Composition and application thereof in preparation of microbial culture medium

Abstract

The invention relates to the technical field of blood culture bottles, in particular to a composition and application thereof in preparation of a microbial culture medium. Yeast extract powder, soy peptone and casein tryptone are combined into a nitrogen source, and an aerobic and anaerobic culture medium for clinical pathogenic microorganism culture is formed. Through comparison verification, in growth experiments of nine aerobic bacteria and seven anaerobic bacteria, the culture speed and the biomass accumulation of the aerobic and anaerobic culture media added with the composition are integrally superior to those of other components excluded in the prior art and screening. Therefore, the composition can effectively promote the performance of a blood culture product, improve the clinical blood flow infection detection capability, inhibit antibiotic abuse and save the life of a patient.

Description

Technical Field

[0001] The present invention relates to the technical field of blood culture bottles, in particular to a composition and application thereof in preparing microbial culture medium. Background Art

[0002] Microorganisms are a group of tiny organisms with simple structures that require a microscope to observe. They include bacteria, fungi, viruses, mycoplasmas, chlamydia, and many other types. Some of these microorganisms can become pathogens and invade the human body, disrupting normal physiological functions and causing various diseases. Bloodstream infections, for example, are systemic infections caused by pathogenic microorganisms and their toxins invading the human circulatory system. Common pathogens include bacteria such as Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus, as well as fungi such as Candida species. Once bloodstream infections occur, patients experience symptoms such as high fever, chills, and fatigue. In severe cases, they can lead to septic shock, multiple organ dysfunction syndrome, and even death, posing a serious threat to human health.

[0003] Among the existing screening methods for pathogenic microorganisms, the culture method still occupies an important position due to its advantage of being able to directly obtain pathogenic microorganisms and provide an accurate basis for clinical diagnosis and treatment. Taking the diagnosis of bloodstream infection as an example, blood culture bottles are a typical application of the culture method. The operating procedure is to inject blood drawn from the patient into a blood culture bottle pre-filled with culture medium, and then place it in a blood culture instrument for culture. If the patient has a bloodstream infection, the pathogens in the blood will grow and multiply in the culture medium, and the carbon dioxide produced by metabolism will change the pH value at the bottom of the blood culture bottle, causing the pre-embedded pH probe to change color or fluorescence. The blood culture instrument will automatically alarm after detecting the signal, and then determine the type and characteristics of the pathogen through subsequent operations such as Gram staining.

[0004] However, the current culture method represented by blood culture bottles has significant drawbacks. The culture time of blood culture bottles is too long, and the detection speed lags far behind the progression of the patient's condition. While waiting for the test report, clinicians often have to blindly use antibiotics to save patients' lives. This not only exacerbates the problem of bacterial resistance, but also increases the patient's treatment costs and risk of adverse reactions. Some patients even die because they cannot receive timely and accurate treatment. How to solve the problems of slow culture speed and high cost has become a difficult problem that needs to be overcome in the field of pathogenic microorganism screening. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a composition and its application in preparing a microbial culture medium.

[0006] The present invention provides the use of at least one of trypticase peptone, soy peptone and yeast extract powder in preparing a culture medium for microorganisms.

[0007] Research has shown that trypticase peptone, soy peptone, and yeast extract powder each have their own advantages as nitrogen sources for culture media. The present invention discovered that when used in a specific ratio, these three ingredients can produce a synergistic effect as nitrogen sources, leading to faster microbial growth. Furthermore, these three raw materials are relatively low in price, reducing the cost of the culture media.

[0008] Experiments in this paper demonstrate that a culture medium prepared using a combination of trypticase peptone, soy peptone, and yeast extract powder in a specific ratio as a nitrogen source can more quickly and accurately culture pathogenic microorganisms, thereby making pathogen identification more timely. Previous experiments using culture media prepared using either peptone or yeast extract powder alone resulted in slower growth of common pathogens.

[0009] The composition provided by the present invention comprises trypticase peptone, soy peptone, and yeast extract powder. Preferably, the mass ratio of trypticase peptone, soy peptone, and yeast extract powder is (10-20):(1-10):(1-10). Preferably, the mass ratio of trypticase peptone, soy peptone, and yeast extract powder is (15-20):(1-5):(3-8). More preferably, the mass ratio of trypticase peptone, soy peptone, and yeast extract powder is 17:3:5.

[0010] The tryptic peptone contains small peptides with a molecular weight of less than 1000 Da, and the mass fraction of the small peptides in the tryptic peptone is not less than 90%.

[0011] The soy peptone has a total nitrogen content of ≥8% and an amino acid nitrogen content of ≥1.8%.

[0012] The yeast extract powder has a total nitrogen content of 10%-13%, an amino acid nitrogen content of ≥5%, a magnesium ion content of ≤500ppm, a calcium ion content of ≤500ppm, a manganese ion content of ≤10ppm, and a copper ion content of ≤5ppm.

[0013] In the composition of the present invention, each component can exist in the form of a powder or a solution. Each component can exist in a mixed form or independently of each other, and the present invention does not limit this.

[0014] Furthermore, the present invention provides a microbial culture medium comprising a carbon source, an inorganic salt, an amino acid, a cofactor and the composition as described above.

[0015] To develop the composition of the present invention, multiple rounds of screening were conducted for the carbon source, inorganic salts, amino acids, and cofactors required for the culture medium. The screening process was validated using 16 pathogenic bacteria. The screening scope mainly involves 43 ingredients, including: peptone (trypticase peptone, soy peptone), animal tissue extract, yeast extract (yeast extract powder, yeast extract), amino acids (L-arginine, L-glutamic acid, L-glutamine, L-isoleucine, L-leucine, L-valine, L-threonine, L-lysine, L-tryptophan), L-cysteine, glucose, sucrose, vitamins (VK3, VB1, VB3, VB4, VB5, VB6, VB7, VB8, VB12), inorganic salts (sodium chloride, dipotassium hydrogen phosphate), sodium pyruvate, sodium citrate, sodium polyanethiosulfonate (SPS), gelatin, nicotinamide adenine dinucleotide (NAD), hemin, saponin, sodium thioglycolate, mannitol, defoaming agent, disodium succinate, dimethylmercaptopropionic acid, betaine, S-methyl-L-methionine.

