterium longum after species-level verification were counted, and the proportion of Bifidobacterium longum positive colonies was calculated.

[0083] The test results are shown in Table 2.

[0084] Table 2. Proportion of Bifidobacterium longum positive colonies in subsequent treatments Example 1 86 28 21 75.0% Example 2 92 35 24 68.6% Example 3 64 25 15 60.0% Comparative Example 1 88 48 18 37.5% Comparative Example 2 90 60 22 36.7% As shown in Table 2, under the same or similar sample processing conditions, this invention improves the proportion of Bifidobacterium longum positive colonies in the colonies entering subsequent processing by matching the separation parameters with the target abundance level and performing micro-sampling judgment before picking and expanding the culture. For low abundance level samples, this invention can also obtain positive colonies that can be used for subsequent purification by adjusting the enrichment inoculum amount, enrichment culture time and separation dilution factor.

[0085] Test Example 2: Test for Invalid Bacterial Picks The number of invalid picks was compared between Example 2 and Comparative Example 2. Invalid picks refer to colonies that, after entering the expansion or purification process, were not confirmed as Bifidobacterium longum upon inoculum-level verification.

[0086] The test results are shown in Table 3.

[0087] Table 3 Comparison of Ineffective Bacterial Picks Example 2 35 24 11 31.4% Comparative Example 2 60 22 38 63.3% As can be seen from Table 3, compared with directly picking suspected colonies and then performing species-level identification, the present invention first screens positive original isolated colonies by micro-sampling and species-level determination, and then performs recovery culture and streak purification. This can reduce the number of non-target suspected colonies entering the subsequent expansion and purification process, thereby reducing the proportion of ineffective colony picking.

[0088] Test Example 3: Separation Process Time Test The time required from sample processing to obtaining pure cultures of *Bifidobacterium longum* with positive results at the species level was recorded for both Example 2 and Comparative Example 2. The test results are shown in Table 4.

[0089] Table 4 Comparison of separation process time Example 2 0.5 days 2.5 days 1 day 2 days 6 days Comparative Example 2 0.5 days 2.5 days 2 days 3 days 8 days As can be seen from Table 4, under the same conditions of sample processing, target detection, enrichment and isolation culture, the present invention reduces the number of non-target suspected colonies entering the expansion culture and subsequent verification process by performing micro-sampling and species-level determination before picking expansion culture, thereby reducing the time consumption of subsequent processing and shortening the cycle from sample to pure culture of Bifidobacterium longum.

[0090] In summary, this invention establishes a complete process for the targeted isolation of Bifidobacterium longum from human intestinal samples by integrating anaerobic protection of samples, species-level target detection of Bifidobacterium longum, matching abundance levels with separation parameters, biased enrichment culture, selective isolation culture, micro-sampling judgment, and recovery culture purification. This process can increase the proportion of positive colonies of the target bacteria, reduce the workload of ineffective bacterial picking and repeated identification, and provide an operable implementation path for obtaining pure cultures of Bifidobacterium longum.

[0091] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for directionally isolating Bifidobacterium longum from human intestinal samples, characterized in that, Includes the following steps: S1. Mix the human intestinal sample with the anaerobic preservation solution to obtain a sample suspension; S2. Take a portion of the sample suspension for species-level target detection of Bifidobacterium longum to obtain the target abundance level of Bifidobacterium longum in the human intestinal sample; S3. Based on the target abundance level of Bifidobacterium longum, determine the enrichment inoculum amount, enrichment culture time, and isolation dilution factor according to the preset correspondence. S4. Inoculate another part of the sample suspension into the Bifidobacterium longum biased enrichment culture system according to the determined enrichment inoculation amount, and carry out enrichment culture under anaerobic conditions for a determined enrichment culture time to obtain the enriched solution. The Bifidobacterium longum biased enrichment culture system contains oligosaccharide carbon sources, slow-release carbon sources, nitrogen sources, and reducing components; S5. The enriched solution is inoculated into a selective separation medium according to the determined separation dilution ratio, and cultured under anaerobic conditions to form isolated colonies that can be picked up individually. S6. Take a small sample of the isolated colonies and determine the species level of Bifidobacterium longum from the sample. Retain the original isolated colonies that were sampled and mark the original isolated colonies that were positive in the species level determination. S7. Select the original isolated colonies that are positive at the species level for recovery culture, streak purification and species level verification to obtain a pure culture of Bifidobacterium longum.

2. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, The human intestinal sample is a fecal sample, intestinal contents sample, or intestinal mucosal swab sample; the time from collection to mixing with the anaerobic protective solution shall not exceed 4 hours.

3. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, The anaerobic protective solution contains a phosphate buffer component, cysteine ​​salt, ascorbate, and glycerol; the mass-to-volume ratio of the human intestinal sample to the anaerobic protective solution is 1 g: (5-20) mL.

4. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, In step S2, the species-level target detection of Bifidobacterium longum adopts species-specific nucleic acid detection of Bifidobacterium longum, and the detection target is selected from one of the 16S rRNA gene, hsp60 gene, groEL gene, and tuf gene.

5. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, The target abundance levels of *Bifidobacterium longum* include high abundance, medium abundance, and low abundance levels. When the target abundance level of *Bifidobacterium longum* is high abundance, the inoculum size is 1%–3% of the enrichment culture system volume, the enrichment culture time is 6–10 hours, and the isolation dilution factor is 10. -5 ~10 -7 A 10-fold dilution was used; when the target abundance level of *Bifidobacterium longum* was medium, the inoculum size was 3%–6% of the enrichment culture system volume, the enrichment culture time was 10–16 hours, and the separation dilution factor was 10. -4 ~10 -6 The dilution factor was 10:1; when the target abundance level of Bifidobacterium longum was low, the inoculum size was 6%–10% of the enrichment culture system volume, the enrichment culture time was 16–24 hours, and the separation dilution factor was 10:

1. -2 ~10 -4 Diluted solution.

6. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, The oligosaccharide carbon source is selected from at least one of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and inulin; the slow-release carbon source is selected from at least one of resistant starch, soluble dietary fiber, and pectin hydrolysate.

7. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, In the Bifidobacterium longum biased enrichment culture system, the mass concentration of oligosaccharide carbon source is 5-20 g / L, the mass concentration of slow-release carbon source is 1-8 g / L, the mass concentration of monosaccharide carbon source does not exceed 2 g / L, and the mass concentration of reducing component is 0.2-1.5 g / L.

8. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, The selective isolation medium contains basic nutrients for Bifidobacteria, blood components, reducing components, and inhibitory components; the inhibitory components include at least two of cephalosporin antibacterial agents, lithium salts, and propionates.

9. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, In step S6, the micro-sampling is performed by taking samples from the edge of the isolated colony. After sampling, the original isolated colony is retained on the selective isolation medium. When the species determination result of the micro-sample is positive for Bifidobacterium longum, the corresponding original isolated colony is marked and picked within 24 hours.

10. The method for directionally isolating Bifidobacterium longum from human intestinal samples according to claim 1, characterized in that, In step S7, the recovery culture is carried out using a low-selectivity pressure recovery medium, which does not contain the inhibitory component of the selective isolation medium in step S5, or the concentration of the inhibitory component in the low-selectivity pressure recovery medium is less than 50% of the concentration of the inhibitory component in the selective isolation medium; after the single colony obtained after streak purification is confirmed to be positive by the Bifidobacterium longum species-level verification, a pure culture of Bifidobacterium longum is prepared.

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