Top-speed ethylene oxide biological indicator and preparation method thereof

Abstract

The invention provides a top-speed ethylene oxide biological indicator, which comprises a biological indicator spore and a recovery medium, the recovery medium comprises 20-25 g / L of a composite carbon source, 2-3 g / L of a composite germinating agent, 8-12 g / L of a composite nitrogen source, 1.5-2.5 g / L of a buffer system, 1-2 ml / L of a color change indicator, 0.5-1 g / L of a substrate, and water as a solvent; the composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source. Compared with the prior art, the top-speed type ethylene oxide biological indicator provided by the invention adopts the recovery culture medium defined by specific components with specific content to realize overall better interaction, and the recovery rate of the ethylene oxide biological indicator is improved through the specific recovery culture medium formula of the ethylene oxide biological indicator. Under the premise of not changing the D value and viable bacteria quantity, the spores can rapidly germinate, and the culture time is shortened to 1 hour, so that the release time of sterilized articles is greatly shortened, the turnover efficiency of the sterilized articles is improved, and the treatment time is won for more patients.

Description

Technical Field

[0001] The present invention relates to the technical field of low-temperature disinfection of medical devices, and more specifically, to an extremely rapid ethylene oxide biological indicator and a preparation method thereof. Background Art

[0002] Sterility assurance of medical devices is the most basic requirement for patients' life and health. The results of sterilization biological monitoring are the most reliable indicators for judging whether medical devices are sterilized. Ethylene oxide can kill various microorganisms, including bacterial propagules, spores, viruses and fungal spores, and is a broad-spectrum sterilant. It is generally believed that it can undergo non-specific alkylation with microbial proteins, DNA and RNA. More importantly, the bactericidal effect of ethylene oxide is irreversible. The 17th edition of the United States Pharmacopoeia first recorded the use of spores as biological monitoring indicators for ethylene oxide and heat sterilization, and the academic term biological indicator appeared. In the 21st edition, biological indicators for various sterilization factors are fully standardized, including the ATCC9372 strain of Bacillus subtilis var. niger spores used for monitoring the sterilization effect of ethylene oxide. The 2017 edition of the "Technical Specifications for Disinfection" of the former Ministry of Health of my country also made clear provisions.

[0003] Biological indicators are a special type of live microbial products that can be used to confirm the performance of sterilization equipment, verify sterilization procedures, monitor the sterilization effect of production processes, etc. Ethylene oxide can be used to sterilize items that are not resistant to high temperatures or moisture, and has the advantages of broad-spectrum sterilization and strong penetration. Moreover, since it kills microorganisms by using the alkylation principle rather than the oxidation process, it causes very little damage to items and has a very wide range of uses for sterilizing heat-sensitive precision instruments. It can be said that ethylene oxide has been, will continue to be, and will continue to be a major low-temperature sterilization method.

[0004] At present, the incubation time of biological indicators used for ethylene oxide sterilization effect detection is generally about 3 hours or 48 hours. For example, patent CN202223465093.4 discloses an ethylene oxide sterilization biological challenge package, which replaces the traditional packaging method of the existing ethylene oxide sterilization biological indicator by combining a plastic shell with a sealing film to form a long channel structure biological challenge package, making it easier to operate and saving time; but the biological indicator contained in the biological challenge package requires a culture time of 3 hours, which cannot be further shortened. It can be seen that there is no ethylene oxide biological indicator in the prior art that can shorten the culture time to 1 hour. A longer culture time will reduce the turnover efficiency of medical equipment, increase the material cost of the hospital, and even delay the rescue time of emergency patients. At the same time, because the sources of carbon and nitrogen sources in the traditional formula are relatively single and will be affected by different environments, simply increasing or decreasing the amount of carbon and nitrogen sources will reduce the stability of the culture medium, and even cause a color reversion (false negative) phenomenon, which will affect the hospital's result interpretation and the normal use of sterilized items, causing economic losses. Summary of the invention

[0005] In view of this, the object of the present invention is to provide an ultra-rapid ethylene oxide biological indicator and a preparation method thereof. The ultra-rapid ethylene oxide biological indicator provided by the present invention can greatly shorten the release time of sterilized items, improve the turnover efficiency of sterilized items, and gain treatment time for more patients.