[0016] After the above screening, with the goal of reducing costs and increasing microbial growth, the culture medium was determined to contain glucose and sodium pyruvate as carbon sources; sodium chloride and dipotassium hydrogen phosphate as inorganic salts; cysteine ​​and tryptophan as amino acids; and hemin, sodium polyanethole sulfonate, sodium citrate, and an antifoaming agent as cofactors. The antifoaming agent was a polyether antifoaming agent. Experiments have shown that using the culture medium of the present invention to cultivate pathogens can achieve higher culture efficiency.

[0017] Preferably, the components of the culture medium include water and trypticase peptone, soy peptone FP408, yeast extract powder, glucose, sodium chloride, dipotassium hydrogen phosphate, hemin, sodium polyanethol sulfonate, sodium citrate, sodium pyruvate, cysteine, antifoaming agent, tryptophan, sucrose, glutamine, sodium glutamate, D-pantothenic acid sodium salt and ammonium sulfate hydrochloride.

[0018] In the culture medium of the present invention, the working concentration of trypticase peptone is 10-20 g / L, the working concentration of soy peptone is 1-10 g / L, and the working concentration of yeast extract powder is 1-10 g / L.

[0019] Preferably, the culture medium comprises water and the following components at working concentrations:

[0020] Trypticase peptone 10-20 g / L, soy peptone 1-10 g / L, yeast extract powder 1-10 g / L, glucose 1-5 g / L, sodium chloride 2-10 g / L, dipotassium hydrogen phosphate 1-4.5 g / L, hemin 0.002-0.015 g / L, sodium polyanethole sulfonate 0.1-1 g / L, sodium citrate 0.05-1 g / L, sodium pyruvate 0.1-2 g / L, cysteine ​​0.1-2 g / L, antifoam 0.05-1 g / L, tryptophan 0.005-1 g / L, sucrose 1-5 g / L, glutamine 0.1-2 g / L, sodium glutamate 0.1-2 g / L, sodium D-pantothenate 0.001-0.015 g / L, and ammonium sulfate hydrochloride 0.001-0.015 g / L.

[0021] More preferably, the culture medium comprises water and 17 g / L tryptone, 3 g / L soy peptone, 5 g / L yeast extract powder, 2.3 g / L anhydrous glucose, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 0.005 g / L hemin, 0.3 g / L sodium polyanethole sulfonate (SPS), 0.2 g / L sodium citrate, 1 g / L sodium pyruvate, 0.6 g / L L-cysteine, 0.15 g / L antifoam, 0.01 g / L L-tryptophan, 2 g / L sucrose, 0.5 g / L glutamine, 0.5 g / L sodium glutamate, 0.002 g / L sodium D-pantothenate, and 0.001 g / L ammonium sulfate hydrochloride.

[0022] Furthermore, when fastidious bacteria are cultured, the culture medium further includes 1-5 g / L of hemolysin. Preferably, the hemolysin is saponin, and its concentration is 2.6 g / L.

[0023] Furthermore, if anaerobic bacteria are cultured, the culture medium further comprises 0.005-0.5 g / L of vitamin B6 and 0.1-1 g / L of sodium thioglycolate. Preferably, the concentration of vitamin B6 in the culture medium is 0.01 g / L, and the concentration of sodium thioglycolate in the culture medium is 0.5 g / L.

[0024] In the present invention, the concentration of each component in the culture medium as described above is the working concentration. During preparation, storage, or transportation, each component can be present in powder form or in solution form. During preparation, storage, or transportation, the concentration of each component can be 2 to 100 times the working concentration. For example, the concentration of each component can independently be 2 times, 5 times, 10 times, 20 times, 50 times, or 100 times.

[0025] Furthermore, the present invention also provides the use of the aforementioned composition or microbial culture medium in preparing a reagent for screening blood influenza pathogens.

[0026] In the present invention, the microorganisms are pathogenic microorganisms of blood flu, including pathogenic microorganisms cultured under aerobic conditions and pathogenic microorganisms cultured under anaerobic conditions.

[0027] As a feasibility case, the pathogenic microorganisms cultured under the aquaculture conditions are Escherichia coli, Streptococcus pyogenes, Alcaligenes faecalis, Candida albicans, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, fastidious Neisseria meningitidis and Haemophilus influenzae.

[0028] As a feasibility case, the pathogenic microorganisms cultured under anaerobic conditions are Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Clostridium histolyticum, Clostridium perfringens, Bacteroides fragilis and Bacteroides vulgaris.

[0029] The blood influenza pathogen screening reagent of the present invention comprises the culture medium and pH probe as described above.

[0030] Furthermore, the present invention also provides a method for culturing pathogens, which comprises inoculating the pathogens into the composition or the microbial culture medium and then culturing them.

[0031] Furthermore, the present invention also provides a method for screening influenza pathogens in blood, which comprises inoculating a blood sample into the aforementioned culture medium and determining whether pathogens are present based on the pH indication.

[0032] The present invention combines yeast extract powder, soy peptone, and trypticase peptone as nitrogen sources to form aerobic and anaerobic culture media for the cultivation of clinical pathogenic microorganisms. Comparative verification demonstrated that aerobic and anaerobic culture media supplemented with the aforementioned combination demonstrated superior growth rates and biomass accumulation compared to existing technologies and other formulations excluded from screening in growth experiments with nine aerobic and seven anaerobic bacteria. This demonstrates that the aforementioned combination can effectively improve the performance of blood culture products, enhance clinical bloodstream infection detection capabilities, curb antibiotic abuse, and save patients' lives. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 (A) Growth curves of Escherichia coli cultured aerobically in eight groups of aerobic blood media containing different ratios of trypticase peptone FP318 and soy peptone FP408, and (B) time-OD at key time points. 600 Histogram;

[0034] Figure 2(A) Growth curves of Staphylococcus aureus cultured aerobically in eight aerobic blood media containing different ratios of trypticase peptone FP318 and soy peptone FP408, and (B) time-OD values ​​at key time points. 600 Histogram;

[0035] Figure 3 (A) Growth curves of Escherichia coli cultured anaerobically in 8 anaerobic blood media containing different ratios of trypticase peptone FP318 and soy peptone FP408, and (B) time-OD values ​​at key time points. 600 Histogram;

