Directional sorting method of gram-positive bacteria

Abstract

The invention provides a directional sorting method of gram-positive bacteria, which comprises the following steps: S1, extracting a human excrement sample to obtain a human bacterium suspension; and S2, treating the human bacterium suspension by using an EDTA-2Na (Ethylene Diamine Tetraacetic Acid) solution and a NaCl solution at the same time. According to the method, the gram-negative bacteria in the intestinal flora can be effectively removed, and meanwhile, the activity of the gram-positive bacteria is kept as much as possible, so that the high-purity gram-positive bacteria flora is obtained.

Description

Technical Field

[0001] The invention relates to a directional sorting method for Gram-positive bacteria, belonging to the technical field of bacteria screening. Background Art

[0002] Studies have shown that Gram-positive bacteria, as part of the intestinal microbiota, may affect mental health states, including depression, through the gut-brain axis. These studies provide new perspectives for understanding the relationship between the intestinal microbiota and depression and provide possible directions for developing new treatment strategies. However, most of these studies are still in the early stages and more research is needed to determine the specific causal relationships and therapeutic potential. Based on the understanding of the relationship between Gram-positive bacteria in the human intestine and health states such as depression, it is necessary to effectively remove Gram-negative bacteria from the intestinal flora while maintaining the activity of Gram-positive bacteria as much as possible, so as to obtain a high-purity Gram-positive bacterial flora for research. However, there is currently no ideal screening method for Gram-positive bacteria in the existing technology. Summary of the invention

[0003] The present invention provides a method for directional sorting of Gram-positive bacteria, which can effectively solve the above problems.

[0004] The present invention is achieved in that:

[0005] A method for directional sorting of Gram-positive bacteria, comprising:

[0006] S1, extracting and processing human fecal samples to obtain human bacterial suspension;

[0007] S2, treating the human bacterial suspension with EDTA-2Na or EDTA-Fe solution and NaCl solution simultaneously.

[0008] In some embodiments, the concentration of the EDTA-2Na or EDTA-Fe solution is 0.1-2M.

[0009] In some embodiments, the concentration of the EDTA-2Na solution is 0.5M.

[0010] In some embodiments, the concentration of the NaCl solution is 2-10 wt %.

[0011] In some embodiments, the concentration of the NaCl solution is 5 wt %.

[0012] In some embodiments, the concentration of the human bacterial suspension is 10 5 -10 10 cells / mL.

[0013] In some embodiments, the treatment time is 1-4 hours.

[0014] In some embodiments, the treatment time is 2 hours.

[0015] In some embodiments, the treatment temperature is 0-30°C.

[0016] In some embodiments, the temperature of the treatment is 4°C.

[0017] The beneficial effects of the present invention are:

[0018] The directional sorting method of Gram-positive bacteria of the present invention can effectively remove Gram-negative bacteria in the intestinal flora, while maintaining the activity of Gram-positive bacteria as much as possible, thereby obtaining a high-purity Gram-positive bacterial flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a diagram of the anaerobic culture of a spore suspension on a Gram-negative bacteria selective solid culture medium according to Example 1 of the present invention.

[0021] Figure 2 This is a diagram of the anaerobic culture of the donor bacterial suspension in Example 1 of the present invention on a Gram-negative bacteria selective solid culture medium.

[0022] Figure 3 This is a Gram staining image of the donor bacterial suspension of Example 1 of the present invention after anaerobically culturing on a Gram-negative bacteria selective solid culture medium.

[0023] Figure 4 This is a comparative bar chart of bacterial genera obtained by different treatments provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] The embodiment of the present invention aims to provide a method for directional sorting of Gram-positive bacteria, which is efficient and practical. The specific steps are as follows:

[0026] First, in step S1, we took measures to extract and process human fecal samples to obtain human bacterial suspension. This process involves a series of pretreatments of fecal samples, including but not limited to centrifugation, washing, and suspension steps, to ensure that the final bacterial suspension contains a rich microbial community.

[0027] Next, in step S2, we use EDTA-2Na or EDTA-Fe solution and NaCl solution to jointly treat the above human bacterial suspension. As a commonly used metal ion chelator, EDTA-2Na or EDTA-Fe solution can effectively remove the outer membrane of Gram-negative bacteria, while NaCl solution helps to maintain the osmotic pressure of the solution to ensure that the cell wall structure of Gram-positive bacteria is not destroyed. Through this combined treatment, we can effectively remove Gram-negative bacteria from the intestinal flora while maximizing the activity and integrity of Gram-positive bacteria.

