CN116948939A - Method for improving bacterial diversity of fecal sample, culture medium and application of culture medium - Google Patents

Method for improving bacterial diversity of fecal sample, culture medium and application of culture medium Download PDF

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CN116948939A
CN116948939A CN202311197586.0A CN202311197586A CN116948939A CN 116948939 A CN116948939 A CN 116948939A CN 202311197586 A CN202311197586 A CN 202311197586A CN 116948939 A CN116948939 A CN 116948939A
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enrichment
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ycfa
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CN116948939B (en
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王琼
余欢
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Sichuan Anaerobic Biotechnology Co ltd
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Abstract

The invention belongs to the field of microorganism separation culture, and particularly relates to a method and a culture medium for improving the bacterial diversity of a fecal sample and application thereof. The method for improving the fungus picking diversity of the fecal sample provided by the invention comprises the following steps: sample processing, enrichment culture, colony isolation, wherein the enrichment culture medium comprises YCFA synthetic medium, cysteine salt, TE141, VFA-mix and high-concentration CuCl 2 Or a hydrate thereof. The enrichment medium provided by the invention can separate more species of culturable microorganisms, and remarkably improves the culturable microorganisms in the fecal sampleThe diversity can provide support for the screening of novel intestinal probiotics.

Description

Method for improving bacterial diversity of fecal sample, culture medium and application of culture medium
Technical Field
The invention belongs to the field of microorganism separation culture, and particularly relates to a method and a culture medium for improving the bacterial diversity of a fecal sample and application thereof.
Background
Microorganisms are the most widely distributed, most abundant species and the largest biomass life forms on earth. Human beings have found microorganisms for over two hundred years, but only for over a hundred years. To date, human cognition and development utilizes less than 1% of the total species of microorganisms, and less than few microorganisms can be cultivated. The development and utilization of the culturable microorganisms can develop into an emerging industry or promote the improvement and upgrade of the traditional industry: currently advanced gene editing techniques, such as CRISPR techniques, were first discovered in microbial cells; the large-scale microorganism manufacturing industry, such as microbiological pharmacy, microbiological fermentation, microbiological transformation and the like, relates to the economic field, and is a large industry closely related to people's life, industrial and agricultural production, national defense construction and the like.
Intestinal microorganisms are important "organs" of the human body, and are closely related to various physiological functions such as immunity, nutrition, metabolism and the like. The complex relationship between intestinal microorganisms and human health has been elucidated using macrogenomic sequencing techniques, but without bacterial isolates, the intrinsic mechanism by which specific bacteria act in the intestinal tract cannot be clarified, thereby further compromising the discovery of "dark matter" in the intestinal tract. The acquisition of the culturable microorganism provides a bacterial sample for deeply researching bacterial phenotype and gene function, and also provides data support for screening novel probiotics in intestinal tracts.
The technology of plate streaking is used as a traditional microorganism separation culture means, and at present, isolated intestinal microorganisms are mostly subjected to plate streaking by using a YCFA culture medium to obtain pure culture strains, and according to researches, the YCFA culture medium can provide 70% of substances required by the growth of human intestinal microorganisms. However, human intestinal culturable microorganisms isolated using only YCFA medium are limited, and some species that grow rapidly and are easy to culture (e.g., enterococcus, E.coli, P.aeruginosa, etc.) can quickly occupy most of the space of the culture plate, making it more difficult to isolate bacteria of other species. The diversity of microorganism species obtained under the same picking workload is lower. Therefore, it is necessary to expand the resources of the microorganisms that can be cultivated by means of improving the medium components and optimizing the cultivation conditions.
The optimization of the culture medium is mainly to adjust the components of the culture medium, and certain special substances are added into the conventional culture medium, for example, an inhibitor (antibiotics, ethanol and metabolic inhibitor) can be added to inhibit the growth of certain non-target bacteria, a signal molecule, namely, cyclic adenosine monophosphate (Cyclic Adenosine Monophosphate, cAMP) and Acyl homoserine lactone (Acyl-Homoserine Lactones, AHL) can be added to improve the microorganism culture efficiency, and certain growth factors (such as amino acids, vitamins and metal ions) can be added to promote the growth and metabolism of microorganisms.