[0036] Figure 4 (A) Growth curves of Staphylococcus aureus cultured in anaerobic blood medium containing eight different ratios of trypticase peptone FP318 and soy peptone FP408, and (B) time-OD values ​​at key time points. 600 Histogram;

[0037] Figure 5 (A) Growth curves and (B) time-OD curves at key time points for Escherichia coli cultured aerobically in the aerobic medium of Comparative Example 1, the aerobic medium of Comparative Example 2, and the aerobic blood culture medium (unaged) of Example 1. 600 Histogram;

[0038] Figure 6 (A) Growth curves of Escherichia coli cultured aerobically in the aerobic medium of Comparative Example 1, the aerobic medium of Comparative Example 2, and the aerobic blood culture medium of Example 1 (aged for 24 hours) and (B) time-OD at each key time point 600 Histogram;

[0039] Figure 7 (A) Growth curves of Escherichia coli cultured aerobically in the aerobic medium of Comparative Example 1, the aerobic medium of Comparative Example 2, and the aerobic blood culture medium of Example 1 (aged for 48 hours) and (B) time-OD at each key time point 600 Histogram;

[0040] Figure 8 (A) Growth curves of Escherichia coli cultured aerobically in the aerobic medium of Comparative Example 1, the aerobic medium of Comparative Example 2, and the aerobic blood culture medium (aged for 72 hours) of Example 1, and (B) time-OD at each key time point. 600 Histogram;

[0041] Figure 9 The growth curves of Escherichia coli cultured aerobically in (A) aerobic culture medium of Comparative Example 1, (B) aerobic culture medium of Comparative Example 2, and (C) aerobic culture medium of Example 1 after aging for different time periods are shown;

[0042] Figure 10(A) growth curves and (B) time-OD at key time points for E. coli grown anaerobically in Comparative Example 1 anaerobic, Comparative Example 2 anaerobic, and Example 1 anaerobic blood culture media (unaged) 600 Bar graphs;

[0043] Figure 11 (A) growth curves and (B) time-OD at key time points for E. coli grown anaerobically in Comparative Example 1 anaerobic, Comparative Example 2 anaerobic, and Example 1 anaerobic blood culture media (aged 24 h) 600 Bar graphs;

[0044] Figure 12 (A) growth curves and (B) time-OD at key time points for E. coli grown anaerobically in Comparative Example 1 anaerobic, Comparative Example 2 anaerobic, and Example 1 anaerobic blood culture media (aged 48 h) 600 Bar graphs;

[0045] Figure 13 (A) growth curves and (B) time-OD at key time points for E. coli grown anaerobically in Comparative Example 1 anaerobic, Comparative Example 2 anaerobic, and Example 1 anaerobic blood culture media (aged 72 h) 600 Bar graphs;

[0046] Figure 14 Growth curves for E. coli grown anaerobically in Comparative Example 1 anaerobic, Comparative Example 2 anaerobic, and Example 1 anaerobic blood culture media aged for different times;

[0047] Figure 15 (A) growth curves and (B) time-OD at key time points for E. coli grown aerobically in the four groups of aerobic blood culture media 600 Bar graphs;

[0048] Figure 16 (A) growth curves and (B) time-OD at key time points for S. aureus grown aerobically in the four groups of aerobic blood culture media 600 Bar graphs;

[0049] Figure 17 (A) growth curves and (B) time-OD at key time points for S. pneumoniae grown aerobically in the four groups of aerobic blood culture media 600 Bar graphs;

[0050] Figure 18 (A) growth curves and (B) time-OD at key time points for S. pyogenes grown aerobically in the four groups of aerobic blood culture media 600 Bar graphs;

[0051] Figure 19 (A) Growth curves of Pseudomonas aeruginosa cultured aerobically in four groups of aerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0052] Figure 20 (A) Growth curves of Candida albicans cultured aerobically in four groups of aerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0053] Figure 21 (A) Growth curves of Alcaligenes faecalis cultured aerobically in four sets of aerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0054] Figure 22 (A) Growth curves of Neisseria meningitidis cultured aerobically in three sets of aerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0055] Figure 23 (A) Growth curves of Haemophilus influenzae cultured aerobically in three sets of aerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0056] Figure 24 (A) Growth curves of Escherichia coli cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0057] Figure 25 (A) Growth curves of Staphylococcus aureus cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0058] Figure 26 (A) Growth curves of Streptococcus pneumoniae cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0059] Figure 27 (A) Growth curves of Clostridium perfringens cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram (C. perfringens will show aggregation phenomenon in the three parallel experiments of anaerobic blood culture medium in comparative example 5, resulting in a sawtooth growth curve (such as Figure 27(A)), making its SD larger);

[0060] Figure 28 (A) Growth curves of Clostridium histolyticum cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram (C. histolytica showed aggregation in the three parallel experiments of anaerobic blood culture medium in Comparative Example 5, resulting in a sawtooth growth curve (such as Figure 28 (A)), making its SD larger);

[0061] Figure 29 (A) Growth curves of Bacteroides fragilis cultured in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Histogram;

[0062] Figure 30 (A) Growth curves of Bacteroides vulgaris cultured anaerobically in four sets of anaerobic blood culture media and (B) time-OD at key time points. 600 Bar chart. DETAILED DESCRIPTION

[0063] The present invention provides compositions and their use in preparing microbial culture media. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0064] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0065] The terms "include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0066] The term "and / or" describes the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0067] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0068] The numerical ranges and parameters involved in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0069] The test materials used in this invention are all commercially available. The trypticase peptone used in the examples is trypticase peptone FP318, the soy peptone is soy peptone FP408, and the yeast extract powder is yeast extract powder FM888. All of these raw materials are from Angel Yeast. Compared to raw materials from other sources, the combination of trypticase peptone FP318, soy peptone FP408, and yeast extract powder FM888 can promote faster microbial growth.