[0028] The Gram-positive bacteria sorting method proposed in the embodiment of the present invention can not only obtain a high-purity Gram-positive bacterial flora, but also has broad application prospects in multiple fields such as medicine, food and agriculture. For example, in the medical field, the method can be used to prepare probiotic preparations to assist in the treatment of depression, irritable bowel syndrome and other diseases; in the food field, it can be used to improve the nutritional value and safety of food, for example, by adding specific Gram-positive bacteria to enhance the health function of food; in the agricultural field, it can be used to improve the yield and quality of crops, for example, by applying biological fertilizers containing specific Gram-positive bacteria to promote plant growth.

[0029] In some specific embodiments, the concentration of EDTA-2Na or EDTA-Fe solution is in the range of 0.1-2M, and more specifically, the treatment effect is particularly significant when the concentration is 0.5M. Similarly, the concentration of NaCl solution in the range of 2-10wt% can achieve the ideal effect, especially when the concentration is 5wt%, it can better maintain the activity of Gram-positive bacteria. In addition, the concentration of human bacterial suspension is 10 5 -10 10 cells / mL range is suitable, and the treatment time is in the range of 1-4 hours, especially 2 hours, to obtain the best sorting effect. Finally, the treatment temperature is feasible in the range of 0-30℃, especially when the temperature is set to 4℃, which can effectively slow down the metabolism of microorganisms, thereby better maintaining the activity and stability of the flora.

[0030] Example 1

[0031] Verification of the effect of selective culture medium for Gram-negative bacteria

[0032] The components of the Gram-negative bacteria selective culture medium are shown in Table 1.

[0033] Table 1 Gram-negative bacteria enrichment solution composition

[0034] Ingredient name g / L Tryptone 20.0g glucose 1.0g Mannitol 2.0g Sodium Citrate 5.0g Sodium deoxycholate 0.5g Dipotassium hydrogen phosphate 4.0g Potassium dihydrogen phosphate 1.5g Sodium chloride 5.0g

[0035] The Gram-negative bacteria selective medium was prepared into a solid medium, and a suspension of Bacillus subtilis (Gram-positive bacteria, Bacillus subtilis, purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.821) was anaerobically cultured on the Gram-negative bacteria selective solid medium for 3 days. Figure 1 As shown, no colonies grew; the donor bacterial suspension (fecal sample from a healthy volunteer) was cultured anaerobically on a solid medium selective for Gram-negative bacteria for 3 days. Figure 2 As shown, the colonies grew in large numbers and after Gram staining, Figure 3 As shown, the colonies were identified as Gram-negative bacteria, indicating that the culture medium can be used to distinguish between Gram-positive and Gram-negative bacteria in intestinal flora.

[0036] Example 2

[0037] The intestinal flora suspension obtained by extracting human feces through an extractor has a concentration (unit bacteria count) of about 10 5 -10 10 cells / mL.

[0038] 5 ml of bacterial suspension was taken and treated with different treatment solutions of equal volume under different conditions, and the overall mortality of bacterial flora and the mortality of Gram-negative bacteria were calculated. The experimental results are shown in Table 2-10. The pH of the solution in each treatment group was 8.

[0039] The data of overall mortality of bacterial colonies is obtained through LIVE / DEAD TM BacLight TM After staining with BacterialViabilityKit live and dead fluorescent dye (Suzhou Thermo Fisher Instrument Co., Ltd.), the results were measured by flow cytometry and calculated according to the following formula.

[0040] Unit viable bacteria count cells / μL = unit bacterial count × activity

[0041]

[0042] Gram-negative bacteria mortality rate: The different treatment groups were diluted in gradients and spread on solid culture medium (only grows Gram-negative bacteria) plates prepared with Gram-negative bacteria selective culture medium, and the spread was counted after anaerobic culture for 24 hours. The Gram-negative bacteria mortality rate calculation formula is as follows:

[0043]

[0044] Table 2 NaCl 4℃ treatment 2h

[0045] NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 2.50％ 58.94 28.98 3％ 69.36 41.76 5％ 88.94 66.87

[0046] Table 3 NaCl 25℃ treatment 2h

[0047] NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 2.50％ 22.28 11.53 3％ 79.37 23.80 5％ 82.41 30.78

[0048] Table 4 NaCl 37℃ treatment 2h

[0049] NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 2.50％ 76.75 17.14 3％ 65.35 14.20 5％ 77.41 27.75

[0050] Table 5 NaCl 0℃ treatment 2h

[0051] NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 2.50％ 13.03 0.59 3％ 20.37 10.18 5％ 74.75 15.89

