Burkholderia cepacia flora detection culture medium for pharmaceutical water system

Abstract

The invention belongs to the field of selective culture of microorganisms, and relates to a Burkholderia cepacia flora detection culture medium for a pharmaceutical water system. The invention discloses a Burkholderia cepacia flora detection culture medium for a pharmaceutical water system. The formula (g / L) of the detection culture medium is as follows: 0.5-2.0 g of peptone, 1-4 g of sodium pyruvate, 1-4 g of a carbon source, 0.5-2.0 g of a yeast extract, 1-4 g of casein amino acid, 0.05-0.2 g of magnesium sulfate and water as a solvent. The inventor obtains the Burkholderia cepacia flora detection culture medium with special components for the pharmaceutical water system through screening, and the Burkholderia cepacia flora culture experiment finds that the Burkholderia cepacia flora can be used as a standard strain or a wild strain separated from the pharmaceutical water system within the same culture time; the OD600 value results of the 1 / 10 TSB liquid culture medium and the R2A liquid culture medium do not exceed 0.5, and the bacterium concentration in the BCB culture medium of a pharmaceutical water system is obviously higher than that of the 1 / 10 TSB liquid culture medium and the R2A liquid culture medium.

Description

Technical Field

[0001] The invention belongs to the field of microbial selection culture and relates to a Burkholderia cepacia detection culture medium for a pharmaceutical water system. Background Art

[0002] The Burkholderia cepacia (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 contamination risk to drugs. There have been many cases of drug recalls due to Bcc contamination at home and abroad, and the pharmaceutical industry is now paying more and more attention to Bcc. Effective identification and monitoring of Bcc in products or their production processes are the key to controlling Bcc contamination risks. The water system is the largest source of Bcc contamination, and a large amount of Bcc can be detected in pharmaceutical water systems. Due to the prominent oligotrophic characteristics of Bcc from the water system, studies have found that some Bcc strains cannot be detected in the pharmacopoeia system culture medium.

[0003] At present, the Bcc test methods in pharmaceutical products in domestic and foreign pharmacopoeias rely on traditional enrichment culture, selection and separation culture to obtain pure cultures of suspected target microorganisms, and further conduct appropriate identification to determine whether the sample is contaminated with Bcc. The microbial detection method of the United States Pharmacopoeia (USP) includes the use of TSB (trypticase soy broth) for microbial enrichment culture, but this method is not suitable for all Bcc strains. The above standard culture method cannot restore the growth of some Bcc strains. Some literature recommends the use of diluted TSB or R2A liquid culture medium to increase the detection rate of Bcc in pharmaceutical water. At present, the Burkholderia onion test methods of the United States Pharmacopoeia and the Chinese Pharmacopoeia both mention that it is recommended to use 10-fold diluted TSB (1 / 10TSB) for enrichment culture when testing Burkholderia onion in pharmaceutical water, but studies have shown that most Bcc strains cannot grow or grow slowly in 1 / 10TSB culture medium.

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

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

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

[0007] A Burkholderia cepacia detection culture medium for a pharmaceutical water system, wherein the detection culture medium formula (g / L) is: 0.5-2.0 g of peptone, 1-4 g of sodium pyruvate, 1-4 g of a carbon source, 0.5-2.0 g of a yeast extract, 1-4 g of casamino acids, 0.05-0.2 g of magnesium sulfate, and the solvent is water.

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

[0009] Preferably, the test medium formula (g / L) is: peptone 0.5-2.0g, sodium pyruvate 4g, carbon source 1-4g, yeast extract 0.5-2.0g, casamino acid 1-4g, magnesium sulfate 0.2g, and the solvent is water.

[0010] Preferably, the test medium formula (g / L) is: 1g peptone, 4g sodium pyruvate, 1-4g carbon source, 0.5-2.0g yeast extract, 4g casamino acid, 0.2g magnesium sulfate, and the solvent is water.

[0011] Preferably, the test medium formula (g / L) is: 1g peptone, 4g sodium pyruvate, 1-4g sucrose, 0.5-2.0g yeast extract, 4g casamino acids, 0.2g magnesium sulfate, and the solvent is water.

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

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

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

[0015] In view of the technical defects of the conventional 1 / 10TSB liquid culture medium for the onion Burkholderia (Bcc) in the prior art, which has poor bacterial growth effect and slow bacterial growth rate, the inventors screened and obtained a special component of the onion Burkholderia detection culture medium for the pharmaceutical water system. The onion Burkholderia culture experiment found that the onion Burkholderia, whether it is a standard strain or a wild strain isolated from the pharmaceutical water system, has an OD of 1 / 10TSB and R2A liquid culture medium within the same culture time. 600The value results did not exceed 0.5, and the bacterial concentration in the Burkholderia cepacia (BCB) culture medium of the pharmaceutical water system was significantly higher than 1 / 10 TSB and R2A liquid culture medium.

