Detection Medium for Burkholderia cepacia Complex in Pharmaceutical Water Systems

Abstract

The present invention belongs to the field of selective culture of microorganisms and relates to a detection medium for Burkholderia cepacia complex in a pharmaceutical water system. A detection medium for Burkholderia cepacia complex in a pharmaceutical water system, the formula (g / L) of the detection medium is: peptone 0.5 - 2.0 g, sodium pyruvate 1 - 4 g, carbon source 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acid 1 - 4 g, magnesium sulfate 0.05 - 0.2 g, and the solvent is water. The inventor has screened a detection medium for Burkholderia cepacia complex in a pharmaceutical water system with special components. Through the culture experiment of Burkholderia cepacia complex, it is found that whether it is a standard strain or a wild strain isolated from a pharmaceutical water system, within the same culture time, the OD 600 value results of 1 / 10 TSB and R2A liquid media do not exceed 0.5, while the bacterial concentration in the detection (BCB) medium for Burkholderia cepacia complex in a pharmaceutical water system is significantly higher than that in 1 / 10 TSB and R2A liquid media.

Description

Technical Field

[0001] The present invention belongs to the field of selective culture of microorganisms, and relates to a detection medium for Burkholderia cepacia complex in a pharmaceutical water system. Background Art

[0002] The Burkholderia cepacia complex (Bcc) belongs to the genus Burkholderia, and there are currently 25 strains in the group. It can survive in low-nutrient environments and harsh conditions, posing a serious pollution risk to drugs. There have been multiple incidents at home and abroad where drugs were recalled due to Bcc contamination. Nowadays, the pharmaceutical industry's attention to Bcc is increasing day by day. Effective identification and monitoring of Bcc in products or their production processes are the key to controlling the Bcc pollution risk. The water production system is the largest source of Bcc pollution, and a large number of Bcc can be detected in the pharmaceutical water system. Due to the prominent oligotrophic characteristics of Bcc from the water production system, it has been found that some Bcc strains cannot be detected in the culture media of the pharmacopoeia system.

[0003] Currently, the Bcc detection methods in drug products of domestic and foreign pharmacopoeias rely on traditional enrichment culture, selection, and isolation culture to obtain pure cultures of suspected target microorganisms, and further conduct appropriate identification to judge whether the sample is contaminated with Bcc. The microbial detection method in the United States Pharmacopeia (USP) includes using TSB (tryptic soy broth) for microbial enrichment culture, but this method is not suitable for all Bcc strains. The above standard culture methods cannot restore the growth of some Bcc strains. Some literature suggests using diluted TSB or R2A liquid medium for enrichment to improve the detection rate of Bcc in pharmaceutical water. Currently, both the United States Pharmacopeia and the Chinese Pharmacopeia's methods for detecting Burkholderia cepacia suggest using 1 / 10 diluted TSB (1 / 10 TSB) for enrichment culture when detecting Burkholderia cepacia in pharmaceutical water. However, research shows that most Bcc strains cannot grow or grow slowly in the 1 / 10 TSB medium.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] Aiming at the technical defects of poor enrichment effect and slow enrichment speed when using 1 / 10 TSB liquid medium for Bcc enrichment culture in the prior art, the present invention provides a detection medium for Burkholderia cepacia complex in a pharmaceutical water system.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] An onion Burkholderia cepacia complex detection medium for a pharmaceutical water system, the formulation of the detection medium (g / L) being: peptone 0.5 - 2.0 g, sodium pyruvate 1 - 4 g, carbon source 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 1 - 4 g, magnesium sulfate 0.05 - 0.2 g, and the solvent being water.

[0008] Preferably, the carbon source is selected from one or both of sucrose and glucose.

[0009] Preferably, the formulation of the detection medium (g / L) is: peptone 0.5 - 2.0 g, sodium pyruvate 4 g, carbon source 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 1 - 4 g, magnesium sulfate 0.2 g, and the solvent being water.