[0052] Table 6 EDTA-2Na 0℃ treatment 2h

[0053] NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.2M 44.52 49.73 0.5M 81.03 72.92

[0054] Table 7 EDTA-2Na 4℃ treatment 2h

[0055] EDTA-2Na concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.2M 66.46 23.60 0.5M 86.24 44.22

[0056] Table 8 EDTA-2Na25℃ treatment 2h

[0057] EDTA-2Na concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.2M 69.52 24.00 0.5M 82.27 37.53

[0058] Table 9 EDTA-Fe 25℃ treatment 15min

[0059] EDTA-Fe concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.5M 90.4 58.1

[0060] Table 10 EDTA-2Na combined with NaCl at 0℃ for 2h

[0061] EDTA-2Na concentration + NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.2MEDTA-2Na+2.5%NaCl 34.77 8.02 0.2MEDTA-2Na+3%NaCl 51.74 17.91 0.2MEDTA-2Na+5%NaCl 76.89 25.20 0.5MEDTA-2Na+2.5%NaCl 78.92 33.24 0.5MEDTA-2Na+3%NaCl 90.68 38.38 0.5MEDTA-2Na+5%NaCl 97.97 44.78

[0062] Table 11 EDTA-2Na combined with NaCl treatment at 4°C for 2h

[0063] EDTA-2Na concentration + NaCl concentration Gram-negative bacteria mortality % Overall mortality of bacterial colony % 0.2MEDTA-2Na+2.5%NaCl 31.96 13.82 0.2MEDTA-2Na+3%NaCl 35.97 22.37 0.2MEDTA-2Na+5%NaCl 73.14 38.84 0.5MEDTA-2Na+2.5%NaCl 57.89 38.11 0.5MEDTA-2Na+3%NaCl 80.53 47.48 0.5MEDTA-2Na+5%NaCl 98.39 56.34

[0064] From the above data, it can be seen that both EDTA-2Na and EDTA-Fe can effectively reduce the number of viable Gram-negative bacteria, and their killing power against Gram-negative bacteria is greater than that against Gram-positive bacteria, and can be used for the sorting of Gram-positive bacteria. When EDTA-2Na is used in combination with NaCl, the two play a synergistic role. On the one hand, they can maintain a high killing power against Gram-negative bacteria, and on the other hand, they can maintain a low overall mortality rate of the bacterial community, which is conducive to the sorting of Gram-positive bacteria. Among them, the treatment group treated with 0.5MEDTA-2Na+5%NaCl at 4℃ for 2h has a sterilization rate of 98.39% for Gram-negative bacteria, and only 56.34% for the overall killing power of the bacterial community, which is a more ideal method for sorting Gram-positive bacteria.

[0065] Example 3

[0066] The intestinal flora suspension obtained by extracting human feces through an extractor has a concentration (unit bacteria count) of about 10 5 -10 10 cells / mL.

[0067] Take 5 ml of bacterial suspension and treat it with equal volumes of 0.5 M EDTA-2 Na, 5% NaCl, and 0.5 M EDTA-2 Na + 5% NaCl at 4°C for 2 h.

[0068] The different treatment groups were diluted in gradients and spread on BHIS plates (purchased from Huankai Microorganisms). After anaerobic culture for 24 hours, the bacteria on the plates were picked and sent for 16S rDNA gene sequencing. The results are as follows: Figure 4 As shown, the percentage of Gram-positive bacteria is circled in red.

[0069] Table 12BHIS medium composition

[0070] Ingredient name g / L Tryptone 10.0g Disodium hydrogen phosphate 2.5g glucose 2.0g Sodium chloride 5.0g Beef heart extract 500mL

[0071] Comparative Example 1

[0072] The intestinal flora suspension of Example 1 was treated with different concentrations of ethanol for 20 minutes and then with 0.5M EDTA-2Na for 15 minutes. The experimental results are shown in Table 13.

[0073] Table 13

[0074]

[0075]

[0076] As shown in Table 13, ethanol and EDTA-2Na have bactericidal effects. When 12.5% ​​ethanol is used first and then EDTA-2Na is used, the killing power to Gram-negative bacteria is only 61.70%, which is not high enough and is not conducive to sorting Gram-positive bacteria. When 28.1% ethanol is used first and then EDTA-2Na is used, the killing power to Gram-negative bacteria is as high as 99.60%, but the killing power to the whole flora is also as high as 71.30%, which is not conducive to sorting Gram-positive bacteria. When only ethanol is used for treatment, the killing power of 12.5% ​​ethanol treatment to Gram-negative bacteria is small, only 40.00%. The killing power of 28.1% ethanol treatment to the whole flora is too high, reaching 95.10%, which is not conducive to obtaining high-purity Gram-positive bacteria flora.