The metal ions are used as important components in the culture medium and participate in various biochemical reactions in cells, and can be used as component parts of enzymes and participate in the metabolic processes of microorganisms. Li Lanxiao et al (study of the influence of Metal ions and growth factors on riboflavin fermentation) report CuSO 4 ·5H 2 O can promote the growth of bacillus subtilis and increase the yield of riboflavin. However, the use of metal ions in the prior art for the purpose of promoting the growth of certain microorganisms has not been used to increase the diversity of culturable microorganisms in fecal samples.
Disclosure of Invention
To solve the unmet practical needs of the prior art, the first aspect of the invention provides a method for improving the diversity of fecal sample picking bacteria.
The specific scheme is as follows:
a method for improving the diversity of fecal sample picking bacteria, comprising:
(1) Sample processing;
(2) Enrichment culture;
(3) Separating bacterial colonies;
wherein: the enrichment medium of step (2), comprising per 1L of medium the following components: 16.5-20.3 g YCFA synthetic culture medium, 0.9-1.1 g cysteine salt, 9-11 mL TE141, 2.9-3.5 mL VFA-mix, characterized in that CuCl is added into the enrichment culture medium 2 Or a hydrate thereof, in CuCl 2 The content of the composition is 140-180 mg/L.
The second aspect of the invention provides CuCl 2 Or a hydrate thereof, in the preparation of an enrichment medium for increasing the diversity of the stool sample, each 1L of medium comprising the following components: 16.5-20.3 g of YCFA synthetic medium, 0.9-1.1 g of cysteine salt, 9-11 mL of TE141 and 2.9-3.5 mL of VFA-mix.
In a third aspect of the invention, an enrichment medium for increasing the diversity of fecal sample picking bacteria is provided, each 1L of the medium comprising the following components: 16.5-20.3 g of YCFA synthetic medium, 0.9-1.1 g of cysteine salt, 9-11 mL of TE141 and 2.9-3.5 mL of VFA-mix.
In some embodiments, cuCl is added to the enrichment medium 2 Or a hydrate thereof.
In some embodiments, in CuCl 2 Based on the amount of CuCl added to the enrichment medium 2 The content is 140-180 mg/L.
In some embodiments, in CuCl 2 Based on the amount of CuCl added to the enrichment medium 2 The content is 160 mg/L.
In some embodiments, the enrichment medium can reduce the segregation duty cycle of pathogenic and/or opportunistic pathogens in the fecal sample.
In some embodiments, the enrichment medium may reduce the amount of a sample of fecal material selected from the group consisting of enterococcus tenuiosusEnterococcus durans）、Paraclostridium dentumBacillus dysenteriaeShigella dysenteriae) Enterococcus faeciumEnterococcus faecium) The separation duty cycle of any one of the species.
In some embodiments, each 1L medium comprises the following components: 18.44 g YCFA synthetic medium, 160 mg CuCl 2 (in CuCl) 2 Meter), 1 g cysteine salt, 10.00 mL of te141, 3.2 mL of VFA-mix.
The invention has the following beneficial effects:
(1) The improved culture medium provided by the invention can effectively reduce dominant species in the fecal sample on the separation plateEnterococcus durans(enterococcus tenaciosus),Paraclostridium dentum、Shigella dysenteriae(shigella dysenteriae),Enterococcus faeciumThe ratio of species such as (enterococcus faecium) is reduced by about 10%, the diversity of the culturable strains of the faeces sample is improved, and 33 species of culturable strains are separated on the premise of less colony count compared with the control group.
(2) The culture medium used in the technical proposal of the invention has simple components, namely, the conventional culture medium is added with CuCl 2 Or a hydrate thereof; the whole experimental process is simple, convenient and easy to get up.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below. It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by a person skilled in the art without making creative efforts based on the embodiments in the present invention shall fall within the protection scope of the present invention.
"diversity" refers to the abundance of a microbial species in a sample. The higher the diversity means the greater the number of species of microorganism in the sample; the lower the diversity means that the fewer the number of species of microorganism in the sample.
"separation ratio" refers to the ratio of the total number of microorganisms of a certain species separated from a sample to the number of all communities separated from the sample.
CuCl of the invention 2 Is an important component for increasing the diversity of the fecal sample, and can be added in any form, such as anhydrate or hydrate, the water content of the hydrate can be any value, the water content does not affect the final effect, and the common CuCl is adopted in one embodiment of the invention 2 ·2H 2 O。
The method for improving the fungus picking diversity of the fecal sample comprises the following steps:
(1) Sample processing;
(2) Enrichment culture;
(3) Separating bacterial colonies;
wherein: adding CuCl into the enrichment medium in the step (2) 2 Or a hydrate thereof.
