CN114907953A - A kind of sterile testing culture bottle and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of sterility detection, and discloses a sterility detection culture bottle and a preparation method thereof. The culture bottle comprises a bottle body and a bottle cap arranged on the top of the bottle body, and the bottle body is sequentially provided with chromaticity sensors from bottom to top , antibiotic adsorption resin and culture medium; the chromaticity sensor is prepared by drying the silica gel dye solution, in parts by weight, the components of the silica gel dye solution include: 0.01-0.1 part of bromothymol blue, 11 parts of water , 0.025-0.3 parts of glacial acetic acid, 4-10 parts of 0.1mol/L sodium hydroxide solution, 2-5 parts of glycerol, 0.02-0.04 parts of emulsifier, and 900-1100 parts of white liquid silica gel. The invention improves the detection sensitivity and the application range of the culture bottle by optimizing the components of the colorimetric sensor in the culture bottle and setting the antibiotic adsorption resin in the culture bottle.

Description

A kind of sterile testing culture bottle and preparation method thereof

technical field

The invention relates to the technical field of sterility detection, in particular to a sterility detection culture bottle and a preparation method thereof.

Background technique

Sterility testing is a method used to check the sterility of pharmaceuticals, biological products, medical devices, raw materials, excipients and other materials required by the Pharmacopoeia to be sterile. Sterility testing is to inoculate the test product or its extract in the medium, and check whether the test product is contaminated by microorganisms by whether there is colony growth. According to GMP regulations, sterility testing is one of the important steps in the release of sterile products. It has the significance of determining whether the sample is contaminated, controlling the quality of the sample, ensuring the effectiveness and safety of the drug, and measuring whether the sanitation level in the whole process of sample production meets the standards. .

The traditional sterility testing methods mainly include direct inoculation method and membrane filtration method, but the traditional sterility testing cycle is as long as 14 days, which is not conducive to enterprises to take corrective measures as soon as possible, and it is not conducive to the early issuance of products with short validity periods. In addition, the traditional sterility detection method generally judges the sterility of the test product by artificially observing the turbidity change of the culture medium brought about by the growth of the bacterial colony in the collection medium. However, relying on manual observation and judgment itself may be misjudged, and the detection accuracy is difficult. ensure.

At present, there have been studies on the detection of microorganisms by sensors, for example, a "culture flask with internal sensor" disclosed in Chinese patent documents, the publication number of which is CN103314097A, in this invention, a color sensor is arranged in the culture flask, The color change of the colorimetric sensor detects microorganisms.

However, after culturing Aspergillus niger and Pseudomonas aeruginosa in the above-mentioned culture flask for 7 days, due to the low pH drop of the medium in the culture flask, the colorimetric sensor at the bottom of the culture flask does not change much, and the detection sensitivity is low; Antibiotic adsorbents cannot detect microorganisms in test samples with antibiotics, which limits their application in sterility testing.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned problems of the culture bottle in the prior art, the present invention provides a sterile detection culture bottle and a preparation method thereof. The components of the colorimetric sensor in the culture bottle are optimized, and antibiotic adsorption resin is arranged in the culture bottle. , which improves the detection sensitivity and the application range of culture flasks.

In order to achieve the above object, the present invention adopts the following technical solutions:

A sterile detection culture bottle, comprising a bottle body and a bottle cap arranged on the top of the bottle body, the bottle body is sequentially provided with a chromaticity sensor, an antibiotic adsorption resin and a culture medium from bottom to top; the chromaticity sensor is dyed by silica gel. It is prepared after the liquid is dried. In parts by weight, the components of the silica gel dyeing solution include: 0.01-0.1 parts of bromothymol blue, 11 parts of water, 0.025-0.3 parts of glacial acetic acid, and 0.1 mol/L sodium hydroxide solution 4~10 parts, 2~5 parts glycerin, 0.02~0.04 parts emulsifier, 900~1100 parts white liquid silica gel.

In the chromaticity sensor in the culture bottle of the present invention, bromothymol blue is used as a dye, and white silica gel is used as a carrier. High sensitivity, even when cultivating microorganisms such as Aspergillus niger and Pseudomonas aeruginosa that reduce the pH value of the medium to a low degree, the colorimetric sensor in the bottle will have a significant color change, and the significant color change is higher than the traditional turbidity. Changes are easier to observe, greatly reducing the possibility of misjudgment by operators through manual observation and judgment. The culture bottle in the present invention can also be used with a sterility detection system, and the color change of the chromaticity sensor can be automatically recognized by the machine to judge the sterility detection result, so as to avoid misjudgment caused by human interpretation. At the same time, the use of the chromaticity sensor of the present invention can greatly shorten the detection period of sterility detection, which can be shortened to 12 hours if it is fast, or 5-7 days if it is slow. The culture bottle of the invention is also provided with an antibiotic adsorbent, so that the microorganisms that are sub-lethal due to antibiotics can also be quickly recovered and grown, thereby expanding the application range of the culture bottle.