[0077] Comparative Example 2

[0078] The intestinal flora suspension of Example 1 was treated with drying (drying in an oven at 37°C for 3 h) and ultrasound (ultrasound at 25°C for 3 h). The experimental results are shown in Table 14.

[0079] Table 14

[0080] Gram-negative bacteria mortality % Overall mortality of bacterial colony % dry 0 0 Ultrasound 0 0

[0081] As shown in Table 14, the overall number of viable bacteria did not decrease after drying and ultrasonic treatment, and the killing rate of Gram-negative bacteria was zero. Both treatments could not obtain a high-purity Gram-positive bacterial flora.

[0082] Comparative Example 3

[0083] The intestinal flora suspension of Example 1 was treated with a hydrochloric acid solution with a pH of 4 and a sodium hydroxide solution with a pH of 10.0 at 25° C. for 15 min. The experimental results are shown in Table 15.

[0084] Table 15

[0085] Gram-negative bacteria mortality % Overall mortality of bacterial colony % pH = 4 0 0.5 pH = 10 0 2.2

[0086] As can be seen from Table 15, the solutions with pH values ​​of 4 and 10 have very low killing rates on the total number of live bacteria, and the killing rate on Gram-negative bacteria is 0, so it is impossible to obtain a high-purity Gram-positive bacterial flora.

[0087] Comparative Example 4

[0088] The intestinal flora suspension of Example 1 was treated with the following processes: 10% LXP resin solution at 25°C for 15 min, 5% potassium citrate at 25°C for 30 min, 8 g / L tea polyphenols at 4°C for 60 min, and 4% phytic acid solution at 25°C for 15 min. The experimental results are shown in Table 16.

[0089] Table 16

[0090] Time / min Temperature / ℃ Gram-negative bacteria mortality % Overall mortality of bacterial colony % 10% LXP resin 15 25 23.4 85 5% Potassium Citrate 30 25 29.4 13.9 8g / L tea polyphenols 60 4 13.0 24.7 4% Phytic Acid 15 25 9.5 2.8

[0091] As shown in Table 16, the killing rates of LXP resin, potassium citrate, tea polyphenols and phytic acid on Gram-negative bacteria and bacterial flora are very low, and it is impossible to obtain high-purity Gram-positive bacterial flora.

[0092] In summary, the present invention sorts intestinal flora through different process treatments, and combines the flow cytometry results and the plate count results to verify whether these process treatments can inactivate Gram-negative bacteria while maintaining the activity of Gram-positive bacteria as much as possible to obtain a high-purity Gram-positive bacterial flora. Among them, EDTA-2Na and EDTA-Fe can both be used for the sorting of Gram-positive bacteria, especially the combination of EDTA-2Na and NaCl has the best effect.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for directional sorting of Gram-positive bacteria, characterized in that: The directional sorting method comprises: S1, extracting and processing human fecal samples to obtain human bacterial suspension; S2, treating the human bacterial suspension with EDTA-2Na or EDTA-Fe solution and NaCl solution simultaneously.

2. The method for directional separation of Gram-positive bacteria according to claim 1, characterized in that: The concentration of the EDTA-2Na or EDTA-Fe solution is 0.1-2M.

3. The method for directional separation of Gram-positive bacteria according to claim 2, characterized in that: The concentration of the EDTA-2Na or EDTA-Fe solution is 0.5M.

4. The method for directional separation of Gram-positive bacteria according to claim 1, characterized in that: The concentration of the NaCl solution is 2-10 wt %.

5. The method for directional separation of Gram-positive bacteria according to claim 4, characterized in that: The concentration of the NaCl solution is 5 wt %.

6. The method for directional separation of Gram-positive bacteria according to claim 1, characterized in that: The concentration of the human bacterial suspension is 10 5 -10 10 cells / mL.

7. The method for directional separation of Gram-positive bacteria according to claim 1, characterized in that: The treatment time is 1-4 hours.

8. The method for directional separation of Gram-positive bacteria according to claim 7, characterized in that: The treatment time is 2 hours.

9. The method for directional separation of Gram-positive bacteria according to claim 5, characterized in that: The treatment temperature is 0-30°C.

10. The method for directional separation of Gram-positive bacteria according to claim 9, characterized in that: The temperature of the treatment was 4°C.

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