The sample treatment in step (1) is to treat the collected fecal sample, which are conventional in the art, including sample weighing, addition of protective agents, shaking, centrifugation, and liquid sample acquisition.
The enrichment culture in the step (2) is to add the sample into an enrichment culture medium and culture under proper conditions (such as 37 ℃ C., anaerobic). Under the condition of no special treatment, the bacteria cultured in the step (2) are mainly dominant bacteria existing in the sample. In order to increase the diversity of the obtained strains, the whole fungus picking process can only be repeated for a plurality of times. Even so, due to the dominanceThe competition of seeds, the growth of strains with small content is inhibited, and finally, the strains are difficult to pick. The invention adds CuCl 2 Or the hydrate thereof can reduce the ratio of a plurality of dominant species and improve the species diversity of the final selected bacteria.
In some embodiments, in CuCl 2 Meter, cuCl 2 Or the addition amount of the hydrate is 140-180 mg/L. Within this range, inhibition of dominant species and normal growth of less-than-species are better balanced, in one particular case, in CuCl 2 The addition amount is 160 mg/L.
The colony isolation in the step (3) is to take and dilute the enriched sample in the step (2) and then culture the diluted enriched sample again so as to obtain a pure culture of single colony.
Based on the discovery of the method, the invention also provides CuCl 2 Or the hydrate thereof, in the preparation of an enrichment medium for improving the diversity of fecal sample picking bacteria.
Based on the findings of the foregoing methods, the present invention further provides an enrichment medium for improving the diversity of fecal sample picking bacteria, the medium comprising CuCl 2 Or a hydrate thereof.
The preparation methods of the culture medium, the reagent, etc. used in the following examples are as follows:
YCFA+160 mg/L CuCl 2 experiment medium: weighing the corresponding amount of the reagent according to the formula dosage of the reagent in the table 1, adding the corresponding amount of boiled pure water into a triangular flask, and stirring and dissolving by a glass rod; boiling, adding two boiling reagents, adjusting pH to 7.0-7.5, and opening Hengalit device to make culture medium in N 2 Boiling again under protection, blowing for about 20 min, and collecting the residue in N 2 Is divided into N parts under protection 2 In the deoxidized anaerobic bottle, the liquid split charging height is not more than 2/3 of the height of the anaerobic bottle; and (5) partially packaging 10-mL into anaerobic tubes, capping plugs, attaching culture medium label paper, and sterilizing at 121 ℃ under moist heat for 15-20 min. Agar powder was added in an amount of 1.5% (mass/volume ratio, 15 g/L) to prepare a solid medium.
TABLE 1 YCFA+160 mg/L CuCl 2 Preparation of experimental culture medium
Reagent name Formulation dosage (1L) Remarks
YCFA synthetic culture medium 18.44 g One-boiling
CuCl 2 ·2H 2 O 0.20 g One-boiling
Cysteine salts 1.00 g Two-stage cooking
TE141 10.00 mL Two-stage cooking
VFA-mix 3.2 mL Two-stage cooking
Note that: YCFA synthetic medium, manufacturer: qingdao sea blogs, goods number: HB9212
YCFA+160 mg/L CuSO 4 Control medium: formulated according to the formulation amounts of the reagents in Table 2, the procedure is referenced YCFA+160 mg/L CuCl 2 Experimental medium preparation step.
TABLE 2 YCFA+160 mg/L CuSO 4 Control Medium formulation
Reagent name Formulation dosage (1L) Remarks
YCFA synthetic culture medium 18.44 g One-boiling
CuSO 4 ·5H 2 O 0.25 g One-boiling
Cysteine salts 1.00 g Two-stage cooking
TE141 10.00 mL Two-stage cooking
VFA-mix 3.2 mL Two-stage cooking
YCFA control medium: formulated according to the formulation amounts of the reagents in Table 3, the procedure is referenced YCFA+160 mg/L CuCl 2 Experimental medium preparation step.
TABLE 3 YCFA control Medium formulation
Reagent name Formulation dosage (1L) Remarks
YCFA synthetic culture medium 18.44 g One-boiling
Cysteine salts 1.00 g Two-stage cooking
TE141 10.00 mL Two-stage cooking
VFA-mix 3.2 mL Two-stage cooking
VFA-mix: acetic acid 27 mL, propionic acid 9 mL, n-valeric acid 3 mL, isobutyric acid 3 mL and butyric acid 3 mL are measured and mixed uniformly for later use.