[0006] The present invention provides an extremely fast ethylene oxide biological indicator, comprising biological indicator spores and a recovery culture medium, wherein the recovery culture medium comprises:

[0007] Composite carbon source 20~25g / L, composite germination agent 2~3g / L, composite nitrogen source 8~12g / L, buffer system 1.5~2.5g / L, color indicator 1~2ml / L, substrate 0.5~1g / L, solvent is water;

[0008] The composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; and the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source.

[0009] Preferably, the biological indicator spores are Bacillus subtilis var. niger ATCC9372 spores.

[0010] Preferably, the mass ratio of glucose, starch, ascorbic acid and cottonseed oil in the composite carbon source is 1:0.5:0.4:0.3.

[0011] Preferably, the composite germination agent consists of L-asparagine, L-cysteine ​​and calcium 2,6-pyridinedicarboxylate.

[0012] Preferably, the mass ratio of L-asparagine, L-cysteine ​​and calcium 2,6-pyridinedicarboxylate in the composite germination agent is 3:1:1.

[0013] Preferably, the composite nitrogen source consists of yeast extract, soy peptone and ammonium sulfate.

[0014] Preferably, the mass ratio of yeast extract, soy peptone and ammonium sulfate in the composite nitrogen source is 1.5:2.5:1.

[0015] Preferably, the buffer system is composed of potassium dihydrogen phosphate and disodium hydrogen phosphate, so that the pH value of the recovery medium is 7.4.

[0016] The color change indicator is 2% bromothymol blue solution;

[0017] The substrate is 4-MUG.

[0018] Preferably, the formula of the recovery medium is specifically:

[0019] Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L, solvent is water.

[0020] The present invention also provides a method for preparing the ultra-fast ethylene oxide biological indicator described in the above technical solution, comprising the following steps:

[0021] The water temperature is raised to 70°C to 80°C, starch is added under stirring, and the temperature is lowered to 20°C to 30°C after dissolution; glucose, ascorbic acid, cottonseed oil, composite germination agent, composite nitrogen source and substrate are then added and stirred continuously; a buffer system and a color change indicator are then added for ultrasonic treatment to obtain a recovery medium;

[0022] The above recovery culture medium is sealed in an ampoule bottle and combined with biological indicator spores to obtain an ultra-rapid ethylene oxide biological indicator.

[0023] The invention provides an ultra-fast ethylene oxide biological indicator, comprising biological indicator spores and a recovery culture medium, wherein the recovery culture medium comprises: 20-25 g / L of a composite carbon source, 2-3 g / L of a composite germination agent, 8-12 g / L of a composite nitrogen source, 1.5-2.5 g / L of a buffer system, 1-2 ml / L of a color change indicator, 0.5-1 g / L of a substrate, and a solvent of water; the composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; and the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source. Compared with the prior art, the ultra-fast ethylene oxide biological indicator provided by the present invention adopts a recovery culture medium limited by a specific component at a specific content to achieve better overall interaction. Through the specific recovery culture medium formula of the above-mentioned ethylene oxide biological indicator, spores can be rapidly germinated without changing its D value (the time or dose required to inactivate 90% of the total number of test microorganisms under specified conditions) and the amount of viable bacteria (the number of actually recoverable colony-forming units or other suitable units), and the culture time can be shortened to 1 hour, thereby greatly shortening the release time of sterilized items, improving the turnover efficiency of sterilized items, and gaining treatment time for more patients.

[0024] In addition, the preparation method provided by the present invention has readily available raw materials, low cost, and easy storage, and the overall process steps are simple, the conditions are mild, and easy to control, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A flowchart of the operation steps of the ultra-fast ethylene oxide biological indicator provided by an embodiment of the present invention;

[0026] Figure 2 A schematic diagram of a reader result interpretation in an embodiment of the present invention;

[0027] Figure 3 A schematic diagram of the incubator result interpretation in an embodiment of the present invention;

[0028] Figure 4 Schematic diagram of the color reversal example in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The present invention provides an extremely fast ethylene oxide biological indicator, comprising biological indicator spores and a recovery culture medium, wherein the recovery culture medium comprises:

[0031] Composite carbon source 20~25g / L, composite germination agent 2~3g / L, composite nitrogen source 8~12g / L, buffer system 1.5~2.5g / L, color indicator 1~2ml / L, substrate 0.5~1g / L, solvent is water;

[0032] The composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; and the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source.