[0070] in,

[0071] FM888 is a powdered product rich in free amino acids, peptides, B vitamins, nucleotides, and trace elements, obtained from purified cultured baker's yeast protein through autolysis, enzymatic hydrolysis, centrifugation, concentration, and spray drying. Its preparation method refers to Chinese patent application CN 115478018 A. The product has a total nitrogen content of 10%-13%, an amino acid nitrogen content of ≥5%, a magnesium ion content of ≤500ppm, a calcium ion content of ≤500ppm, a manganese ion content of ≤10ppm, a copper ion content of ≤5ppm, and an endotoxin content of <100EU / g. It has high clarity and excellent microbial cultivation ability, can significantly promote microbial growth, and does not produce phosphate precipitation, which can improve the stability and culture effect of blood culture medium.

[0072] FP408 is produced using an ultrafiltration process, with a total nitrogen content ≥8%, an amino acid nitrogen content ≥1.8%, and an endotoxin content ≤100 EU / g. It does not produce phosphate precipitation, avoiding the adverse effects of other soy peptones on microbial growth. Its richer and more stable nutritional profile provides a more suitable growth environment for microorganisms, significantly improving blood culture effectiveness.

[0073] FP318 is a protein hydrolysate obtained by hydrolyzing animal casein with trypsin. It is rich in peptides and amino acids, with small peptides below 1000Da accounting for more than 90%. It is sterilized and free of phosphate precipitation.

[0074] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0075] In the examples, the CI value was calculated according to the Bliss independence model, with Example 1 being the combined drug, and Comparative Example 1 and Comparative Example 2 being single drugs A and B, respectively, to calculate the synergy coefficient using the formula:

[0076]

[0077] CI>1 indicates synergy, CI=1 indicates additiveness, and CI<1 indicates antagonism.

[0078] The present invention will be further described below in conjunction with the embodiments:

[0079] Example 1

[0080] Aerobic culture medium formula:

[0081] Tryptic peptone FP318 17 g / L, soy peptone FP408 3 g / L, yeast extract powder FM888 5 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium polyanethole sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine ​​0.6 g / L, polyether antifoaming agent 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, and ammonium sulfate hydrochloride 0.001 g / L.

[0082] Aerobic culture medium + hemolysin formula (suitable for fastidious bacteria):

[0083] Tryptic peptone FP318 17 g / L, soy peptone FP408 3 g / L, yeast extract powder FM888 5 g / L, saponin 2.6 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium polyanethole sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine ​​0.6 g / L, polyether antifoaming agent 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, and ammonium sulfate hydrochloride 0.001 g / L.

[0084] Anaerobic culture medium formula:

[0085] Tryptic peptone FP318 17 g / L, soy peptone FP408 3 g / L, yeast extract powder FM888 5 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium polyanethole sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine ​​0.6 g / L, polyether antifoaming agent 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, ammonium sulfate hydrochloride 0.001 g / L, VB6 0.01 g / L, sodium thioglycolate 0.5 g / L.

[0086] Anaerobic culture medium + hemolysin formula (suitable for fastidious bacteria):

[0087] Trypticase peptone FP318 17g / L, soy peptone FP408 3g / L, yeast extract powder FM888 5g / L, saponin 2.6g / L, anhydrous glucose 2.3g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, hemin 0.005g / L, sodium polyanethole sulfonate (SPS) 0.3g / L, sodium citrate 0.2g / L, sodium pyruvate 1g / L, L-cysteine ​​0.6g / L, polyether antifoaming agent 0.15g / L, L-tryptophan 0.01g / L, sucrose 2g / L, glutamine 0.5g / L, sodium glutamate 0.5g / L, D-pantothenic acid sodium salt 0.002g / L, ammonium sulfate hydrochloride 0.001g / L, VB6 0.01g / L, sodium thioglycolate 0.5g / L.

[0088] Comparative Example 1

[0089] Aerobic culture medium formula:

[0090] Yeast extract powder FM888 5g / L, anhydrous glucose 2.3g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, hemin 0.005g / L, sodium polyanethole sulfonate (SPS) 0.3g / L, sodium citrate 0.2g / L, sodium pyruvate 1g / L, L-cysteine ​​0.6g / L, polyether antifoaming agent 0.15g / L, L-tryptophan 0.01g / L, sucrose 2g / L, glutamine 0.5g / L, sodium glutamate 0.5g / L, D-pantothenic acid sodium salt 0.002g / L, ammonium sulfate hydrochloride 0.001g / L.

[0091] Anaerobic medium formulation:

[0092] Yeast extract powder FM888 5 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium poly-m-cresol sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine 0.6 g / L, polyether antifoam 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, ammonium sulphate hydrochloride 0.001 g / L, VB6 0.01 g / L, sodium thio glycolate 0.5 g / L.

[0093] Medium for obligate anaerobes (both aerobic and anaerobic) as in Example 1 with saponin added to a concentration of 2.6 g / L.

[0094] Comparative Example 2

[0095] Aerobic medium formulation:

[0096] Tryptone peptone FP318 17 g / L, soya peptone FP408 3 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium poly-m-cresol sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine 0.6 g / L, polyether antifoam 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, ammonium sulphate hydrochloride 0.001 g / L.

[0097] Anaerobic medium formulation:

[0098] Tryptone peptone FP318 17 g / L, soya peptone FP408 3 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hemin 0.005 g / L, sodium poly-m-cresol sulfonate (SPS) 0.3 g / L, sodium citrate 0.2 g / L, sodium pyruvate 1 g / L, L-cysteine 0.6 g / L, polyether antifoam 0.15 g / L, L-tryptophan 0.01 g / L, sucrose 2 g / L, glutamine 0.5 g / L, sodium glutamate 0.5 g / L, D-pantothenic acid sodium salt 0.002 g / L, ammonium sulphate hydrochloride 0.001 g / L, VB6 0.01 g / L, sodium thio glycolate 0.5 g / L.

[0099] The culture medium for fastidious bacteria (including aerobic and anaerobic) was the same as in Example 1, and saponin was added to a concentration of 2.6 g / L.