[0016] The cepacia Burkholderia flora can grow well in BCB medium, and the bacterial growth ability of BCB medium is significantly better than that of 1 / 10TSB and R2A liquid culture medium, indicating that BCB medium has a good growth-promoting ability for cepacia Burkholderia flora and is more suitable as a culture medium for the detection and growth of cepacia Burkholderia flora in pharmaceutical water systems. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 This is the single factor test result of the culture medium components in Example 1;

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

[0020] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of 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 by weight, 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 examples, unless otherwise specified, can be purchased from conventional biochemical reagent stores.

[0023] Yeast extract, sodium pyruvate, and magnesium sulfate were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; peptone and casamino acids were purchased from Beijing Coolaibo Technology Co., Ltd.; sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd.; tryptone soy broth (TSB) was purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; and R2A liquid culture 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, casamino acid 4 g, magnesium sulfate 0.2 g.

[0025] Example 1

[0026] 1. Screening of carbon sources

[0027] The basic culture medium consists of 0.5 g / L yeast extract, 0.5 g / L peptone, 0.5 g / L casamino acid, 0.024 g / L anhydrous magnesium sulfate, and 0.3 g / L sodium pyruvate. The carbon source was changed, and five culture media containing glucose, lactose, maltose, fructose, and sucrose were added respectively, and the concentration of the above sugar substances was 2.0 g / L. All the ingredients were dissolved in purified water and sterilized at 121°C for 15 minutes before use.

[0028] Three representative standard strains and wild strains of the cepacia group were selected for testing, including Burkholderia ambifaria and Burkholderia contaminans, which have strict requirements on 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 testing.

[0029] After the above strains were revived, they were streaked onto R2A medium and cultured for 24 hours. Fresh colonies on the R2A medium were scraped and added to R2A liquid medium to make a 0.5 McFarland turbidity bacterial solution. The inoculation volume was 1% and inoculated into the above 5 liquid culture media with different culture medium components. Three parallel samples were made for each culture medium. After culturing at 33°C for 24 hours, 200 μl of the bacterial solution was taken and its OD was measured using an ELISA reader. 600 The numerical value.

[0030] The test results are as follows Figure 1 When sucrose was used as the carbon source, the OD value of the three bacterial strains was the highest and the bacterial concentration was the highest after cultivation.

[0031] 2 Culture medium single factor test investigation

[0032] Based on the above research results, sucrose was used as the carbon source, and the concentrations of sodium pyruvate, casamino acids, peptone, magnesium sulfate, sucrose, and yeast extract were designed (see Table 1). Three parallel samples were made for each component concentration, and the wild-type BCSA-6T6-1 Burkholderia cenocepacia, which grows relatively slowly, was used as a representative for the experiment. Fresh colonies on the R2A medium were scraped and made into a 0.5 McFarland turbidity bacterial solution in the R2A liquid medium. The inoculation amount was 1% and inoculated into liquid culture media with different concentrations of culture media components. After culturing at 33°C for 24 hours, 200 μl of the bacterial solution was taken and its OD was measured using an ELISA reader. 600 The single factor experimental analysis was used to investigate the effects of different concentrations of culture medium components on the growth of Burkholderia cepacia.

[0033] Table 1 Design of medium component concentration

[0034]

[0035] The results of single factor experiments with sodium pyruvate, casamino acids, peptone, magnesium sulfate, sucrose, and yeast extract are as follows: Figure 2 As shown, the factor levels of the orthogonal experiment for medium component optimization were confirmed, and the relevant factor levels are shown in Table 2.

[0036] Table 2 Factor levels of orthogonal experiment for optimization of culture medium composition

[0037]

[0038] 3. Orthogonal experimental design of culture medium

[0039] According to the results of the single-factor test of the culture medium, the three levels of each factor were determined. SPSS was used to design L18(3 6 ) orthogonal table, design an orthogonal experiment with 6 factors and 3 levels. The orthogonal experiment design is shown in Table 3. Scrape the fresh colonies of BCSA-6T6-1 Burkholderia cenocepacia on R2A medium and make a 0.5 McFarland turbidity bacterial solution in R2A liquid medium. Inoculate it in liquid medium at different levels with an inoculum of 1%, design 3 parallel samples, and take 200 μl of the bacterial solution after 48 hours to measure its OD using an enzyme reader 600 The best formula was selected through range variance analysis, and the measurement results were analyzed according to orthogonal experimental design.

