Compositions and their use in the preparation of microbial media

Abstract

The present application relates to blood culture bottle technical field, especially to composition and its application in the preparation of microbial culture medium, the present application combines yeast extract powder, soybean peptone and pancreatic casein peptone into nitrogen source, forming aerobic and anaerobic culture medium for clinical pathogenic microorganism culture, through comparison and verification, the aerobic and anaerobic culture medium added with the composition as described above is superior to prior art and other formula in culture speed and biomass accumulation in the growth experiment of 9 aerobic bacteria and 7 anaerobic bacteria, which shows that the composition as described above can effectively promote the performance of blood culture product, improve the ability of clinical blood stream infection detection, curb antibiotic abuse and save patients' lives.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of blood culture bottle, and particularly relates to a composition and application thereof in preparation of a microbial culture medium. BACKGROUND

[0002] Microorganisms are a kind of microorganism population with small individual and simple structure, which can be observed only by using a microscope, and cover various types such as bacteria, fungi, viruses, mycoplasma and chlamydia. Among these microorganisms, part of them as pathogenic microorganisms will invade the human body, destroy the normal physiological function of the body, and cause various diseases. Taking blood stream fever (i.e. blood stream infection) as an example, it is a systemic infectious disease caused by pathogenic microorganisms and their toxins invading the human blood circulation system. Common pathogenic agents include Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus and other bacteria, and Candida and other fungi. Once blood stream fever occurs, the patient will have symptoms such as high fever, chills, fatigue, and in severe cases can lead to septic shock, multiple organ dysfunction syndrome, and even death, which poses a serious threat to human health.

[0003] Among the existing screening methods of pathogenic microorganisms, the culture method has the advantage of being able to directly obtain pathogenic microorganisms, and still occupies an important position. Taking the diagnosis of blood stream fever as an example, the blood culture bottle is a typical application of the culture method. The operation process is to inject the blood drawn from the patient into the blood culture bottle preloaded with culture medium, and then place it in the blood culture instrument for culture. If the patient has blood stream infection, the pathogenic bacteria in the blood will grow and reproduce in the culture medium, and the carbon dioxide metabolized will change the pH value at the bottom of the blood culture bottle, prompting the pre-embedded pH probe to change color or fluorescence. After the blood culture instrument detects the signal, it automatically alarms, and then determines the type and characteristics of the pathogenic bacteria through subsequent gram staining and other operations.

[0004] However, the current culture method represented by the blood culture bottle has significant drawbacks. The culture time of the blood culture bottle is too long, and the detection speed lags far behind the development speed of the patient's condition. In the process of waiting for the detection report, the clinician often has to blindly use antibiotics to save the patient's life, which not only aggravates the problem of bacterial drug resistance, but also increases the patient's treatment cost and risk of adverse reactions, and some patients even die because they cannot receive precise treatment in time. How to solve the problems of slow culture speed and high cost has become a difficult problem to be solved in the field of screening of pathogenic microorganisms. SUMMARY

[0005] Therefore, the present application aims to solve the technical problem of providing a composition and application thereof in preparation of a microbial culture medium.

[0006] The present application provides application of at least one of pancreas casein peptone, soybean peptone and yeast extract powder in preparation of a microbial culture medium.

[0007] Studies have shown that pancreas casein peptone, soybean peptone and yeast extract powder each has advantages as a nitrogen source of culture medium, and the present application finds that the three can produce synergies as a nitrogen source under a specific ratio, so that the microorganism grows more quickly. Meanwhile, the prices of the three raw materials are low, which reduces the cost of the culture medium.

[0008] The present application experiment shows that the culture medium prepared by using the composition composed of pancreas casein peptone, soybean peptone and yeast extract powder under a specific ratio as a nitrogen source can more quickly and accurately culture pathogenic microorganisms, so that the identification of pathogens is more time-effective. In the early experiment, the culture medium obtained by using single peptone or yeast extract powder for compounding has a slower growth rate of pathogenic bacteria when culturing common pathogenic bacteria.