[0033] In the present invention, the ultra-rapid ethylene oxide biological indicator comprises biological indicator spores and a recovery medium, preferably consists of biological indicator spores and a recovery medium, and is obtained by a combination method well known to those skilled in the art; wherein the biological indicator spores are preferably Bacillus subtilis black variant ATCC9372 spores; the present invention adopts its adapted recovery medium formula, which can extremely shorten the spore recovery time.

[0034] In the present invention, the recovery medium comprises:

[0035] Composite carbon source 20~25g / L, composite germination agent 2~3g / L, composite nitrogen source 8~12g / L, buffer system 1.5~2.5g / L, color indicator 1~2ml / L, substrate 0.5~1g / L, solvent is water;

[0036] Preferably:

[0037] Composite carbon source 22g / L, composite germination agent 2.5g / L, composite nitrogen source 10g / L, buffer system 2.1979g / L, color change indicator 1.6ml / L, substrate 0.8735g / L, solvent is water.

[0038] In the present invention, the composite carbon source is composed of glucose, starch, ascorbic acid and cottonseed oil; the mass ratio of glucose, starch, ascorbic acid and cottonseed oil in the composite carbon source is preferably 1:0.5:0.4:0.3. The present invention has no special restrictions on the sources of the glucose, starch, ascorbic acid and cottonseed oil, and commercially available products well known to those skilled in the art can be used.

[0039] In the present invention, the composite germination agent is preferably composed of L-asparagine, L-cysteine ​​and calcium 2,6-pyridinedicarboxylate; the mass ratio of L-asparagine, L-cysteine ​​and calcium 2,6-pyridinedicarboxylate in the composite germination agent is preferably 3:1:1. 2+ The source of DPA is not particularly limited, and a commercially available product known to those skilled in the art may be used.

[0040] In the present invention, the composite nitrogen source includes an organic nitrogen source and an inorganic nitrogen source, preferably composed of yeast extract (organic nitrogen source), soy peptone (organic nitrogen source) and ammonium sulfate (inorganic nitrogen source); the mass ratio of yeast extract, soy peptone and ammonium sulfate in the composite nitrogen source is preferably 1.5:2.5:1. The present invention has no special restrictions on the sources of the yeast extract, soy peptone and ammonium sulfate, and commercially available products known to those skilled in the art can be used.

[0041] In the present invention, the buffer system is preferably composed of potassium dihydrogen phosphate and disodium hydrogen phosphate, so that the pH value of the recovery medium is preferably 7.4; the color change indicator is preferably a 2% bromothymol blue solution; and the substrate is preferably 4-MUG. The present invention has no particular restrictions on the sources of the potassium dihydrogen phosphate, disodium hydrogen phosphate, 2% bromothymol blue solution and 4-MUG, and commercially available products known to those skilled in the art can be used.

[0042] In the present invention, the water is preferably deionized water well known to those skilled in the art.

[0043] In the present invention, the formula of the recovery medium is preferably as follows:

[0044] Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L, solvent is water.

[0045] At present, many hospitals use ethylene oxide sterilizers. However, how to ensure the sterilization effect of ethylene oxide sterilizers requires the use of sterilization effect monitoring products - ethylene oxide biological indicators. The ultra-fast ethylene oxide biological indicators can greatly shorten the release time of sterilized items, improve the turnover efficiency of sterilized items, and gain treatment time for more patients; the present invention aims to provide a new formula of ultra-fast ethylene oxide biological indicators. The ethylene oxide biological indicators on the market have a culture time of 48 hours or 3 hours under the specified culture conditions (a combination of growth medium and inoculation method used to promote microbial recovery, growth and (or) reproduction). During the peak period of the number of operating tables in the hospital, the pressure of instrument turnover is often too high, resulting in the postponement of the operation time. Based on a large number of experiments, the present invention found that the factor affecting the culture time of ethylene oxide biological indicators is mainly the composition of the recovery culture medium. On this basis, the present invention takes shortening the sterilization monitoring process time as the starting point, and obtains an ultra-fast ethylene oxide biological indicator with a culture time of 1 hour through an innovative formula.