[0100] Comparative Example 3

[0101] Aerobic culture medium formula:

[0102] Tryptic peptone FP318 0-20g / L, soy peptone FP408 0-20g / L, yeast extract powder FM888 5g / L, anhydrous glucose 2.3g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, hemin 0.005g / L, sodium polyanethole sulfonate (SPS) 0.3g / L, sodium citrate 0.2g / L, sodium pyruvate 1g / L, L-cysteine ​​0.6g / L, polyether antifoaming agent 0.15g / L, L-tryptophan 0.01g / L, sucrose 2g / L, glutamine 0.5g / L, sodium glutamate 0.5g / L, D-pantothenic acid sodium salt 0.002g / L, and ammonium sulfate hydrochloride 0.001g / L.

[0103] Anaerobic culture medium formula:

[0104] Tryptic peptone FP318 0-20g / L, soy peptone FP408 0-20g / L, yeast extract powder FM888 5g / L, anhydrous glucose 2.3g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 2.5g / L, hemin 0.005g / L, sodium polyanethole sulfonate (SPS) 0.3g / L, sodium citrate 0.2g / L, sodium pyruvate 1g / L, L-cysteine ​​0.6g / L, polyether antifoaming agent 0.15g / L, L-tryptophan 0.01g / L, sucrose 2g / L, glutamine 0.5g / L, sodium glutamate 0.5g / L, D-pantothenic acid sodium salt 0.002g / L, ammonium sulfate hydrochloride 0.001g / L, VB6 0.01g / L, sodium thioglycolate 0.5g / L.

[0105] Among them, the proportions of trypsin casein FP318 and soy peptone FP408 were set to (20 g / L: 0 g / L), (17 g / L: 3 g / L), (14 g / L: 6 g / L), (11 g / L: 9 g / L), (8 g / L: 12 g / L), (5 g / L: 15 g / L), (2 g / L: 18 g / L), and (0 g / L: 20 g / L) according to experimental requirements.

[0106] The culture medium for fastidious bacteria (including aerobic and anaerobic) was the same as in Example 1, and saponin was added to a concentration of 2.6 g / L.

[0107] Comparative Example 4

[0108] Imported product M aerobic blood culture medium instructions formula:

[0109] The culture medium consists of casein peptone (1.0% w / v), yeast extract (0.45% w / v), soy peptone (0.3% w / v), meat peptone (0.1% w / v), sodium polyanethol sulfonate (0.083% w / v), menadione (0.00005% w / v), hemin (0.0005% w / v), L-cysteine ​​(0.03% w / v), pyruvate (0.1% w / v), pyridoxine hydrochloride (0.001% w / v), niacin (0.0002% w / v), pantothenic acid (0.0002% w / v), thiamine hydrochloride (0.0001% w / v), as well as purified water, other complex amino acids, and carbohydrate substrates.

[0110] Imported product M anaerobic blood culture medium instructions formula:

[0111] The culture medium consists of peptone (1.48% w / v), yeast extract (0.5% w / v), sodium polyanethol sulfonate (0.083% w / v), menadione (0.00005% w / v), hemin (0.001% w / v), pyridoxine hydrochloride (0.0008% w / v), pyruvate (0.1% w / v), reducing agent (0.38% w / v), as well as purified water, other complex amino acids, and carbohydrate substrates.

[0112] Comparative Example 5

[0113] Imported product B aerobic blood culture medium instructions formula:

[0114] Soybean-casein decomposition broth (3% w / v), yeast extract (0.25% w / v), amino acids (0.05% w / v), sugars (0.2% w / v), sodium polyanethole xanthocyanate (0.05% w / v), vitamins (0.025% w / v), antioxidant / reducing agent (0.005% w / v).

[0115] Imported product B anaerobic blood culture medium instructions formula:

[0116] Soybean-casein digest broth (2.75% w / v), yeast extract (0.2% w / v), animal tissue digest (0.05% w / v), glucose (0.2% w / v), hemin (0.0005% w / v), vitamin K3 (0.00005% w / v), sodium citrate (0.02% w / v), thiols (0.1% w / v), sodium pyruvate (0.1% w / v), saponin (0.26% w / v), defoaming agent (0.01% w / v), sodium polyanethole xanthocyanate (0.035% w / v).

[0117] Efficacy verification:

[0118] 1. Aerobic culture of Escherichia coli and Staphylococcus aureus using the aerobic blood culture medium prepared in Example 1 and Comparative Example 3 was performed, and the culture effects were comprehensively evaluated;

[0119] 1.1 Preparation of working bacterial suspension

[0120] Inoculate standard strains of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) into Columbia blood agar medium, incubate aerobically at 35°C for 18 hours, and then dilute with 0.9% sterile sodium chloride solution to make 10 4 cfu / mL concentration of working bacterial suspension.

[0121] 1.2 Growth curve determination

[0122] Working suspensions of the aforementioned test bacteria were inoculated into 800 µl of aerobic blood culture media containing various ratios of tryptone FP318 to soy peptone FP408 (20:0, 17:3, 14:6, 11:9, 8:12, 5:15, 2:18, and 0:20, respectively). The inoculum size ranged from 100 to 1000 cfu / mL. The culture was aerobically incubated at 35°C and 500 rpm for 24 hours in a growth curve analyzer. The growth curves of Escherichia coli and Staphylococcus aureus in these eight aerobic blood cultures containing various ratios of tryptone FP318 to soy peptone FP408 were determined.

[0123] 1.3 Experimental Results

[0124] Growth curves of Escherichia coli and Staphylococcus aureus cultured aerobically in 8 groups of aerobic blood media containing different ratios of trypticase peptone FP318 and soy peptone FP408 and their OD values ​​at key time points 600 Value Figure 1-Figure 2The lag phase of E. coli in the eight groups of aerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 was similar, and the proportion of pancreas tryptone and soybean peptone had little effect on the growth of E. coli; the lag phase of S. aureus in the eight groups of aerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 was significantly different, and the lag phase was the shortest in the pancreas tryptone: soybean peptone (17:3), and the logarithmic phase growth was faster; based on the above experimental results, the aerobic blood culture medium with a pancreas tryptone: soybean peptone ratio of 17:3 was selected.

[0125] 2. The anaerobic blood culture medium prepared in Example 1 and Comparative Example 3 was used for anaerobic culture of E. coli and S. aureus, and the culture effect was comprehensively evaluated.