[0040] Table 3 Orthogonal test design table

[0041]

[0042] 4.3.4 Orthogonal test results

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

[0044] Table 4 Orthogonal test results

[0045]

[0046] Table 5 Test of between-subject effects

[0047]

[0048]

[0049] Note: aR square = 0.989 (adjusted R square = 0.963)

[0050] From the orthogonal test results in Table 4, it can be seen that the influence of culture medium components is from large to small, sodium pyruvate>magnesium sulfate>casamino acid>peptone>sucrose>yeast extract. According to the variance analysis results in Table 5, it can be seen that the culture results of sodium pyruvate, magnesium sulfate, casamino acid and peptone are statistically different (P<0.05), and there is no significant difference in sucrose and yeast extract (P>0.05). The analysis selection level A3B3C2D3E3F1 determines the formula (g / l) of the enrichment culture medium: peptone 1g, sodium pyruvate 4g, sucrose 4g, yeast extract powder 0.5g, casamino acid 4g, magnesium sulfate 0.2g, and the enrichment culture medium is named BCB (Burkholderia cepacia Broth) culture medium.

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

[0052] The Burkholderia cepacia flora test in pharmaceutical water systems is usually carried out with 1 / 10 TSB culture medium for bacterial enrichment. Therefore, the inventors conducted a comparative study on BCB, 1 / 10 TSB culture medium, and R2A liquid culture medium commonly used in water system microbial testing.

[0053] 21 Burkholderia cepacia strains (10 standard strains and 11 wild strains) stored in the laboratory were inoculated into 1 / 10 TSB, BCB, and R2A liquid culture media to investigate and compare the bacterial growth capacity of BCB. 1 mL of bacterial solution was diluted to about 10% with 0.9% sterile sodium chloride solution. 5 cfu / mL, 20 μL of bacterial solution was mixed with 180 μL of liquid culture medium (1 / 10 TSB, BCB and R2A liquid culture medium), three parallel samples were set up, cultured at 33°C for 24 h, and the OD of 96-well plate was measured by microplate reader 600 The results are shown in Table 6.

[0054] Table 6 OD of bacterial growth in different culture media 600 value

[0055]

[0056]

[0057]

[0058] Growth of Burkholderia cepacia in three liquid media Figure 3 As shown in the figure, it was found that the OD of 1 / 10 TSB and R2A liquid culture medium was the same for both standard strains and wild strains isolated from pharmaceutical water system. 600 The values ​​did not exceed 0.5, and the bacterial concentration in BCB medium was significantly higher than 1 / 10 TSB and R2A liquid culture medium.

[0059] According to the results in Table 6, the growth-promoting ability of different culture media on Burkholderia cepacia is shown in Table 7.

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

[0061]

[0062] According to Table 7, the growth-promoting ability of different liquid culture media on Burkholderia cepacia is BCB>R2A liquid culture medium>1 / 10TSB. Burkholderia cepacia can grow well in BCB medium, and the bacterial growth ability of BCB medium is significantly better than that of 1 / 10TSB and R2A liquid culture medium, indicating that BCB medium has a good growth-promoting ability for Burkholderia cepacia and is more suitable as a culture medium for the detection and growth of Burkholderia cepacia in pharmaceutical water systems.

[0063] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred 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 the relevant parts can be referred to the method part.

[0064] The above is a detailed introduction to the Burkholderia cepacia detection medium for the pharmaceutical water system provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A Burkholderia cepacia detection medium for a pharmaceutical water system, characterized in that The test medium formula (g / L) is: 0.5-2.0 g of peptone, 1-4 g of sodium pyruvate, 1-4 g of carbon source, 0.5-2.0 g of yeast extract, 1-4 g of casamino acid, 0.05-0.2 g of magnesium sulfate, and the solvent is water.

2. The Burkholderia cepacia detection medium according to claim 1, characterized in that: The carbon source can be sucrose or glucose or both.

3. The Burkholderia cepacia detection medium according to claim 1, characterized in that: The test medium formula (g / L) is: 0.5-2.0 g of peptone, 4 g of sodium pyruvate, 1-4 g of carbon source, 0.5-2.0 g of yeast extract, 1-4 g of casamino acid, 0.2 g of magnesium sulfate, and the solvent is water.

4. The Burkholderia cepacia detection medium according to claim 1, characterized in that: 1g of peptone, 4g of sodium pyruvate, 1-4g of carbon source, 0.5-2.0g of yeast extract, 4g of casamino acids, 0.2g of magnesium sulfate, and the solvent is water.

5. The Burkholderia cepacia detection medium according to claim 1, characterized in that: 1g of peptone, 4g of sodium pyruvate, 1-4g of sucrose, 0.5-2.0g of yeast extract, 4g of casamino acids, 0.2g of magnesium sulfate, and the solvent is water.

6. The Burkholderia cepacia detection medium according to claim 1, characterized in that: 1g of peptone, 4g of sodium pyruvate, 4g of sucrose, 0.5-2.0g of yeast extract, 4g of casamino acids, 0.2g of magnesium sulfate, and the solvent is water.

7. The Burkholderia cepacia detection medium according to claim 1, characterized in that The culture conditions of the detection culture medium are: 1 mL of bacterial solution is diluted stepwise with 0.9% sterile sodium chloride solution to about 10 5 cfu / mL, mix 20 μL of bacterial solution with 180 μL of culture medium and culture at 33℃±2℃ for 24 h.

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