[0046] On the one hand, the present invention constructs a composite multi-carbon source structure, which improves the recovery speed of spores while avoiding the problem of color reversion. During the recovery process, spores use carbon sources to produce acidic metabolites, which reduces the pH value of the culture medium, but after the carbon source is exhausted, they also use nitrogen sources to produce alkaline metabolites, which can make the pH value rise again, affect color changes, and ultimately affect the interpretation of the results of biological indicator culture; and the composite multi-carbon source species defined in the present invention, sugars as the first carbon source, glucose as a fast-acting carbon source, so that spores can recover quickly; starch as a slow-acting carbon source, continuous carbon supply, prevent spores from producing too many alkaline metabolites to affect the results; cottonseed oil as the second carbon source, grease can also play a role in defoaming in the culture medium, ensure that the spores are fully in contact with the recovery culture medium, and at the same time, the utilization rate of cottonseed oil by Bacillus subtilis black variant ATCC9372 spores is high, which can greatly improve the speed of spore recovery. However, due to the introduction of oil, the oxygen demand in the culture medium increases, and the oxygen consumption increases. If there is too much organic acid during the hypoxic process, the pH value of the culture medium will decrease, affecting the interpretation of the results; if there is sufficient oxygen, the possibility of oil spoilage will increase, and peroxides will be produced. Peroxides will produce trace amounts of hydroxyl radicals (·OH) under the catalysis of light. Hydroxyl radicals have an antibacterial effect and will hinder the recovery of spores. Therefore, the third carbon source, ascorbic acid, is introduced. Ascorbic acid has a certain reducing property. It can inhibit the production of peroxides while supplying carbon. When the carbon source utilization is close to the end, the pH value of the culture medium is maintained stable to prevent the culture medium from recoloring due to the increase of alkaline metabolites.

[0047] On the other hand, the present invention also constructs a binary composite nitrogen source structure, including an organic nitrogen source and an inorganic nitrogen source; the organic nitrogen source and the inorganic nitrogen source are two pathways for spore absorption and recovery, and the composite nitrogen source increases the recovery speed, enhances the stability of the culture medium, and avoids the problem of color reversion.

[0048] Compared with the prior art (the culture time of the existing ethylene oxide sterilization biological indicators on the market is generally about 3 hours or 48 hours; there are almost no reports on ethylene oxide sterilization 1-hour biological indicators), the ethylene oxide 1-hour ultra-fast biological indicator of the present invention can greatly shorten the release time of sterilized items, improve the turnover efficiency of sterilized items, and gain treatment time for more patients. At the same time, it greatly improves the stability of the culture solution. The newly constructed diversified composite carbon source and composite nitrogen source can shorten the culture time and completely solve the problem of indicator color reversion (false negative).

[0049] The present invention also provides a method for preparing the ultra-fast ethylene oxide biological indicator described in the above technical solution, comprising the following steps:

[0050] The water temperature is raised to 70°C to 80°C, starch is added under stirring, and the temperature is lowered to 20°C to 30°C after dissolution; glucose, ascorbic acid, cottonseed oil, composite germination agent, composite nitrogen source and substrate are then added and stirred continuously; a buffer system and a color change indicator are then added for ultrasonic treatment to obtain a recovery medium;

[0051] The above recovery culture medium is sealed in an ampoule bottle and combined with biological indicator spores to obtain an ultra-rapid ethylene oxide biological indicator.

[0052] In the present invention, the raw material components used in the preparation method are the same as those in the above technical solution and will not be repeated here.

[0053] In the present invention, the stirring speed of the stirring condition is preferably 100 rpm to 500 rpm, more preferably 300 rpm.

[0054] In the present invention, the dissolution method is preferably ultrasound, and the dissolution time is preferably 5 min to 15 min.

[0055] In the present invention, the ultrasonic treatment time is preferably 5 min to 15 min, more preferably 10 min.

[0056] The preparation method provided by the present invention has readily available raw materials, low cost, and is easy to store, and the overall process steps are simple, the conditions are mild, and are easy to control, and has broad application prospects.

[0057] The invention provides an ultra-fast ethylene oxide biological indicator, comprising biological indicator spores and a recovery culture medium, wherein the recovery culture medium comprises: 20-25 g / L of a composite carbon source, 2-3 g / L of a composite germination agent, 8-12 g / L of a composite nitrogen source, 1.5-2.5 g / L of a buffer system, 1-2 ml / L of a color change indicator, 0.5-1 g / L of a substrate, and a solvent of water; the composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; and the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source. Compared with the prior art, the ultra-fast ethylene oxide biological indicator provided by the present invention adopts a recovery culture medium limited by a specific component at a specific content to achieve better overall interaction. Through the specific recovery culture medium formula of the above-mentioned ethylene oxide biological indicator, spores can be rapidly germinated without changing its D value (the time or dose required to inactivate 90% of the total number of test microorganisms under specified conditions) and the amount of viable bacteria (the number of actually recoverable colony-forming units or other suitable units), and the culture time can be shortened to 1 hour, thereby greatly shortening the release time of sterilized items, improving the turnover efficiency of sterilized items, and gaining treatment time for more patients.