In the silica gel dye solution used in the preparation of the chromaticity sensor, the bromothymol blue is dissolved with sodium hydroxide solution, the pH of the solution is adjusted with glacial acetic acid, and glycerol and an emulsifier are added at the same time, which can increase the solubility of the dye and make the The silica gel dye solution can form a uniform emulsion, avoid the existence of solid dye particles in the colorimetric sensor and improve the uniformity of dye distribution, which is beneficial to improve the sensitivity and accuracy of the colorimetric sensor.

Preferably, the white liquid silica gel includes liquid silica gel E600A and liquid silica gel E600B with a mass ratio of 1:0.8-1.2. The chromaticity sensor of the present invention adopts the compound of liquid silica gel E600A and liquid silica gel E600B as the carrier of the dye, which is white silica gel after curing, which is easier to reflect the color change of the dye than transparent silica gel, and the compound of liquid silica gel E600A and liquid silica gel E600B The resulting silica gel is highly viscous, and degumming is not easy to occur in the culture bottle.

Preferably, the emulsifier is Tween 80.

Preferably, the culture medium is a sterile tryptone liquid medium or a sterile thioglycolate fluid medium; when the medium is a sterile thioglycolate fluid medium, the bottle The body is filled with nitrogen as a protective gas. The aerobic culture flask is prepared by using tryptone soy liquid medium, and the anaerobic culture flask is prepared by using thioglycolate fluid medium, which can meet the detection requirements of various microorganisms.

Preferably, the ratio of the added amount of bromothymol blue to the antibiotic adsorption resin and the culture medium in the color sensor is 0.01-0.1 g:1-2 g:30 mL.

Preferably, the bottle body is a PC plastic bottle.

The invention also discloses a preparation method of the above-mentioned sterile detection culture bottle, comprising the following steps:

(1) Prepare silica gel dye solution;

(2) Preparation of chromaticity sensor: inject silica gel dye solution into the bottle, vacuumize and defoam, dry and sterilize to obtain a chromaticity sensor;

(3) Inject sterile antibiotic adsorption resin and sterile culture medium into the bottle;

(4) The bottle body is sealed with a cap, and the culture bottle is vacuum-packed;

(5) Pre-culture.

Preferably, the preparation method of the silica gel dye solution in step (1) is as follows: adding bromothymol blue into sodium hydroxide solution and water, stirring and dissolving, adding glacial acetic acid, then adding glycerol and emulsifier, and stirring to obtain the dye solution; The dyeing liquid is mixed with white liquid silica gel, and the silica gel dyeing liquid is obtained after stirring evenly.

Preferably, step (3) and step (4) are carried out under sterile conditions; when the medium is a sterile thioglycolate fluid medium, after the medium is injected, the bottle body is filled with nitrogen, and then Then do the gland sealing.

Preferably, the pre-cultivation time in step (5) is 14-15 days; when the culture medium is sterile tryptone soy liquid medium, the pre-culture temperature is 20-24°C; the culture medium is sterile thioethanol When using saline fluid medium, the pre-incubation temperature is 31-35°C.

Therefore, the present invention has the following beneficial effects:

(1) The colorimetric sensor uses bromothymol blue as the dye and white silica gel as the carrier, which has high discoloration sensitivity. Chroma sensors also experience significant color changes;

(2) The pH of the solution is adjusted with glacial acetic acid in the silica dye solution, and glycerol and emulsifier are added at the same time, which can increase the solubility of the dye and make the silica dye solution form a uniform emulsion, which is beneficial to improve the sensitivity of the chromaticity sensor. Accuracy;

(3) There is also an antibiotic adsorbent in the culture bottle, which can quickly recover and grow microorganisms that are sub-lethal due to antibiotics, and expand the application range of the culture bottle.