[0009] The composition provided by the present application is composed of pancreas casein peptone, soybean peptone and yeast extract powder. As preferred, the mass ratio of the pancreas casein peptone, soybean peptone and yeast extract powder is (10-20):(1-10):(1-10). Preferably, the mass ratio of the pancreas casein peptone, soybean peptone and yeast extract powder is (15-20):(1-5):(3-8). More preferably, the mass ratio of the pancreas casein peptone, soybean peptone and yeast extract powder is 17:3:5.

[0010] The pancreas casein peptone is a small peptide with a molecular weight of 1000 Da or less in pancreas casein peptone, and the mass fraction is not less than 90%.

[0011] The soybean peptone is a soybean peptone with a total nitrogen content of ≥8% and an amino acid nitrogen content of ≥1.8%.

[0012] The yeast extract powder is a yeast extract powder with a total nitrogen content of 10%-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.

[0013] In the composition of the present application, each component can exist in the form of powder or solution. Each component can exist in a mixed form or independently, and the present application does not make any limitation in this regard.

[0014] Further, the present application provides a microbial culture medium, which comprises a carbon source, inorganic salt, amino acid, auxiliary factor and the composition as described above.

[0015] To match the composition of the present application, further rounds of screening of the carbon source, inorganic salt, amino acid and cofactor required by the culture medium are carried out. The screening process is verified by using 16 pathogenic bacteria. The screening range mainly involves 43 components, including: proteose peptone (tryptone, soybean peptone), animal tissue extract, yeast extract (yeast extract powder, yeast extract), amino acid (L-arginine, L-glutamic acid, L-glutamine, L-isoleucine, L-leucine, L-valine, L-threonine, L-lysine, L-tryptophan), L-cysteine, glucose, sucrose, vitamin (VK3, VB1, VB3, VB4, VB5, VB6, VB7, VB8, VB12), inorganic salt (sodium chloride, dipotassium hydrogen phosphate), sodium pyruvate, sodium citrate, sodium polyanethole sulfonate (SPS), gelatin, nicotinamide adenine dinucleotide (NAD), hemin, saponin, sodium thio glycolate, mannitol, antifoam agent, disodium succinate, dimethyl mercaptopropionic acid, betaine, S-methyl-L-methionine.

[0016] After the above screening, in order to reduce the cost and improve the growth rate of microorganisms, it is determined that the carbon source in the culture medium is glucose and sodium pyruvate; the inorganic salt is sodium chloride and dipotassium hydrogen phosphate; the amino acid is cysteine and tryptophan; and the cofactor includes hemin, sodium polyanethole sulfonate, sodium citrate and antifoam agent. Among them, the antifoam agent is a polyether antifoam agent. Experiments show that the culture efficiency of pathogenic bacteria can be higher by using the culture medium of the present application.

[0017] As preferred, the components of the culture medium include water and tryptone, soybean peptone FP408, yeast extract powder, glucose, sodium chloride, dipotassium hydrogen phosphate, hemin, sodium polyanethole sulfonate, sodium citrate, sodium pyruvate, cysteine, antifoam agent, tryptophan, sucrose, glutamine, sodium glutamate, D-pantothenate sodium salt and ammonium sulfate hydrochloride.

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

[0019] Preferably, the culture medium includes water and the following working concentration of components:

[0020] Pancreatic tryptone FP 10-20 g / L, soybean 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, hematin 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 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, D-pantothenic acid sodium salt 0.001-0.015 g / L, and ammonium thiocyanate hydrochloride 0.001-0.015 g / L.

[0021] More preferably, the culture medium comprises water and pancreatic tryptone 17 g / L, soybean peptone 3 g / L, yeast extract powder 5 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2.5 g / L, hematin 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, antifoam 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 thiocyanate hydrochloride 0.001 g / L.

[0022] Further, when culturing fastidious bacteria, the culture medium further comprises hemolysin 1-5 g / L, and preferably, the hemolysin is saponin, and the concentration of the saponin is 2.6 g / L.

[0023] Still further, when culturing anaerobic bacteria, the culture medium further comprises vitamin B6 0.005-0.5 g / L and sodium thioglycolate 0.1-1 g / L. 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 application, the concentrations of the components in the culture medium as described above are working concentrations. During preparation or storage and transportation, the components can exist in the form of powder or solution. During preparation, storage or transportation, the concentrations of the components can be 2-100 times of the working concentrations. For example, the concentrations of the components can be independently 2 times, 5 times, 10 times, 20 times, 50 times or 100 times of the working concentrations.