[0058] In addition, the preparation method provided by the present invention has readily available raw materials, low cost, and easy storage, and the overall process steps are simple, the conditions are mild, and easy to control, and has broad application prospects.

[0059] In order to further illustrate the present invention, the following examples are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available.

[0060] Example

[0061] The biological indicator spores provided by the present invention are Bacillus subtilis black variant ATCC9372 spores, and the recovery culture medium formula adapted thereto is used, which is specifically as follows:

[0062] Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0063] Preparation process: First, 1L of deionized water was heated to 75°C, and 5g of soluble starch was added. The soluble starch was easily dissolved at this temperature. Then, ultrasonic treatment was performed for 10 minutes, and the temperature was lowered to room temperature. Then, 10g of glucose, 4g of ascorbic acid, 3g of cottonseed oil, 1.5g of L-asparagine, 0.5g of L-cysteine, and calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) 0.5g, yeast extract 3g, soy peptone 5g, ammonium sulfate 2g, substrate 4-MUG 0.8735g; then add potassium dihydrogen phosphate 0.2245g, disodium hydrogen phosphate 1.9734g to form a buffer system; then add 2% bromothymol blue solution 1.6ml; finally ultrasonic for 10min, the final pH value of the prepared solution will be fixed at 7.4, and the pH value is verified with a pH detector. The stirring state should be maintained throughout the process except for the ultrasonic step, and the speed is 300rpm.

[0064] The culture medium was then sealed in an ampoule with a capacity of 0.45 mL.

[0065] Culture results determination:

[0066] 1h culture result: Xinhua brand reader JS-1006-S.

[0067] 3h culture result: Xinhua brand reader KS-0303-C.

[0068] 24h culture results: constant temperature incubator (37℃).

[0069] For operation steps, see Figure 1shown.

[0070] Step 1: Press the cover: press the cover of the rapid biological indicator;

[0071] Step 2: Use a special clamp to break the ampoule inside the rapid biological indicator;

[0072] Step 3: Shake: Pinch the upper cover of the rapid biological indicator and shake it 3-4 times to ensure that the bacterial sheet is moistened;

[0073] Step 4 Culture: The sterilization group and the positive control group are simultaneously inserted into the ethylene oxide biological reader culture wells, and the results are read within the corresponding culture time.

[0074] Reader result interpretation see Figure 2 As shown; among them: the number keys represent the corresponding culture well numbers; the yellow bar line and time represent the countdown, and the initial value is 180 minutes; the icon in the lower left corner indicates that the test is in progress; the plus icon in the middle indicates a positive result; the minus icon in the lower right corner indicates a negative result.

[0075] Incubator results interpretation Figure 3 As shown; observe the color to judge, yellow is positive and blue is negative.

[0076] Training reference: GB18281.1-2015 Part 1: General principles; no further details will be given here.

[0077] Experimental results:

[0078] (1) During the experiment, it was found that when the other components of the culture medium remained unchanged, 10 g / L glucose, 5 g / L starch, 4 g / L ascorbic acid, and 3 g / L cottonseed oil formed a stable system, which greatly shortened the culture time.

[0079] This experiment consists of three groups of comparative examples and one group of embodiments. Please refer to the above records for the embodiments.

[0080] Experiment 1: Proportional formula:

[0081] ① Glucose 30g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0082] ② Glucose 10g / L, starch 10g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate (Ca2+ -DPA) 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0083] ③ Glucose 10g / L, starch 6g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0084] The culture operation and environment are as described above.

[0085] (2) During the experiment, it was found that in the germination agent formula (L-asparagine, L-cysteine, calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) can quickly induce spore germination and meet the time requirements of the ultra-rapid ethylene oxide biological indicator.

[0086] This experiment is a group of comparative examples and a group of embodiments, and the embodiments are the same as the previous experiment.

[0087] Experiment 2 comparative formula:

[0088] ① Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0089] The culture operation and environment were the same as above.