Description of drawings

Fig. 1 is the cross-sectional schematic diagram of the culture flask of the present invention;

In the picture: 1 bottle body, 2 bottle caps, 3 color sensor; 4 antibiotic adsorption resin, 5 culture medium;

Fig. 2 is the change curve of RGB value of chromaticity sensor in 7 days in the aerobic culture bottle that Aspergillus niger makes in Example 1;

Fig. 3 is the RGB value change curve of colorimetric sensor in the aerobic culture bottle that Candida albicans made in Example 1 within 7 days;

Fig. 4 is the change curve of RGB value of colorimetric sensor in 7 days in the aerobic culture bottle obtained by Bacillus subtilis in Example 1;

Fig. 5 is the RGB value change curve of colorimetric sensor in 7 days in the anaerobic culture bottle that Escherichia coli made in Example 2;

Fig. 6 is the RGB value change curve of colorimetric sensor in 7 days in the anaerobic culture bottle obtained by Clostridium sporogenes in Example 2;

Fig. 7 is the RGB value change curve of colorimetric sensor in 7 days in the anaerobic culture bottle that Staphylococcus aureus made in Example 2;

Fig. 8 is the RGB value change curve of the colorimetric sensor in the anaerobic culture flask prepared by Pseudomonas aeruginosa in Example 2 within 7 days;

Fig. 9 is the color change situation of the color sensor after 7 days in the culture bottle and the negative culture bottle where 7 kinds of quality control bacteria are located;

In the figure, (a) negative anaerobic bottle, (b) positive Escherichia coli, (c) positive Clostridium sporogenes, (d) positive Pseudomonas aeruginosa, (e) positive Staphylococcus aureus, (f) positive ) negative aerobic bottle, (g) positive Aspergillus niger, (h) positive Candida albicans, (i) positive Bacillus subtilis;

Fig. 10 is the physical map of the silica gel dye liquor prepared in Comparative Example 1;

Fig. 11 is the physical picture of the colorimetric sensor in the negative aerobic bottle made in Comparative Example 2;

FIG. 12 is a physical view of the colorimetric sensor in the culture flask prepared in Comparative Example 3. FIG.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or commonly used in the industry, and all methods are conventional methods in the art unless otherwise specified.

Example 1:

As shown in Figure 1, a sterile testing culture bottle includes a bottle body 1 and a bottle cap 2 arranged on the top of the bottle body. The bottle body adopts a PC plastic bottle. The bottle body is provided with a chromaticity sensor 3, an antibiotic adsorption bottle from bottom to top. Resin 4 and Medium 5.

Among them, the culture medium adopts sterile tryptone soy liquid medium; the color sensor is prepared by drying the silica gel dye solution. In parts by weight, the components of the silica gel dye solution include: 0.05 part of bromothymol blue, purified water 11 parts, 0.1 part of glacial acetic acid, 6 parts of 0.1mol/L sodium hydroxide solution, 3 parts of glycerol, 0.03 part of Tween 80, 500 parts of liquid silica gel E600A, and 500 parts of liquid silica gel E600B.

Its preparation method is:

(1) Preparation of silica gel dye solution: add bromothymol blue to sodium hydroxide solution and purified water, stir and dissolve, add glacial acetic acid, then add glycerin and emulsifier, stir evenly to obtain dye solution; mix liquid silica gel E600A and liquid silica gel E600B is mixed evenly to obtain white liquid silica gel; the dye liquor is mixed with white liquid silica gel, and the silica gel dye liquor is obtained after stirring evenly;

(2) Preparation of chromaticity sensor: use a glue dispenser to inject the silica gel dye solution into the bottle body, after vacuuming and defoaming, place the bottle body in a drying oven at 70 °C for 30 minutes, and then perform moist heat sterilization to obtain color. degree sensor;

(3) Transfer the sterilized bottle body into the aseptic filling isolator, weigh the sterile antibiotic adsorption resin (Tianjin Yunkai resin, YKC107) and inject it into the bottle body, and then fill the bottle with aseptic filling technology. Sterile tryptic soy liquid medium was injected into the body, and the ratio of bromothymol blue in the colorimetric sensor to antibiotic adsorption resin and medium was 0.05g:1.5g:30mL;

(4) The bottle body is plugged and capped in the aseptic filling isolator, and the culture bottle is vacuum-packed in a vacuum packaging machine;

(5) Pre-culture: place the culture flask in a 22°C incubator for 14 days of pre-culture to obtain the desired culture flask.

Example 2:

A sterile testing culture bottle includes a bottle body and a bottle cap arranged on the top of the bottle body.