[0025] Furthermore, the present invention also provides the use of the composition or the microbial culture medium described above in the preparation of reagents for screening influenza pathogens.

[0026] In this invention, the microorganisms are pathogenic microorganisms of influenza, including pathogenic microorganisms cultured under aerobic conditions and pathogenic microorganisms cultured under anaerobic conditions.

[0027] As a feasibility example, the pathogenic microorganisms cultured under the required nutrient 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 example, the pathogenic microorganisms cultured under anaerobic conditions are Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Clostridium histolytica, Clostridium perfringens, Bacteroides fragilis, and Bacteroides vulgaris.

[0029] The screening reagent for influenza pathogens described in this invention includes the culture medium and pH probe as previously described.

[0030] Furthermore, the present invention also provides a method for culturing pathogens, which includes 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. This method includes inoculating a blood sample into the culture medium as described above, and determining the presence of pathogens based on pH indicators.

[0032] This invention combines yeast extract powder, soybean peptone, and tryptone as nitrogen sources to form aerobic and anaerobic culture media for culturing clinical pathogenic microorganisms. Comparative verification showed that the aerobic and anaerobic culture media containing the aforementioned composition exhibited superior overall growth rates and biomass accumulation compared to existing technologies and other formulations excluded during screening in growth experiments of 9 aerobic and 7 anaerobic bacteria. This demonstrates that the aforementioned composition can effectively enhance the performance of blood culture products, improve the ability to detect bloodstream infections in clinical settings, curb antibiotic overuse, and save patient lives. Attached Figure Description

[0033] Figure 1 (A) Growth curves of Escherichia coli in aerobic blood culture media with eight different ratios of tryptone FP318 and soybean peptone FP408, and (B) Time-OD at each key time point. 600 Bar chart;

[0034] Figure 2(A) growth curves and (B) time-OD at key time points for S. aureus in aerobic blood culture media with 8 different ratios of pancreatic tryptone FP318 and soy peptone FP408 600 bar graphs;

[0035] Figure 3 (A) growth curves and (B) time-OD at key time points for E. coli in anaerobic blood culture media with 8 different ratios of pancreatic tryptone FP318 and soy peptone FP408 600 bar graphs;

[0036] Figure 4 (A) growth curves and (B) time-OD at key time points for S. aureus in anaerobic blood culture media with 8 different ratios of pancreatic tryptone FP318 and soy peptone FP408 600 bar graphs;

[0037] Figure 5 (A) growth curves and (B) time-OD at key time points for E. coli in aerobic blood culture media of Comparative Example 1 aerobic, Comparative Example 2 aerobic and Example 1 aerobic (not aged) 600 bar graphs;

[0038] Figure 6 (A) growth curves and (B) time-OD at key time points for E. coli in aerobic blood culture media of Comparative Example 1 aerobic, Comparative Example 2 aerobic and Example 1 aerobic (aged 24h) 600 bar graphs;

[0039] Figure 7 (A) growth curves and (B) time-OD at key time points for E. coli in aerobic blood culture media of Comparative Example 1 aerobic, Comparative Example 2 aerobic and Example 1 aerobic (aged 48h) 600 bar graphs;

[0040] Figure 8 (A) growth curves and (B) time-OD at key time points for E. coli in aerobic blood culture media of Comparative Example 1 aerobic, Comparative Example 2 aerobic and Example 1 aerobic (aged 72h) 600 bar graphs;

[0041] Figure 9 Growth curves for E. coli in aerobic blood culture media of (A) Comparative Example 1 aerobic, (B) Comparative Example 2 aerobic and (C) Example 1 aerobic aged for different times

[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 and (B) time-OD at key time points for Pseudomonas aeruginosa grown aerobically in four sets of aerobic blood culture media 600 Bar graphs;

[0052] Figure 20 (A) Growth curves and (B) time-OD at key time points for Candida albicans grown aerobically in four sets of aerobic blood culture media 600 Bar graphs;