TE141: the reagents were weighed according to the amounts in the formulations of Table 4. In the first step, nitrilotriacetic acid (Nnitrilotriacetic acid) is added into 800 mL pure water, the pH is regulated to 7.0, after the mixture is stirred until the mixture is clarified, water is continuously added to 1000 mL, and finally the pH is regulated to 6.5. The other components were added sequentially in the order listed, each step was stirred until clear, and if clear was not achieved for a long period of time, the pH was adjusted to 6.5. Weighing Na 2 SeO 3 ·5H 2 O0.12508 g, adding 12.5. 12.5 mL pure water, dissolving completely to obtain Na 2 SeO 3 ·5H 2 And using O mother liquor. Weighing Na 2 WO 4 ·2H 2 O0.14735 g, adding 14.7. 14.7 mL pure water, dissolving completely to obtain Na 2 WO 4 ·2H 2 And using O mother liquor. Finally, the clarified TE141 is packaged and labeled, and the product is placed and stored at room temperature.
Table 4 formulation of te141
Reagent name Formulation dosage (1L)
Nnitrilotriacetic acid 1.50324 g
MgSO 4 ·7H 2 O 3.00384 g
MnSO 4 ·H 2 O 0.50442 g
NaCl 1.00415 g
FeSO 4 ·7H 2 O 0.10378 g
CoSO 4 ·7H 2 O 0.18171 g
CaCl·2H 2 O 0.11388 g
CuSO 4 ·5H 2 O 0.01331 g
ZnSO 4 ·7H 2 O 0.18426 g
KAI(SO4) 2 ·12H 2 O 0.02003 g
H 2 BO 3 0.01391 g
Na 2 MO 4 ·2H 2 O 0.01096 g
NiCL·6H 2 O 0.0339 g
Na 2 SeO 3 ·5H 2 O 0.03 mL mother liquor
Na 2 WO 4 ·2H 2 O 0.04 mL mother liquor
NaOH solution Adjusting pH
HCl solution Adjusting pH
Preparing a flat plate: YCFA+160 mg/LCuCl 2 Solid medium, YCFA+160 mg/LCuSO 4 The solid culture medium and the YCFA solid culture medium are heated at 104 ℃ for 25-30 min respectively, and then are dissolved, and are placed in a water bath kettle at 50 ℃. Mix sugar was filtered through a 0.22 μm filter and added to the medium, 9.6 ml Mix sugar was added per 360. 360 ml medium, the plates were poured into a biosafety cabinet, air dried for 20 minutes, and transferred to an anaerobic glove box overnight for deoxygenation.
Mix sugar: the reagents were weighed according to the amounts in the formulations of Table 5. Mixing the boiled deoxidized components in proportion, proportionally metering to a proper volume, introducing nitrogen to deoxidize for 30 min, packaging and sealing, and sterilizing at 115 ℃ for 20 min under high pressure. 0.5% Vitamin K1 mother liquor: 0.1 mlailamin K1+20ml 95% ethanol. 2 mg/mL of a Heamin mother liquor: 200 mg of Heamin+4 mL of 1M NaOH was fixed to a volume of 100 mL. The filter sterilized components were added in proportion, added to the sterilized sugar solution using a syringe and needle filter, and the filtration was performed in an anaerobic glove box. And 4. After the filtration is completed, the mixture is stored at a temperature of 4 ℃ in a dark place.
TABLE 5 preparation of Mix sugar
Reagent name Conversion amount Remarks
Glucose（50%） 5.00 g Boiling deoxidization
Maltose（10%） 0.50 g Boiling deoxidization
Cellobiose（10%） 0.50 g Boiling deoxidization
Pure water Constant volume 20 mL Boiling deoxidization
10·V141 1 mL Filter sterilization
Vitamin K1 0.13 mL Filter sterilization
Heamin 3.27 mL Filter sterilization
Totalizing 24.4 mL
10.v 141: after all the components in Table 6 were dissolved, a 0.22 μm filter was used for sterilization and sealed for use.