Wherein, the culture medium is sterile thioglycolate fluid medium; the colorimetric sensor is prepared by drying the silica gel dye solution, and the components of the silica gel dye solution in parts by weight include: 0.1 part of bromothymol blue, purified water 11 parts, 0.3 parts of glacial acetic acid, 10 parts of 0.1mol/L sodium hydroxide solution, 2 parts of glycerol, 0.04 parts of Tween 80, 450 parts of liquid silica gel E600A, and 450 parts of liquid silica gel E600B.

Its preparation method is:

(1) Preparation of silica gel dye solution: add bromothymol blue to sodium hydroxide solution and water, stir and dissolve, add glacial acetic acid, then add glycerin and emulsifier, stir evenly to obtain dye solution; mix liquid silica gel E600A and liquid silica gel E600B Mix evenly to obtain white liquid silica gel; mix the dye solution with white liquid silica gel, and stir well to obtain silica gel dye solution;

(2) Preparation of chromaticity sensor: use a glue dispenser to inject the silica gel dye solution into the bottle body, after vacuuming and defoaming, place the bottle body in a drying oven at 70 °C for 30 minutes, and then perform moist heat sterilization to obtain color. degree sensor;

(3) Transfer the sterilized bottle body into the aseptic filling isolator, weigh the sterile antibiotic adsorption resin (Tianjin Yunkai resin, YKC107) and inject it into the bottle body, and then fill the bottle with aseptic filling technology. Inject sterile thioglycolate fluid medium into the body, and fill the culture bottle with nitrogen as a protective gas. The ratio of bromothymol blue in the color sensor to antibiotic adsorption resin and medium is 0.1g :2g:30mL;

(4) The bottle body is plugged and capped in the aseptic filling isolator, and the culture bottle is vacuum-packed in a vacuum packaging machine;

(5) Pre-culture: place the culture flask in a 33°C incubator for 14 days of pre-culture to obtain an anaerobic culture flask.

Comparative Example 1 (without adding glycerin and emulsifier):

In the culture flask of Comparative Example 1, the components of the silica gel dye solution for preparing the colorimetric sensor in parts by weight include: 0.05 part of bromothymol blue, 11 parts of purified water, 0.1 part of glacial acetic acid, and 0.1 mol/L of sodium hydroxide 6 parts of the solution, 500 parts of liquid silica gel E600A, 500 parts of liquid silica gel E600B, the rest are the same as in Example 1.

Comparative Example 2 (using transparent silicone):

In the culture flask of Comparative Example 2, the components of the silica gel dye solution for preparing the colorimetric sensor in parts by weight include: 0.05 part of bromothymol blue, 11 parts of purified water, 0.1 part of glacial acetic acid, and 0.1 mol/L of sodium hydroxide 6 parts of solution, 3 parts of glycerin, 800.03 parts of Tween, liquid silica gel (Shenzhen Kejia Adhesive Material Co., Ltd., KJ-K6363T) 600 parts of A glue, 200 parts of B glue.

Comparative Example 3 (replace acetic acid with potassium dihydrogen phosphate):

In the culture flask of Comparative Example 1, the components by weight of the silica gel dye solution for preparing the colorimetric sensor included: 0.05 part of bromothymol blue, 11 parts of purified water, 0.1 part of potassium dihydrogen phosphate, and 0.1 mol/L hydrogen 6 parts of sodium oxide solution, 3 parts of glycerin, 0.03 parts of Tween 80, 500 parts of liquid silica gel E600A, 500 parts of liquid silica gel E600B.

Seven kinds of quality control bacteria stipulated in pharmacopoeia sterility inspection were cultured using the culture flasks prepared in the above examples and comparative examples. The culture flasks were cultured, and Escherichia coli, Clostridium sporogenes, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured with the anaerobic culture flasks prepared in Example 2, and the chromaticity sensor in each culture flask was recorded at 7. The change curve of RGB value within the day, the results are shown in Figure 2~8. Figure 9 shows the color changes of the colorimetric sensor after 7 days of testing in the culture flasks and negative culture flasks of the seven quality control bacteria.

As can be seen from Fig. 2 to Fig. 8, using the culture bottle of the present invention, the fastest detectable quality control bacteria specified in the Pharmacopoeia sterility test is 0.5 days, and the slowest detection time is 3 days, which greatly shortens the sterility. detection time. As can be seen from FIG. 9 , the colorimetric sensor in the culture flask prepared by the present invention is sensitive to discoloration, and the color change can also be directly observed with the naked eye.