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

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

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

[0056] Figure 24 (A) Growth curves and (B) time-OD at key time points for Escherichia coli grown anaerobically in four sets of anaerobic blood culture media 600 Bar graphs;

[0057] Figure 25 (A) Growth curves and (B) time-OD at key time points for Staphylococcus aureus grown anaerobically in four sets of anaerobic blood culture media 600 Bar graphs;

[0058] Figure 26 (A) Growth curves and (B) time-OD at key time points for Streptococcus pneumoniae grown anaerobically in four sets of anaerobic blood culture media 600 Bar graphs;

[0059] Figure 27 (A) Growth curves and (B) time-OD at key time points for Clostridium perfringens grown anaerobically in four sets of anaerobic blood culture media 600 Bar graphs (Clostridium perfringens growth curves appear jagged due to flocculation in three replicates of Comparative Example 5 anaerobic blood culture media Figure 27(As shown in (A)), making its SD larger);

[0060] Figure 28 (A) Growth curves of Clostridium histolytica in four groups of anaerobic blood culture media and (B) Time-OD at each key time point. 600 The bar chart shows that Clostridium histolyticum exhibited aggregation in three parallel experiments on anaerobic blood medium (Comparative Example 5), resulting in a sawtooth-shaped growth curve (e.g., ...). Figure 28 (As shown in (A)), making its SD larger);

[0061] Figure 29 (A) Growth curves of Bacteroides fragilis in four groups of anaerobic blood culture media and (B) Time-OD at each key time point. 600 Bar chart;

[0062] Figure 30 (A) Growth curves of *Bacteroides spp.* in four groups of anaerobic blood culture media and (B) Time-OD at each key time point. 600 Bar chart. Detailed Implementation

[0063] This invention provides compositions and their application in the preparation of microbial culture media. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0064] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0065] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

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

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

[0068] The numerical ranges and parameters involving in the present application have been presented as precisely as possible in the specific examples. However, any numerical value inherently involves a standard deviation due to the individual test method. Therefore, unless otherwise explicitly stated, all numerical ranges or specific data used in the present disclosure can have a certain reasonable deviation within a certain range, for example: ±10%, ±5%, ±1% or ±0.5%.

[0069] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The pancreas casein peptone involved in the examples is pancreas casein peptone FP318, the soybean peptone is soybean peptone FP408, and the yeast extract powder is yeast extract powder FM888. All the above raw materials come from Angel Yeast. Compared with raw materials from other sources, the combination of pancreas casein peptone FP318, soybean peptone FP408 and yeast extract powder FM888 can make the microorganism grow more rapidly.

[0070] wherein,

[0071] FM888 is a powder product rich in free amino acids, polypeptides, B vitamins, nucleotides and trace elements, which is prepared from purified cultured bread yeast protein by autolysis, centrifugal separation, concentration and spray drying. The preparation method is referred to Chinese patent application CN 115478018 A. The total nitrogen content of the product prepared is 10%-13%, the amino acid nitrogen content is ≥5%, the magnesium ion content is ≤500ppm, the calcium ion content is ≤500ppm, the manganese ion content is ≤10ppm, the copper ion content is ≤5ppm, and the endotoxin content is <100 EU / g. FM888 has high clarity and excellent microbial culture ability, can significantly promote the growth of microorganisms, and does not produce phosphate precipitation, which can improve the stability and culture effect of blood culture medium.

[0072] FP408 is prepared by ultrafiltration process, wherein the total nitrogen content is ≥8%, the amino acid nitrogen content is ≥1.8%, and the endotoxin content is ≤100 EU / g. It does not produce phosphate precipitation, avoiding the adverse effects of other soybean peptones on microbial growth. Its nutrient components are more abundant and stable, which can provide a more suitable growth environment for microorganisms, significantly improving the effect of blood culture.

[0073] FP318 is a protein hydrolysate obtained by hydrolyzing animal casein with trypsin, which is rich in peptides and amino acids, and more than 90% of small peptides below 1000 Da. It is sterilized and does not produce phosphate precipitation.