[0126] 2.1 Preparation of working bacterial suspension

[0127] The standard strains E. coli (ATCC 25922) and S. aureus (ATCC 25923) were inoculated into Columbia blood agar medium and incubated aerobically at 35°C for 18 hours, and then diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension with a concentration of 10 4 cfu / mL.

[0128] 2.2 Growth curve determination

[0129] The working bacterial suspension of the above test bacteria was inoculated into the anaerobic blood culture medium (800 μl) with different proportions of pancreas tryptone FP318 and soybean peptone FP408, and the proportions of pancreas tryptone FP318 and soybean peptone FP408 were 20:0, 17:3, 14:6, 11:9, 8:12, 5:15, 2:18, and 0:20, respectively, and the inoculation amount was 100-1000 cfu / mL. The growth curves of E. coli and S. aureus in the eight groups of anaerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 were determined by placing them in a growth curve determination instrument and incubating anaerobically at 35°C and 500 rpm for 24 hours.

[0130] 2.3 Experimental results

[0131] The growth curves of E. coli and S. aureus in the eight groups of anaerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 and their OD 600 values at key time points are shown in the following table: Figure 3-Figure 4The lag phase of E. coli in the anaerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 was similar, and the proportion of pancreas tryptone and soybean peptone had little effect on the lag phase of E. coli, but the biomass accumulation was significantly reduced in the anaerobic blood culture medium with a pancreas tryptone: soybean peptone ratio of 20:0; the lag phase of S. aureus in the anaerobic blood culture medium with different proportions of pancreas tryptone FP318 and soybean peptone FP408 was similar, but the growth rate in the logarithmic phase increased with the increase of the proportion of pancreas tryptone FP318; based on the above results, the anaerobic blood culture medium with a pancreas tryptone: soybean peptone ratio of 17:3 can meet the needs of E. coli and S. aureus for growth rate and biomass accumulation, and reduce the cost of nutrient solution.

[0132] 3. Example 1, Comparative Example 1 and Comparative Example 2, aerobic blood culture medium prepared by, comprehensive evaluation of the culture effect;

[0133] 3.1 Preparation of working bacterial suspension

[0134] The standard strain E. coli (ATCC 25922) was inoculated into Columbia blood agar medium and incubated aerobically at 35°C for 18 hours, then diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension with a concentration of 10 4 cfu / mL.

[0135] 3.2 Growth curve determination

[0136] The working bacterial suspension of the test bacteria was inoculated into three groups of aerobic blood culture medium with different main component combinations and aerobic blood culture medium with different main component combinations after aging experiment (the prepared medium was not inoculated, and was placed at 37°C for 24h, 48h, 72h, and then inoculated), and the three groups of different main component combinations were aerobic blood culture medium containing yeast extract powder FM888, pancreas tryptone FP318 + soybean peptone FP408, and pancreas tryptone FP318 + soybean peptone FP408 + yeast extract powder FM888, and the inoculation amount was 100-1000 cfu / mL. The growth curves of E. coli in the three groups of different main component combinations were determined by placing them in a growth curve determination instrument and incubating aerobically at 35°C and 500 rpm for 24 hours.

[0137] 3.3 Experimental results

[0138] The growth curves of E. coli in aerobic blood culture medium with different main component combinations and aerobic blood culture medium with different main component combinations after aging experiment and the OD 600 values at key time points are as follows: Figure 5-Figure 9As shown in Table 1, although the colony density of each group of bacteria can be obtained at the end of the culture, it can be seen from the curves that the E. coli cultured in Example 1 grows faster and reaches the inflection point in a shorter time.

[0139] According to Bliss independent model, the synergistic coefficient of yeast extract powder FM888, pancreas tryptone FP318 and soybean protein peptone FP408 (FM888 was used in Comparative Example 1, FP318+FP408 was used in Comparative Example 2 as the effect in the case of being used alone, and Example 1 as the effect in the case of being used in combination) was calculated, and the results showed that the yeast extract powder FM888, pancreas tryptone FP318 and soybean protein peptone FP408 used in combination can produce a significant synergistic effect in the logarithmic phase of E. coli in aerobic culture. And similar effects cannot be obtained when other components are used.

[0140] Regardless of whether it is not aged or aged for different times, the performance of the aerobic blood culture medium of Example 1 is better than that of Comparative Examples 1 and 2, and the performance of the aerobic blood culture medium does not decrease significantly after being aged for different times.

[0141] Table 1 OD values of E. coli at key time points during aerobic culture 600 and synergistic coefficient (CI)

[0142]

[0143] * The key time points are the times at each stage of the growth of E. coli, including: the starting time point (0.5h), the initial stage of the logarithmic phase (9h), the logarithmic phase (10h) and the stationary phase (12h).

[0144] 4. The anaerobic blood culture medium prepared by Example 1, Comparative Example 1 and Comparative Example 2 was used for anaerobic culture of E. coli, and the culture effect was comprehensively evaluated;

[0145] 4.1 Preparation of working bacteria suspension

[0146] The standard strain E. coli (ATCC 25922) was inoculated into Columbia blood agar medium and cultured aerobically at 35°C for 18 hours, and then diluted with 0.9% sterile sodium chloride solution to prepare a working bacteria suspension with a concentration of 10 4 cfu / mL.

[0147] 4.2 Growth curve determination

[0148] Working suspensions of the aforementioned test bacteria were inoculated into 800 µL of anaerobic blood culture media containing different combinations of principal components, as well as anaerobic blood culture media (800 µL) containing different principal component combinations after aging (prepared culture media were not inoculated but incubated at 37°C for 24, 48, and 72 hours before inoculation). The inoculum size ranged from 100 to 1000 cfu / mL. The cultures were incubated anaerobically at 35°C and 500 rpm for 24 hours in a growth curve analyzer. The growth curves of E. coli in the three principal component combinations were determined.

[0149] 4.3 Experimental Results

[0150] Growth curves of Escherichia coli cultured anaerobically in anaerobic blood medium containing different main component combinations and anaerobic blood medium containing different main component combinations after aging experiments, as well as their OD values ​​at key time points 600 Value Figure 10-14 and as shown in Table 2.