[0010] Preferably, the formulation of the detection medium (g / L) is: peptone 1 g, sodium pyruvate 4 g, carbon source 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 4 g, magnesium sulfate 0.2 g, and the solvent being water.

[0011] Preferably, the formulation of the detection medium (g / L) is: peptone 1 g, sodium pyruvate 4 g, sucrose 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 4 g, magnesium sulfate 0.2 g, and the solvent being water.

[0012] Preferably, the formulation of the detection medium (g / L) is: peptone 1 g, sodium pyruvate 4 g, sucrose 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 4 g, magnesium sulfate 0.2 g, and the solvent being water.

[0013] Preferably, the culture conditions of the detection medium are: take 1 mL of the bacterial solution and serially dilute it with 0.9% sterile sodium chloride solution to about 10 5 cfu / mL, mix 20 μL of the bacterial solution with 180 μL of the medium, and culture at 33°C ± 2°C for 24 h.

[0014] The beneficial effects of the present invention are:

[0015] Aiming at the technical defects in the prior art that the enrichment effect of the onion Burkholderia cepacia complex (Bcc) with the conventional 1 / 10 TSB liquid medium is poor and the enrichment speed is slow, the inventor screened an onion Burkholderia cepacia complex detection medium for a pharmaceutical water system with special components. Through the culture experiment of the onion Burkholderia cepacia complex, it was found that whether it is a standard strain or a wild strain isolated from the pharmaceutical water system of the onion Burkholderia cepacia complex, within the same culture time, the OD of the 1 / 10 TSB and R2A liquid media 600The value results are all not more than 0.5, while the bacterial concentration in the Burkholderia cepacia complex (BCB) medium for the detection of Burkholderia cepacia in the pharmaceutical water system is significantly higher than that in 1 / 10 TSB and R2A liquid media.

[0016] Burkholderia cepacia can grow well in the BCB medium, and the enrichment ability of the BCB medium is significantly better than that of 1 / 10 TSB and R2A liquid media, indicating that the BCB medium has good growth-promoting ability for Burkholderia cepacia and is more suitable as an enrichment medium for the detection of Burkholderia cepacia in the pharmaceutical water system. Description of the Drawings

[0017] Figure 1 is the screening result of the carbon source of the medium in Example 1;

[0018] Figure 2 is the result of the single-factor experiment on the components of the medium in Example 1;

[0019] Figure 3 is the enrichment culture result of Bcc using 1 / 10 TSB, BCB and R2A liquid media. Detailed Embodiments

[0020] The technical solutions of the present invention will be further specifically described below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0021] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0022] The reagents used in the following embodiments can be purchased from conventional biochemical reagent stores unless otherwise specified.

[0023] Yeast extract, sodium pyruvate, and magnesium sulfate were purchased from Shanghai Macklin Biochemical Co., Ltd.; peptone and casein amino acids were purchased from Beijing Coolaber Technology Co., Ltd.; sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd.; tryptic soy broth medium (TSB) was purchased from Guangdong Huankai Microbial Technology Co., Ltd.; R2A liquid medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0024] BCB medium (g / L): peptone 1 g, sodium pyruvate 4 g, sucrose 4 g, yeast extract 0.5 g, casein amino acids 4 g, magnesium sulfate 0.2 g.

[0025] Example 1

[0026] Screening of carbon sources

[0027] The basal medium components are 0.5 g / L of yeast extract, 0.5 g / L of peptone, 0.5 g / L of casein amino acids, 0.024 g / L of anhydrous magnesium sulfate, and 0.3 g / L of sodium pyruvate. The type of carbon source was changed, and five media containing glucose, lactose, maltose, fructose, and sucrose were added respectively. The concentration of the above saccharide substances was 2.0 g / L. All components were dissolved in purified water and used after sterilization at 121 °C for 15 min.