TABLE 6 preparation of 10V 141
Reagent name Dosage of the formula
Biotin 20.00 mg
Folic acid 20.00 mg
Pyridoxine-HCl 100.00 mg
Thiamine-HCl·2H 2 O 50.00 mg
Riboflavin 50.00 mg
Nicotinic acid 50.00 mg
Ca-pantothenate 50.00 mg
Vitamin B 12 1.00 mg
p-Aminobenzoic acid 50.00 mg
Lipoic acid 50.00 mg
Purified water 1000.00 mL
And (3) a protective agent: formulated according to the formulation amounts of the reagents in Table 7, the procedure is referenced YCFA+160mg/L CuCl 2 And (5) preparing an experiment culture medium.
Table 7 protectant formulation
Reagent name Formulation dosage (1L) Remarks
Na 2 HPO 4 ·12H 2 O 3.85 g One-boiling
KH 2 PO 4 0.27 g One-boiling
NaCl 8.00 g One-boiling
Glycerol 200 mL One-boiling
Cysteine salts 1.00 g Two-stage cooking
Resazurin (L.) Kuntze 1 mL 0.1% aqueous solution
Lysate: mixing pure formic acid and pure acetonitrile according to the proportion of 4:3, and uniformly mixing.
Matrix liquid: uniformly mixing 5% trifluoroacetic acid (spectrum level) and acetonitrile according to a ratio of 1:1 to obtain a matrix solvent; alpha-cyano-4-hydroxy cinnamic acid is added into a matrix solvent to prepare a matrix solution with the concentration of 10 mg/ml.
Example 1 sample processing, enrichment culture and separation
(1) Sample processing
a. Weighing and recording color, character and other information after the original fecal sample arrives at a laboratory;
b. taking 1 branch of clean 50 mL centrifuge tubes, and adding 6-7 sterilized and dried glass beads into the centrifuge tubes;
c. picking 0.5-1 g of sample in a 50 mL anaerobic sample tube in an anaerobic glove box, adding 10 times of volume protective agent, covering a spiral cover, after rotating and oscillating for 5 minutes, subpackaging a Pasteur pipette with about 1 mL/spiral cap tube, and pasting corresponding labels on each tube;
d. putting the split-packed samples into a 9*9 freezing box, then putting into a music buckle box, putting into 2 anaerobic deoxidizing bags, and preserving at a low temperature of minus 80 ℃;
(2) Enrichment culture
Randomly picking up a frozen sample at the temperature of minus 80 ℃, thawing in an anaerobic glove box, respectively taking 0.1 mL sample, adding the sample into an anaerobic tube of 10 mL for enrichment, and adding 160 mg/L CuCl 2 The YCFA medium of (2) was set as an experimental group, the YCFA medium without copper ions was set as a control group 1, and 160 mg/L CuSO was added 4 Is set as control group 2. Anaerobic culture was performed at 37℃for 14 days with shaking of the anaerobic tube every 2-3 days in the middle.
(3) Colony isolation
a. Sample coating: taking enriched samples of 0.5 mL each in 4.5 ml diluted anaerobic PBS, mixing uniformly and recording as 10 -1 Gradient, then sequentially carrying out 10-time gradient dilution; take 10 -3 To 10 -4 Diluting gradient liquid 100 μl each, coating onto corresponding plate, placing coated plate in a music buckle box, adding deoxidizingBags, anaerobic culture for 4-5 days.
b. Picking a bacterial colony: filtering and sterilizing Mix sugar, adding YCFA+160 mg/L CuCl respectively 2 Liquid culture medium, YCFA+160 mg/L CuSO 4 Liquid medium and YCFA liquid medium (9.6 ml Mix sugar is added into 360. 360 ml medium), and then the culture medium is respectively split into 384 plates 1 and 2 (80 mu L per well); according to different properties of bacterial colony such as morphology, size, color and dryness, bacterial colony is picked into 384-well plate 1, 10 mu L of liquid is inoculated into 384-well plate 2 corresponding hole sites after blowing and mixing uniformly, and 96 bacterial colonies are picked for each sample.
EXAMPLE 2 colony identification
(1) Autof MS2000 weight reduction
Taking out 384 plates 2 of example 1, centrifuging at 5000 rpm for 10 min, discarding the supernatant, washing with ultra pure water (80. Mu.L/well), centrifuging at 5000 rpm for 10 min, discarding the supernatant, and repeating the above steps once; adding 10 mu L/hole lysate into 384-well plate, mixing, blowing, taking 1 mu L supernatant to spot target, air drying at room temperature, and spot-adding 1 mu L matrix solution; performing mass spectrum identification after airing at room temperature; and (3) carrying out cluster analysis according to the protein patterns of the bacterial colonies, randomly selecting a hole site from 5 identical patterns, picking the hole site to a new 96-well plate 1 and 2 with corresponding liquid culture medium, culturing for 2-3 days at 37 ℃, and then carrying out PCR sequencing by using the 96-well plate 1.