[0151] The synergistic coefficients of yeast extract FM888, trypticase peptone FP318, and soy peptone FP408 were calculated using the Bliss independence model (Comparative Example 1 used FM888, and Comparative Example 2 used FP318 + FP408, respectively, as the effects of using them alone, while Example 1 showed the effects of using them in combination). The results showed that the combination of yeast extract FM888, trypticase peptone FP318, and soy peptone FP408 produced significant synergistic effects during the logarithmic and stationary phases of anaerobic Escherichia coli growth. Previous attempts to use other components had failed to achieve similar results.

[0152] The performance of the anaerobic blood culture medium in Example 1 was better than that of the anaerobic blood culture medium in Comparative Examples 1 and 2, regardless of whether it was not aged or aged for different periods of time. Moreover, the performance of the anaerobic blood culture medium did not decrease significantly after aging for different periods of time.

[0153] Table 2 OD of Escherichia coli at key time points during anaerobic culture growth 600 Values ​​and synergy coefficients (CI)

[0154]

[0155] * The key time points are the times at various stages of E. coli growth, including: starting time point (0.5h), initial logarithmic phase (8h), logarithmic phase (9h) and stable phase (12h).

[0156] 5. The aerobic blood culture medium prepared in Example 1 and two imported products were used to aerobically culture 9 strains, and the culture effects were comprehensively evaluated;

[0157] 5.1 Preparation of working bacterial suspension

[0158] Inoculate standard strains of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, and Alcaligenes faecalis onto Columbia blood agar, inoculate Candida albicans onto YPD agar, and inoculate Neisseria meningitidis and Haemophilus influenzae onto Haemophilus chocolate agar selective medium. Incubate at 35°C for 18 hours and then dilute with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the desired concentration.

[0159] 5.2 Growth curve determination

[0160] Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (ATCC 19615), Pseudomonas aeruginosa (ATCC 27853), Alcaligenes faecalis (ATCC 8750), and Candida albicans (ATCC 90028): Inoculate working suspensions of these seven strains into home-produced aerobic blood medium and aerobic blood medium containing hemolysin, as well as into two competitive aerobic blood medium products (800 µl) at an inoculum size of 100-1000 cfu / mL.

[0161] Neisseria meningitidis (ATCC 13090) and Haemophilus influenzae (ATCC 19418) are fastidious bacteria. The growth factors (such as X-Factor and V-Factor) required for their growth are usually obtained by destroying red blood cells with hemolysin. The addition of sheep blood makes the absorbance OD 600 Exceeding the upper limit of the instrument's measurement would affect the determination of the microbial growth curve. Therefore, when culturing these two fastidious bacteria, NAD was directly added to replace the process of hemolysis to release growth factors. Neisseria meningitidis and Haemophilus influenzae were inoculated into aerobic blood culture medium containing NAD and two competing aerobic blood culture media (800µl). The target inoculum size for Neisseria meningitidis was 100 cfu / mL, and the target inoculum size for Haemophilus influenzae was 1000 cfu / mL.

[0162] The nutrient solution of the above-mentioned inoculated strains was placed in a growth curve measuring instrument and aerobically cultured at 35°C and 500 rpm for 24-48 hours. The growth curves of these nine bacteria in the aerobic blood culture medium of Example 1, the aerobic blood culture medium containing hemolysin Example 1, and the two competing nutrient solutions were measured.

[0163] 5.3 Experimental Results

[0164] Growth curves of nine standard strains cultured aerobically in four sets of aerobic blood culture media and their OD at key time points 600 Value Figure 15-Figure 23 As shown, in comprehensive comparison, the performance of the aerobic blood culture medium of Example 1 is not inferior to the aerobic blood culture medium of the two imported products, and the addition of hemolysin can effectively hemolyze and has little effect on bacterial growth.

[0165] The performance of the self-produced aerobic blood culture medium and the competing aerobic blood culture medium each have their own advantages in culturing different bacteria. Among them, Escherichia coli, Streptococcus pyogenes, Alcaligenes faecalis and Candida albicans grew the fastest in the aerobic blood culture medium of Example 1; the growth of Staphylococcus aureus and Streptococcus pneumoniae was slightly slower than that of the competing products; the growth lag phase of Pseudomonas aeruginosa in the aerobic blood culture medium of Example 1 was faster than that of the aerobic medium of Comparative Example 5 (imported product B), and slightly slower than that of Comparative Example 4 (imported product M), but the logarithmic growth phase was faster than that of the imported product M; the growth rate of the fastidious bacterium Neisseria meningitidis in the aerobic blood culture medium of Example 1 was slower than that of the imported product M, and the growth lag phase of Haemophilus influenzae in the aerobic blood culture medium of Example 1 was consistent with that of the imported product M. Both fastidious bacteria did not grow in the imported product B within 48 hours.

[0166] Without adding sheep blood, we tested whether hemolysin would affect the growth of the above seven bacteria except Haemophilus influenzae and Neisseria meningitidis. The experimental results showed that hemolysin only had a significant inhibitory effect on the growth of Candida albicans. Since the experiment was conducted without adding sheep blood, its growth process lacked the growth factors released by hemolysis compared with the culture containing sheep blood. Overall, the addition of hemolysin can effectively hemolyze and has little effect on bacterial growth.

[0167] 6. The anaerobic blood culture medium prepared in Example 1 and two competing products were used to simultaneously culture seven anaerobic bacteria, and the culture effects were comprehensively evaluated;

[0168] 6.1 Preparation of working bacterial suspension

[0169] Standard strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Clostridium histolyticum (ATCC 19401), Clostridium perfringens (ATCC 13124), Bacteroides fragilis (ATCC 25285) and Bacteroides vulgaris (ATCC 8482) were inoculated into Columbia blood agar medium, incubated anaerobically at 35°C for 18 hours, and then diluted with 0.9% sterile sodium chloride solution to prepare 10 4 cfu / mL concentration of working bacterial suspension.

[0170] 6.2 Growth curve determination

[0171] Working suspensions of the aforementioned test bacteria were inoculated into 800 µl of self-produced anaerobic blood medium, anaerobic blood medium containing hemolysin, and two competing anaerobic blood medium products at a concentration of 100-1000 cfu / mL. The suspensions were incubated anaerobically in a growth curve analyzer at 35°C and 500 rpm for 48 hours. The growth curves of the seven bacterial strains in the self-produced anaerobic blood medium, anaerobic blood medium containing hemolysin, and the two competing nutrient solutions were measured.