[0074] It should be understood that the size of the sequence number of the above processes does not mean the order of execution in various embodiments of the present application, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to 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 embodiments, the calculation of the CI value is based on the Bliss independent model, and the synergistic coefficient is calculated for the combination of Example 1, and single drugs A and B for Comparative Examples 1 and 2, respectively, and the formula is:

[0076]

[0077] CI>1 is synergistic, CI=1 is additive, and CI<1 is antagonistic.

[0078] The present application is further described below in conjunction with the embodiments:

[0079] Example 1

[0080] Aerobic medium formula:

[0081] Trypticase peptone FP318 17 g / L, soybean peptone FP408 3 g / L, yeast extract powder FM888 5 g / L, anhydrous glucose 2.3 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, hematin 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 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, and ammonium sulfate hydrochloride 0.001 g / L.

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

[0083] Trypticase peptone FP318 17 g / L, soybean 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, potassium phosphate dibasic 2.5 g / L, hematin 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 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, and ammonium sulfate hydrochloride 0.001 g / L.

[0084] Anaerobic medium formula:

[0085] Trypticase 17 g / L, soy peptone 3 g / L, yeast extract 5 g / L, glucose 2.3 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, hematin 0.005 g / L, sodium poly-m-cresulfate (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 sulfate hydrochloride 0.001 g / L, VB6 0.01 g / L, sodium thio glycolate 0.5 g / L.

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

[0087] Trypticase 17 g / L, soy peptone 3 g / L, yeast extract 5 g / L, saponin 2.6 g / L, glucose 2.3 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, hematin 0.005 g / L, sodium poly-m-cresulfate (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 sulfate hydrochloride 0.001 g / L, VB6 0.01 g / L, sodium thio glycolate 0.5 g / L.

[0088] Comparative Example 1

[0089] Aerobic medium formula:

[0090] Yeast extract 5 g / L, glucose 2.3 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 2.5 g / L, hematin 0.005 g / L, sodium poly-m-cresulfate (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 sulfate hydrochloride 0.001 g / 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 thioglycolate 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 thioglycolate 0.5 g / L.

[0099] The medium for the fastidious bacteria (both aerobic and anaerobic) was as in Example 1 with the addition of saponin to a concentration of 2.6 g / L.

[0100] Comparative Example 3

[0101] Aerobic medium formulation:

[0102] Tryptone FP318 0-20 g / L, soya peptone FP408 0-20 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, hematin 0.005 g / L, sodium poly-anisole 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.

[0103] Anaerobic medium formulation:

[0104] Tryptone FP318 0-20 g / L, soya peptone FP408 0-20 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, hematin 0.005 g / L, sodium poly-anisole 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.

[0105] The tryptone FP318 and soya peptone FP408 were set in the following proportions according to the experiment: (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), (0 g / L: 20 g / L).

[0106] The medium for the fastidious bacteria (both aerobic and anaerobic) was as in Example 1 with the addition of saponin to a concentration of 2.6 g / L.

[0107] Comparative Example 4

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

[0109] The medium is composed of casein peptone (1.0% w / v), yeast extract (0.45% w / v), soybean peptone (0.3% w / v), meat peptone (0.1% w / v), sodium poly-m-cresol sulfonate (0.083% w / v), menadione (0.00005% w / v), hemin chloride (0.0005% w / v), L-cysteine (0.03% w / v), pyruvic acid (0.1% w / v), pyridoxine hydrochloride (0.001% w / v), nicotinic acid (0.0002% w / v), pantothenic acid (0.0002% w / v), thiamine hydrochloride (0.0001% w / v), and purified water, other complex amino acids and carbohydrate substrates.

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

[0111] The medium is composed of peptone (1.48% w / v), yeast extract (0.5% w / v), sodium poly-m-cresol sulfonate (0.083% w / v), menadione (0.00005% w / v), hemin chloride (0.001% w / v), pyridoxine hydrochloride (0.0008% w / v), pyruvic acid (0.1% w / v), reducing agent (0.38% w / v), and purified water, other complex amino acids and carbohydrate substrates.