(2) PCR sequencing
Sample washing before PCR: centrifuging 96-well plate 1 at 5300 rpm for 10 min, and discarding supernatant; adding 30-100 μl of 1×TE resuspended thallus per well according to fungus concentration, centrifuging at 5300 rpm for 10 min, and discarding supernatant; the resuspended cells were stored at-80℃overnight.
PCR detection: taking out the frozen overnight bacterial liquid plate, breaking the walls by microwaves for 3-4 times, each time for 1 minute and 30 seconds, and placing the bacterial liquid plate on ice for complete cooling after each time of microwave heating and then carrying out the next microwave operation; the PCR reaction system was prepared according to the sample amount (see Table 8). The 96-well PCR plate was placed on a PCR instrument and the amplification procedure was set: 98℃for 5 min,98℃for 10 s,58℃for 10 s,72℃for 20 s,30 cycles, 72℃for 2 min,4℃for the end of amplification; the PCR product was sent to sequencing systems, inc. of Programme technologies for sequencing.
TABLE 8 PCR reaction System
Composition of the components Volume of
2×T5 Supr PCR Mix(Colony) 12.5 μL
5×Enhacer Buffer 5 μL
27-F 1 μL
1492-R 1 μL
PCR water (sterilized ultrapure water) 4.5 μL
DNA template 1 μL
c. Analysis of results: and uploading the 16S sequence fed back by the sequencing company to a refseq library of NCBI for sequence comparison, and obtaining the Latin name corresponding to the sequence.
TABLE 9 isolation and identification of microorganisms in stool samples of experimental and control groups
Experimental group Experiment Group of Experimental group Control group 1 Control group 1 Control group 1 Control group 2 Control group 2 Control group 2
Species Quantity of Split duty cycle （%） Species Quantity of Split duty cycle （%） Species Quantity of Split duty cycle （%）
Enterococcus durans 28 11.38 Enterococcus durans 63 19.27 Peptoniphilus tyrrelliae 19 26.76
Clostridium butyricum 23 9.35 Paraclostridium dentum 62 18.96 Enterococcus mundtii 17 23.94
Enterococcus faecalis 19 7.72 Shigella dysenteriae 51 15.60 Peptoniphilus indolicus 10 14.08
Enterococcus avium 17 6.91 Enterococcus faecium 41 12.54 Finegoldia magna 9 12.68
Shigella dysenteriae 16 6.50 Enterococcus innesii 30 9.17 Ruthenibacterium lactatiformans 7 9.86
Shigella sonnei 9 3.66 Shigella sonnei 20 6.12 Clostridium lactatifermentans 3 4.23
Enterococcus innesii 9 3.66 Enterococcus gallinarum 17 5.20 Bacillus licheniformis 2 2.82
Bacteroides uniformis 8 3.25 Clostridium baratii 14 4.28 Enterococcus casseliflavus 2 2.82
Finegoldia magna 7 2.85 Enterococcus avium 11 3.36 Enterococcus faecium 1 1.41
Phocaeicola vulgatus 7 2.85 Clostridium tertium 6 1.83 Clostridium sordellii 1 1.41
Bacteroides thetaiotaomicron 6 2.44 Enterococcus faecalis 5 1.53
Thomasclavelia ramosa 6 2.44 Citrobacter braakii 5 1.53
Enterococcus hirae 5 2.03 [Clostridium] symbiosum 1 0.31
Clostridium baratii 5 2.03 Enterococcus hirae 1 0.31
Parabacteroides distasonis 5 2.03
Eubacterium maltosivorans 5 2.03
Enterococcus gilvus 5 2.03
Enterococcus xiangfangensis 4 1.63
Paraclostridium dentum 4 1.63
Intestinibacter bartlettii 4 1.63
Lacticaseibacillus paracasei 4 1.63
Enterococcus gallinarum 4 1.63
Enterococcus faecium 3 1.22
Lentilactobacillus buchneri 3 1.22
Loigolactobacillus coryniformis 3 1.22
Bacteroides kribbi 3 1.22
Enterococcus thailandicus 3 1.22
[Clostridium] innocuum 3 1.22
Clostridium tertium 2 0.81
Cutibacterium avidum 2 0.81
Bacteroides fragilis 2 0.81
Enterococcus hulanensis 2 0.81
[Clostridium] symbiosum 2 0.81
Lacticaseibacillus rhamnosus 2 0.81
Bittarella massiliensis 2 0.81
Blautia producta 2 0.81
Peptoniphilus tyrrelliae 2 0.81
Phocaeicola dorei 1 0.41