[0172] 6.3 Experimental Results

[0173] Growth curves of 7 standard strains cultured anaerobically in four sets of anaerobic blood culture media and their OD at key time points 600 Value Figures 24-30 As shown, the performance of the self-produced anaerobic blood culture medium and the competitor's anaerobic blood culture medium for the cultivation of the seven bacteria each has its own advantages. A comprehensive comparison shows that the performance of the anaerobic blood culture medium in Example 1 is consistent with or even faster than that of the competitor's anaerobic blood culture medium, and the hemolysin has no inhibitory effect on the growth of the seven bacteria, but has a significant growth-promoting effect on Clostridium perfringens.

[0174] The growth hysteresis period of Escherichia coli, Staphylococcus aureus, and Bacteroides vulgaris in the anaerobic blood culture medium of Example 1 was consistent with that of the two imported products, and the bioaccumulation of Escherichia coli during the stable growth period was higher than that of the imported products; the growth of Streptococcus pneumoniae in the anaerobic blood culture medium of Example 1 was slightly slower than that in the anaerobic culture medium of Comparative Example 4 (imported product M) and consistent with that of the anaerobic culture medium of Comparative Example 5 (imported product B); the growth of Clostridium perfringens in the anaerobic blood culture medium of Example 1 was faster than that of imported product M and slower than that of imported product B; the growth of Clostridium histolyticum in the anaerobic blood culture medium of Example 1 was faster than that of imported product M and consistent with that of imported product B; the growth of Bacteroides fragilis in the anaerobic blood culture medium of Example 1 was faster than that of imported product B, had the same hysteresis period as that of imported product M, and grew faster in the anaerobic blood culture medium of Example 1 during the logarithmic phase.

[0175] The effect of hemolysin on the growth of the seven bacteria mentioned above was tested without adding sheep blood. The experimental results showed that hemolysin had no inhibitory effect on the seven bacteria, but had a significant promoting effect on Clostridium perfringens.

[0176] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition comprising trypticase peptone, soy peptone and yeast extract powder; the mass ratio of the trypticase peptone, soy peptone and yeast extract powder is (10-20): (1-10): (1-10).

2. The composition according to claim 1, characterized in that The mass ratio of the tryptic peptone, soy peptone and yeast extract powder is (15-20): (1-5): (3-8).

3. The composition according to claim 2, characterized in that The mass ratio of the tryptic peptone, soy peptone and yeast extract powder is 17:3:

5.

4. The composition according to claim 3, characterized in that The tryptic peptone contains small peptides with a molecular weight of less than 1000 Da, and the mass fraction of the small peptides in the tryptic peptone is not less than 90%.

5. The composition according to claim 3, characterized in that The soy peptone has a total nitrogen content of ≥8% and an amino acid nitrogen content of ≥1.8%.

6. The composition according to claim 3, characterized in that The yeast extract powder has a total nitrogen content of 10% to 13%, an amino acid nitrogen content of ≥5%, a magnesium ion content of ≤500 ppm, a calcium ion content of ≤500 ppm, a manganese ion content of ≤10 ppm, and a copper ion content of ≤5 ppm.

7. A microbial culture medium comprising a carbon source, an inorganic salt, an amino acid, a vitamin, a cofactor and the composition according to any one of claims 1 to 6.

8. The microbial culture medium according to claim 7, characterized in that The carbon sources are glucose, sucrose and sodium pyruvate.

9. The microbial culture medium according to claim 7, characterized in that The inorganic salts are sodium chloride and dipotassium hydrogen phosphate.

10. The microbial culture medium according to claim 7, characterized in that The amino acids are cysteine, glutamine, sodium glutamate and tryptophan.

11. The microbial culture medium according to claim 7, characterized in that The vitamins are D-pantothenic acid sodium salt and ammonium sulfate hydrochloride.

12. The microbial culture medium according to claim 7, characterized in that The cofactors include hemin, sodium polyanethole sulfonate, sodium citrate and antifoaming agents.

13. The microbial culture medium according to claim 12, characterized in that The antifoaming agent is a polyether antifoaming agent.

14. The microbial culture medium according to claim 7, characterized in that The solution includes water, tryptone 10-20 g / L, soy peptone 1-10 g / L, yeast extract 1-10 g / L, glucose 1-5 g / L, sodium chloride 2-10 g / L, dipotassium hydrogen phosphate 1-4.5 g / L, hemin 0.002-0.015 g / L, sodium polyanethole sulfonate 0.1-1 g / L, sodium citrate 0.05-1 g / L, sodium pyruvate 0.1-2 g / L, cysteine ​​0.1-2 g / L, polyether antifoaming agent 0.05-1 g / L, tryptophan 0.005-1 g / L, sucrose 1-5 g / L, glutamine 0.1-2 g / L, sodium glutamate 0.1-2 g / L, sodium D-pantothenate 0.001-0.015 g / L, and ammonium sulfate hydrochloride 0.001-0.015 g / L.

15. The microbial culture medium according to claim 14, characterized in that It also includes hemolysin 1~5 g / L.

16. The microbial culture medium according to claim 15, characterized in that The hemolysin is saponin.

17. The microbial culture medium according to claim 15 or 16, characterized in that It also includes 0.005~0.5 g / L of vitamin B6 and 0.1~1 g / L of sodium thioglycolate.

18. Use of the composition according to any one of claims 1 to 6 or the microbial culture medium according to any one of claims 7 to 17 in preparing a reagent for screening influenza pathogens in blood.

19. The use according to claim 18, characterized in that The pathogens include at least one of Escherichia coli, Streptococcus pyogenes, Alcaligenes faecalis, Candida albicans, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, fastidious Neisseria meningitidis, Haemophilus influenzae, Clostridium histolyticum, Clostridium perfringens, Bacteroides fragilis or Bacteroides vulgaris.

20. A method for culturing pathogenic bacteria, comprising inoculating the pathogenic bacteria into the composition according to any one of claims 1 to 6 or the microbial culture medium according to any one of claims 7 to 17 and then culturing the pathogenic bacteria.

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