[0112] Comparative Example 5

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

[0114] Soybean-casein digest broth (3% w / v), yeast extract (0.25% w / v), amino acids (0.05% w / v), sugar (0.2% w / v), sodium poly-m-cresol sulfonate (0.05% w / v), vitamins (0.025% w / v), antioxidant / reducing agent (0.005% w / v).

[0115] Imported product B anaerobic blood culture medium instruction 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), Hematin (0.0005% w / v), Vitamin K3 (0.00005% w / v), Sodium citrate (0.02% w / v), Mercaptoalcohol (0.1% w / v), Sodium pyruvate (0.1% w / v), Saponin (0.26% w / v), Antifoam agent (0.01% w / v), Polyanethol sulfonate (0.035% w / v).

[0117] Efficacy verification:

[0118] 1. Aerobic blood culture medium prepared by Example 1 and Comparative Example 3 was used for aerobic culture of Escherichia coli and Staphylococcus aureus, and the culture effect was comprehensively evaluated.

[0119] 1.1 Preparation of working bacterial suspension

[0120] Standard strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were 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.

[0121] 1.2 Growth curve determination

[0122] The working bacterial suspension of the above test bacteria was inoculated into aerobic blood culture medium (800 μl) with different proportions of tryptone peptone FP318 and soybean peptone FP408, and the proportions of tryptone peptone 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. Incubate aerobically at 35°C and 500 rpm for 24 hours, and determine the growth curves of Escherichia coli and Staphylococcus aureus in eight groups of aerobic blood culture medium with different proportions of tryptone peptone FP318 and soybean peptone FP408.

[0123] 1.3 Experimental results

[0124] The growth curves of Escherichia coli and Staphylococcus aureus in eight groups of aerobic blood culture medium with different proportions of tryptone peptone FP318 and soybean peptone FP408 and the OD 600 values at key time points are as follows: Figures 1-2The 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: Figures 3-4The 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: Figures 5-9As 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] The working bacterial suspension of the above-mentioned test bacteria was inoculated into anaerobic blood culture medium containing different combinations of main components and anaerobic blood culture medium containing different combinations of main components subjected to aging experiment (the prepared culture medium was not inoculated, and was placed at 37°C for 24 h, 48 h, 72 h, and then inoculated), three groups of different combinations of main components were anaerobic blood culture medium containing yeast extract powder FM888, pancreatic casein peptone FP318+soybean peptone FP408, and pancreatic casein peptone FP318+soybean peptone FP408+yeast extract powder FM888, and the inoculation amount was 100-1000 cfu / mL. The growth curves of Escherichia coli in the three groups of different combinations of main components were determined by placing them in a growth curve determination instrument and anaerobically culturing at 35°C and 500 rpm for 24 hours.

[0149] 4.3 Experimental results

[0150] The growth curves of Escherichia coli in anaerobic blood culture medium containing different combinations of main components and anaerobic blood culture medium containing different combinations of main components subjected to aging experiment and the OD values at key time points 600 are shown in Table 1 and Table 2. Figures 10-14

[0151] According to the Bliss independent model, the synergistic coefficients of yeast extract powder FM888 and pancreatic casein peptone FP318 and soybean peptone FP408 were calculated (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 was used as the effect of using components in combination), and the results showed that the combination of yeast extract powder FM888 and pancreatic casein peptone FP318 and soybean peptone FP408 could produce significant synergistic effect in the logarithmic phase and stationary phase of Escherichia coli during anaerobic culture. And similar effects could not be obtained when other components were tried before.

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

[0153] Table 2 OD values of Escherichia coli at key time points during anaerobic culture 600 and synergistic coefficients (CI)

[0154]

[0155] * The key time points are the times at each stage of the growth of Escherichia coli, including: the initial time point (0.5 h), the initial stage of the logarithmic phase (8 h), the logarithmic phase (9 h), and the stationary phase (12 h). ​

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

[0157] 5.1 Preparation of working bacterial suspension

[0158] Standard strains of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa and Alcaligenes faecalis were inoculated into Columbia blood agar medium, Candida albicans was inoculated into YPD agar medium, and Neisseria meningitidis and Haemophilus influenzae were inoculated into Haemophilus chocolate agar selective medium. After incubation at 35°C for 18 hours, the working bacterial suspension was prepared by diluting with 0.9% sterile sodium chloride solution to the required 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): working bacterial suspensions of the above 7 strains were inoculated into self-produced aerobic blood culture medium and hemolysin-containing aerobic blood culture medium, and simultaneously inoculated into two competitive aerobic blood culture medium products (800 μl), with an inoculation amount of 100-1000 cfu / mL.