[0028] Three representative standard strains and wild-type strains in Burkholderia cepacia complex were selected for the experiment, including Burkholderia ambifaria and Burkholderia contaminans, which have relatively strict requirements for carbon sources. The standard strain CMCC23005 Burkholderia cepacia, the wild strain WYH10-3-4 Burkholderia ambifaria, and the wild strain HZwyh01T1-1 Burkholderia contaminans were used for the experiment.

[0029] After the above strains were revived, they were streaked on R2A medium and cultured for 24 hours. Then, fresh colonies on the R2A medium were scraped and made into a bacterial suspension with a 0.5 McFarland turbidity in R2A liquid medium, and inoculated into the liquid media with the above 5 different medium components at an inoculation amount of 1%. Three parallel samples were made for each medium. After culturing at 33 °C for 24 h, 200 μl of the bacterial suspension was taken and the OD 600 value at that point was measured using a microplate reader.

[0030] The experimental results are as Figure 1 , when sucrose was used as the carbon source, the OD values of the three strains after cultivation were the highest and the bacterial concentration was the highest.

[0031] 2 Investigation of single-factor experiments on the medium

[0032] Based on the above research results, using sucrose as the carbon source, the concentrations of sodium pyruvate, casein amino acids, peptone, magnesium sulfate, sucrose, and yeast extract were designed (see Table 1). Three parallel samples were prepared for each component concentration. The experiment was conducted using the relatively slow-growing wild-type Burkholderia cenocepacia BCSA-6T6-1 as a representative. Fresh colonies on the R2A medium were scraped and made into a bacterial suspension with a turbidity of 0.5 McFarland in R2A liquid medium, and then inoculated into the liquid medium with different concentrations of medium components at an inoculation amount of 1%. After culturing at 33°C for 24 h, 200 μl of the bacterial suspension was taken and the OD value was measured using a microplate reader. 600 The value at this point was used to analyze the effect of different concentrations of medium components on the growth of Burkholderia cenocepacia through a single-factor experiment.

[0033] Table 1 Design of medium component concentrations

[0034]

[0035] The results of the single-factor experiments on sodium pyruvate, casein amino acids, peptone, magnesium sulfate, sucrose, and yeast extract are as Figure 2 shown, which confirmed the factor levels for the orthogonal experiment of medium component optimization. The relevant factor levels are shown in Table 2.

[0036] Table 2 Factor levels for the orthogonal experiment of medium component optimization

[0037]

[0038] 3 Orthogonal experiment design of the medium

[0039] Based on the results of the single-factor experiment of the medium, three levels of each factor were determined, and an L18(3 6 ) orthogonal table was designed using SPSS to conduct an orthogonal experiment with 6 factors and 3 levels. The orthogonal experiment design is shown in Table 3. Fresh colonies of Burkholderia cenocepacia BCSA-6T6-1 on the R2A medium were scraped and made into a bacterial suspension with a turbidity of 0.5 McFarland in R2A liquid medium, and then inoculated into the liquid medium at different levels at an inoculation amount of 1%. Three parallel samples were designed. After 48 h, 200 μl of the bacterial suspension was taken and the OD value was measured using a microplate reader. 600 The value at this point was used to screen the optimal formula through range and variance analysis, and the measurement results were analyzed according to the orthogonal experiment design.

[0040] Table 3 Orthogonal experiment design table

[0041]

[0042] 4.3.4 Results of the orthogonal experiment

[0043] The orthogonal experiment is shown in Table 4, and Table 5 shows the results of variance analysis.

[0044] Table 4 Results of the orthogonal experiment

[0045]

[0046] Table 5 Tests of between-subjects effects

[0047]

[0048]

[0049] Note: a. R-squared = 0.989 (Adjusted R-squared = 0.963)

[0050] It can be seen from the results of the orthogonal experiment in Table 4 that the order of influence of the culture medium components from large to small is sodium pyruvate > magnesium sulfate > casein amino acids > peptone > sucrose > yeast extract. According to the results of the variance analysis in Table 5, there are statistical differences in the culture results of sodium pyruvate, magnesium sulfate, casein amino acids, and peptone (P < 0.05), while there are no significant differences in sucrose and yeast extract (P > 0.05). After analysis, the selected levels are A3B3C2D3E3F1, and the formula of the enrichment medium (g / l) is determined as follows: peptone 1g, sodium pyruvate 4g, sucrose 4g, yeast extract powder 0.5g, casein amino acids 4g, magnesium sulfate 0.2g. And this enrichment medium is named BCB (Burkholderia cepacia Broth) medium.