Klebsiella oxytoca 1 0.41
Enterococcus raffinosus 1 0.41
Parabacteroides merdae 1 0.41
Eggerthella lenta 1 0.41
Eubacterium callanderi 1 0.41
Bacteroides nordii 1 0.41
Flavonifractor plautii 1 0.41
Streptococcus anginosus 1 0.41
Citrobacter braakii 1 0.41
Totals to 246 1 Totals to 327 1 Totals to 71 1
As shown in table 9, the experimental group had 47 isolated culturable strain species, and the control group 1 had only 14 and the control group 2 had 10. The culture medium of the experimental group can effectively reduce dominant species in the fecal sample on the separation plateEnterococcus durans(enterococcus tenaciosus),Paraclostridium dentum、 Shigella dysenteriae(shigella dysenteriae),Enterococcus faeciumThe ratio of species such as (enterococcus faecium) is reduced by about 10 percent. The experimental group had 33 more species than the control group of culturable strains isolated with a smaller colony count. Adding the same amount of CuSO 4 A similar effect cannot be achieved. To sum up, 160 mg/L CuCl is added 2 The improved YCFA culture medium of (2) can effectively improve the diversity of the culturable microorganisms in the fecal sample.

Claims (7)

1. A method for improving the diversity of fecal sample picking bacteria, comprising:
(1) Sample processing;
(2) Enrichment culture;
(3) Separating bacterial colonies;
the enrichment medium of step (2), comprising per 1L of medium the following components: 16.5-20.3 g YCFA synthetic culture medium, 0.9-1.1 g cysteine salt, 9-11 mL TE141, 2.9-3.5 mL VFA-mix, characterized in that CuCl is added into the enrichment culture medium 2 Or a hydrate thereof, in CuCl 2 The content of the composition is 140-180 mg/L.
2.CuCl 2 Or a hydrate thereof, in the preparation of an enrichment medium for increasing the diversity of the stool sample, each 1L of medium comprising the following components: 16.5-20.3 g YCFA synthetic culture medium, 0.9-1.1 g cysteine salt, 9-11 mL TE141, 2.9-3.5 mL VFA-mix, characterized in that CuCl is added into the enrichment culture medium 2 Or a hydrate thereof, in CuCl 2 The content of the composition is 140-180 mg/L.
3. An enrichment medium for improving the diversity of fecal sample picking bacteria, wherein each 1L of medium comprises the following components: 16.5-20.3 g YCFA synthetic culture medium, 0.9-1.1 g cysteine salt, 9-11 mL TE141, 2.9-3.5 mL VFA-mix, characterized in that CuCl is added into the enrichment culture medium 2 Or a hydrate thereof, in CuCl 2 The content of the composition is 140-180 mg/L.
4. The method according to claim 1 or the use according to claim 2 or the enrichment medium according to claim 3, characterized in that the step of reducing the concentration of the enriched medium is performed in CuCl 2 Counting, adding CuCl into the enrichment medium 2 Or its hydrate content is 160 mg/L.
5. The method or use or enrichment medium according to claim 4, wherein the enrichment medium reduces the separation ratio of pathogenic and/or opportunistic pathogens in a fecal sample.
6. The method or use or enrichment medium according to claim 4, wherein the enrichment medium reduces the amount of enterococcus tenuiosus selected from the group consisting of enterococcus tenuiosus in a fecal sampleEnterococcus durans）、Paraclostridium dentumBacillus dysenteriaeShigella dysenteriae) Enterococcus faeciumEnterococcus faecium) The separation duty cycle of any one of the species.
7. The method or use or enrichment medium according to claim 4, wherein each 1L of medium comprises the following components: 18.44 g YCFA synthetic medium, 160 mg CuCl 2 1 g cysteine salt, 10 mL TE141, 3.2 mL VFA-mix.
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