[0161] Neisseria meningitidis (ATCC 13090) and Haemophilus influenzae (ATCC 19418) are fastidious bacteria, and the growth factors required for their growth, such as X-Factor and V-Factor, are usually obtained by releasing hemolysin to destroy red blood cells. The addition of sheep blood makes the optical density OD 600 exceeds the upper limit of the instrument, affecting the determination of the growth curve of the microorganism, so when culturing these two fastidious bacteria, NAD is directly added instead of the process of releasing growth factors by hemolysis. Neisseria meningitidis and Haemophilus influenzae were inoculated into NAD-containing aerobic blood culture medium and two competitive aerobic blood culture media (800 μl), with a target inoculation amount of 100 cfu / mL for Neisseria meningitidis and 1000 cfu / mL for Haemophilus influenzae.

[0162] The nutrient solution of the inoculated strains was placed in a growth curve determination instrument and incubated aerobically at 35°C and 500 rpm for 24-48 hours. The growth curves of the 9 strains in the aerobic blood culture medium of Example 1, the hemolysin-containing aerobic blood culture medium of Example 1 and the two competitive nutrient solutions were determined.

[0163] 5.3 Experimental results

[0164] Growth curves of 9 standard strains in four groups of aerobic blood culture media and OD at each key time point 600 Values as shown in Table 1, in general, the performance of the aerobic blood culture medium of Example 1 is not inferior to that of two imported products, and the addition of hemolysin can effectively hemolyze and has little effect on the growth of bacteria. Figures 15-23

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

[0166] The effect of hemolysin on the growth of the above-mentioned 7 bacteria except Haemophilus influenzae and Neisseria meningitidis was tested without the addition of sheep blood. The experimental results showed that hemolysin only had a significant inhibitory effect on the growth of Candida albicans. Since the experiment was carried out without the addition of sheep blood, the growth factor released by hemolysis was lacking compared with the culture containing sheep blood. In general, the addition of hemolysin can effectively hemolyze and has little effect on the growth of bacteria.

[0167] 6. The anaerobic blood culture medium prepared in Example 1 was used to culture 7 anaerobic bacteria synchronously with two competitive products, and the culture effect was comprehensively evaluated.

[0168] 6.1 Preparation of working bacterial suspension

[0169] The standard strains 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 vulgatus (ATCC 8482) were inoculated into Columbia blood agar medium and incubated anaerobically 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.

[0170] 6.2 Growth curve determination ​

[0171] Working bacterial suspensions of the above-mentioned test bacteria were inoculated into self-produced anaerobic blood medium, anaerobic blood medium containing hemolysin, and two competing anaerobic blood medium products (800 µl), with an inoculation volume of 100-1000 CFU / mL. The cultures were placed in a growth curve analyzer and anaerobically cultured at 35℃ and 500 rpm for 48 hours, respectively. The growth curves of these seven bacteria in self-produced anaerobic blood medium, anaerobic blood medium containing hemolysin, and the two competing nutrient solutions were then measured.

[0172] 6.3 Experimental Results

[0173] Growth curves of seven standard strains cultured anaerobically in four groups of anaerobic blood media and their OD values ​​at key time points. 600 Values ​​such as Figures 24-30 As shown, the performance of the self-produced anaerobic blood culture medium and the competing anaerobic blood culture medium each have their own advantages in the cultivation of 7 kinds of bacteria. In a comprehensive comparison, the performance of the anaerobic blood culture medium in Example 1 is consistent with or even faster than that of the competing anaerobic blood culture medium. Moreover, the hemolysin has no inhibitory effect on the growth of the seven kinds of bacteria and has a significant growth-promoting effect on Clostridium perfringens.