As shown in Figure 10, in the process of preparing the chromaticity sensor in Comparative Example 1, glycerin and emulsifier were not added to the silica gel dye solution, so that the dye solution could not be completely dissolved in the glue, and the dye was dispersed in droplets in the colloid. Only the upper layer of the colloid is slightly dyed, with a small amount of dye solution dissolved, and the lower layer is milky white, and no dye solution is dissolved in it. The colorimetric sensor prepared with this formula is prone to misjudgment during instrument detection due to uneven color distribution.

As shown in FIG. 11 , in Comparative Example 2, transparent silica gel was used as the dye carrier, and the obtained colorimetric sensor would transmit light, so that the yellow color of the medium would be transmitted through the bottom of the bottle. Because the detection mechanism of the present invention is that when the chromaticity sensor at the bottom of the bottle is blue, the detection sample is judged as negative, and when the chromaticity sensor at the bottom of the bottle is yellow, the detection sample is judged as positive; therefore, the light-transmitting chromaticity sensor in Comparative Example 2 The yellow color of the medium penetrates through the bottom of the bottle, which will easily misjudge a negative as a positive when testing with an instrument.

As shown in Figure 12, in Comparative Example 3, potassium dihydrogen phosphate was used instead of acetic acid to adjust the pH of the solution, the dye was not completely dissolved, and there were solid dye particles in the prepared color sensor. Deviations will occur.

Claims (10)

1. a sterile detection culture bottle, comprising a bottle body and a bottle cap arranged on top of the bottle body, is characterized in that, in the described bottle body, from bottom to top is sequentially provided with chromaticity sensor, antibiotic adsorption resin and culture medium; The chromaticity sensor is prepared by drying a silica gel dye solution. In parts by weight, the components of the silica gel dye solution include: 0.01-0.1 parts of bromothymol blue, 11 parts of water, 0.025-0.3 parts of glacial acetic acid, 0.1 mol/ L of sodium hydroxide solution 4~10 parts, glycerin 2~5 parts, emulsifier 0.02~0.04 parts, white liquid silica gel 900~1100 parts. 2. a kind of aseptic testing culture bottle according to claim 1, is characterized in that, described white liquid silica gel comprises liquid silica gel E600A and liquid silica gel E600B that mass ratio is 1:0.8～1.2. 3. a kind of aseptic testing culture bottle according to claim 1 and 2 is characterized in that, described emulsifying agent is Tween 80. 4. a kind of aseptic detection culture bottle according to claim 1, is characterized in that, described culture medium is aseptic tryptone soy peptone liquid culture medium or aseptic thioglycolate fluid culture medium; When the culture medium is a sterile thioglycolate fluid medium, the bottle body is filled with nitrogen gas as a protective gas. 5. a kind of aseptic detection culture bottle according to claim 1 is characterized in that, the ratio of the addition amount of bromothymol blue in the described chromaticity sensor and antibiotic adsorption resin and culture medium is 0.01～0.1g : 1~2g: 30mL. 6. a kind of aseptic testing culture bottle according to claim 1 is characterized in that, described bottle body adopts PC plastic bottle. 7. a preparation method of the sterile detection culture bottle as described in any one of claims 1～6, is characterized in that, comprises the steps: (1) Prepare silica gel dye solution; (2) Preparation of chromaticity sensor: inject silica gel dye solution into the bottle, vacuumize and defoam, dry and sterilize to obtain a chromaticity sensor; (3) Inject sterile antibiotic adsorption resin and sterile culture medium into the bottle; (4) The bottle body is sealed with a cap, and the culture bottle is vacuum-packed; (5) Pre-culture. 8. The preparation method of a sterile testing culture bottle according to claim 7, wherein the preparation method of the silica gel dye solution in step (1) is: adding bromothymol blue to sodium hydroxide solution and water, After stirring and dissolving, adding glacial acetic acid, then adding glycerin and an emulsifier, and stirring uniformly to obtain a dye liquor; mixing the dye liquor with white liquid silica gel, and stirring uniformly to obtain the silica gel dye liquor. 9. The preparation method of a sterile testing culture bottle according to claim 7, wherein step (3) and step (4) are carried out under aseptic conditions; the culture medium is aseptic sulfur In the case of glycolate fluid medium, the bottle is filled with nitrogen after the medium is injected, and then the cap is sealed. 10 . The preparation method of a sterile testing culture bottle according to claim 7 , wherein the pre-cultivation time in step (5) is 14-15 days; the culture medium is sterile tryptone soy liquid. 11 . When the medium is a medium, the pre-culture temperature is 20-24°C; when the medium is a sterile thioglycolate fluid medium, the pre-culture temperature is 31-35°C.

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