[0051] Example 2 Investigation of the growth-promoting ability of BCB

[0052] In the inspection of Burkholderia cepacia in the pharmaceutical water system, enrichment is usually carried out with 1 / 10 TSB medium. Therefore, the present inventors compared and studied BCB with 1 / 10 TSB medium and R2A liquid medium commonly used in the inspection of water system microorganisms.

[0053] Twenty-one strains of Burkholderia cepacia (10 standard strains and 11 wild strains) preserved in the laboratory were respectively inoculated into 1 / 10 TSB, BCB, and R2A liquid media to investigate and compare the enrichment ability of BCB. Take 1 mL of the bacterial solution and serially dilute it with 0.9% sterile sodium chloride solution to about 10 5 cfu / mL. Mix 20 μL of the bacterial solution with 180 μL of the liquid medium (1 / 10 TSB, BCB, and R2A liquid media), set 3 parallel samples, and culture at 33 °C for 24 h. Use an enzyme-linked immunosorbent assay reader to measure and read the OD 600 value, and the results are shown in Table 6.

[0054] Table 6 Enrichment OD 600 values of different media

[0055]

[0056]

[0057]

[0058] The growth of Burkholderia cepacia complex in three liquid media is as Figure 3 shown. It is found that whether it is a standard strain or a wild strain isolated from a pharmaceutical water system of Burkholderia cepacia complex, within the same cultivation time, the OD 600 value results in 1 / 10 TSB and R2A liquid media do not exceed 0.5, while the bacterial concentration in BCB medium is significantly higher than that in 1 / 10 TSB and R2A liquid media.

[0059] Calculated according to the results in Table 6, the growth-promoting abilities of different media for Burkholderia cepacia complex are shown in Table 7 in detail.

[0060] Table 7 Growth-promoting abilities of different liquid media

[0061]

[0062] As can be seen from Table 7, the growth-promoting abilities of different liquid media for Burkholderia cepacia complex are BCB > R2A liquid medium > 1 / 10 TSB. Burkholderia cepacia complex can grow well in BCB medium, and the enrichment ability of BCB medium is significantly better than that of 1 / 10 TSB and R2A liquid media, indicating that BCB medium has good growth-promoting ability for Burkholderia cepacia complex and is more suitable as an enrichment medium for the detection of Burkholderia cepacia complex in a pharmaceutical water system.

[0063] In this specification, each embodiment is described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and reference can be made to the description in the method part for related parts.

[0064] The above has introduced in detail the detection medium for Burkholderia cepacia complex in a pharmaceutical water system provided by the present invention. Specific examples are used herein to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An onion Burkholderia cepacia group detection medium for a pharmaceutical water system, characterized in that The formula of the detection medium (g / L) is as follows: peptone 1 g, sodium pyruvate 4 g, sucrose 1 - 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 4 g, magnesium sulfate 0.2 g, and the solvent is water.

2. The Burkholderia cepacia complex detection medium according to claim 1, wherein: peptone 1 g, sodium pyruvate 4 g, sucrose 4 g, yeast extract 0.5 - 2.0 g, casein amino acids 4 g, magnesium sulfate 0.2 g, and the solvent is water.

3. The Burkholderia cepacia complex detection medium according to claim 1, wherein The culture conditions of the detection medium are as follows: Take 1 mL of the bacterial solution and serially dilute it with 0.9% sterile sodium chloride solution to 10 5 cfu / mL, mix 20 μL of the bacterial solution with 180 μL of the medium, and culture at 33 °C ± 2 °C for 24 h.

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