[0174] The lag phases of *Escherichia coli*, *Staphylococcus aureus*, and *Bacteroides* in the anaerobic blood medium of Example 1 were consistent with those of the two imported products, and the bioaccumulation of *Escherichia coli* during the stationary phase was higher than that of the imported products. *Streptococcus pneumoniae* grew slightly slower in the anaerobic blood medium of Example 1 compared to Comparative Example 4 (imported product M), but consistent with Comparative Example 5 (imported product B). *Clostridium perfringens* grew faster in the anaerobic blood medium of Example 1 than imported product M, but slower than imported product B. *Clostridium histolyticum* grew faster in the anaerobic blood medium of Example 1 than imported product M, and consistent with imported product B. *Bacteroides fragilis* grew faster in the anaerobic blood medium of Example 1 than imported product B, with a lag phase consistent with imported product M, and grew even faster in the anaerobic blood medium of Example 1 during the logarithmic phase.

[0175] The experiment tested whether hemolysin would affect the growth of the seven bacteria without the addition of sheep blood. The results showed that hemolysin had no inhibitory effect on the seven bacteria and had a significant promoting effect on Clostridium perfringens.

[0176] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microbial culture medium comprising a carbon source, inorganic salts, amino acids, vitamins, cofactors, and a nitrogen source; wherein the nitrogen source is composed of tryptone FP318, soybean peptone FP408, and yeast extract FM888; wherein the mass ratio of tryptone, soybean peptone, and yeast extract in the nitrogen source is (10~20):(1~10):(1~10).

2. The microbial culture medium according to claim 1, characterized in that, The mass ratio of tryptone FP318, soybean peptone FP408 and yeast extract FM888 in the nitrogen source is (15~20):(1~5):(3~8).

3. The microbial culture medium according to claim 2, characterized in that, The nitrogen source comprises tryptone FP318, soybean peptone FP408, and yeast extract FM888 in a mass ratio of 17:3:

5.

4. The microbial culture medium according to any one of claims 1 to 3, characterized in that, The mass fraction of small peptides with a molecular weight of less than 1000 Da in the tryptone FP318 is not less than 90%.

5. The microbial culture medium according to any one of claims 1 to 3, characterized in that, The soybean peptone FP408 contains a total nitrogen content of ≥8% and an amino acid nitrogen content of ≥1.8%.

6. The microbial culture medium according to any one of claims 1 to 3, characterized in that, The yeast extract powder FM888 has a total nitrogen content of 10%~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. The microbial culture medium according to claim 1, characterized in that, The carbon source is glucose, sucrose, and sodium pyruvate.

8. The microbial culture medium according to claim 1, characterized in that, The inorganic salts are sodium chloride and dipotassium hydrogen phosphate.

9. The microbial culture medium according to claim 1, characterized in that, The amino acids are cysteine, glutamine, monosodium glutamate, and tryptophan.

10. The microbial culture medium according to claim 1, characterized in that, The vitamins are sodium D-pantothenate and ammonium sulfate hydrochloride.

11. The microbial culture medium according to claim 1, characterized in that, The cofactors include hemolysin, sodium polyanisole sulfonate, sodium citrate, and an antifoaming agent.

12. The microbial culture medium according to claim 11, characterized in that, The antifoaming agent is a polyether-based antifoaming agent.

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

14. The microbial culture medium according to claim 13, characterized in that, It also includes hemolysin 1~5 g / L.

15. The microbial culture medium according to claim 14, characterized in that, The hemolysin is a saponin.

16. The microbial culture medium according to claim 14 or 15, characterized in that, It also includes 0.005~0.5 g / L of vitamin B6 and 0.1~1 g / L of sodium thioglycolate.

17. The use of the microbial culture medium according to any one of claims 1 to 16 in the preparation of a reagent for screening influenza pathogens; wherein 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 histolytica, Clostridium perfringens, Bacteroides fragilis, or Bacteroides commonis.

18. A method for culturing pathogenic bacteria, comprising inoculating the pathogenic bacteria into a microbial culture medium according to any one of claims 1 to 16 and then culturing it; wherein the pathogenic bacteria 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 histolytica, Clostridium perfringens, Bacteroides fragilis, or Bacteroides vulgaris.

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