[0090] (3) During the experiment, it was found that when the other components of the culture medium remained unchanged, 3 g / L yeast extract, 5 g / L soy peptone, and 2 g / L ammonium sulfate formed a stable system that greatly shortened the culture time.

[0091] The experiment consists of two groups of comparative examples and one group of embodiments, and the embodiments are the same as the above two experiments.

[0092] Experiment 3 comparative formula:

[0093] ① Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, yeast extract 10g / L, potassium dihydrogen phosphate 0.2245g / L, sodium dihydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0094] ② Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) 0.5g / L, yeast extract 5g / L, soy peptone 5g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L.

[0095] The culture operation and environment were the same as above.

[0096] (4) Color reversion experiment:

[0097] See the color reversion example Figure 4 As shown: Starting from the upper part of the indicator, it changes from yellow to green and finally back to blue.

[0098] The above experiments and effect data are shown in Tables 1 to 3 below.

[0099] Table 1 Comparison of effect data of Experiment 1

[0100] Table 2 Comparison of effect data of Experiment 2

[0101] L-Asparagine, L-Cysteine, Calcium 2,6-pyridinedicarboxylate (Ca 2+ -DPA) is used as a germination agent to induce spore germination. The germination agent can quickly cross the spore coat and cortex to reach the surface of the spore inner membrane in a very short time after contacting the spore, interact with the receptor protein located on the spore inner membrane, transmit the germination signal, and then the spore begins to enter the germination stage. After testing, the germination agent is only an inducing factor. After the spore enters the germination stage, the germination agent loses its effect, and even if it is completely consumed, it will not affect the germination process of the spore.

[0102] Table 3 Comparison of effect data of Experiment 3

[0103] The present invention constructs a binary composite nitrogen source, with soy peptone and yeast extract as organic nitrogen sources and ammonium sulfate as inorganic nitrogen source. The nitrogen (trivalent) in the ammonium salt is at the same oxidation level as the nitrogen in the organic nitrogen compounds in the cells, and can be directly absorbed and utilized by the bacteria. After the ammonia nitrogen (ammonium sulfate) is absorbed by the spores, a trace amount of acidic free sulfate is produced, which can offset the effect of alkaline metabolites on the pH value of the culture medium to a certain extent, increase stability, and prevent the culture medium from recoloring.

[0104] In addition, potassium dihydrogen phosphate and disodium hydrogen phosphate form a buffer system with a pH of 7.4 to maintain the pH stability of the culture medium; 2% bromothymol blue solution is a color-changing indicator, and the presence or absence of bacterial colony growth can be determined by the color change of the culture medium; finally, the substrate 4-MUG needs to be added, and the indicator rapid reading result is based on the fluorescence reaction. The growth of spores produces corresponding enzymes, and enzymatic hydrolysis occurs to decompose the substrate and produce the fluorescent substance 4-MU, which is excited by the 365nm ultraviolet light of the indicator reader and emits fluorescence with a wavelength of 465nm, and the reader receives the judgment result.

[0105] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An ultra-rapid ethylene oxide biological indicator, comprising biological indicator spores and a recovery medium, characterized in that: The recovery medium comprises: Composite carbon source 20~25g / L, composite germination agent 2~3g / L, composite nitrogen source 8~12g / L, buffer system 1.5~2.5g / L, color indicator 1~2ml / L, substrate 0.5~1g / L, solvent is water; The composite carbon source consists of glucose, starch, ascorbic acid and cottonseed oil; and the composite nitrogen source comprises an organic nitrogen source and an inorganic nitrogen source.

2. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The biological indicator spores are Bacillus subtilis black variant ATCC9372 spores.

3. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The mass ratio of glucose, starch, ascorbic acid and cottonseed oil in the composite carbon source is 1:0.5:0.4:0.

3.

4. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The composite germination agent consists of L-asparagine, L-cysteine ​​and 2,6-pyridinedicarboxylic acid calcium.

5. The ultra-rapid ethylene oxide biological indicator according to claim 4, characterized in that: The mass ratio of L-asparagine, L-cysteine ​​and calcium 2,6-pyridinedicarboxylate in the composite germination agent is 3:1:

1.

6. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The composite nitrogen source consists of yeast extract, soybean peptone and ammonium sulfate.

7. The ultra-rapid ethylene oxide biological indicator according to claim 6, characterized in that: The mass ratio of yeast extract, soy peptone and ammonium sulfate in the composite nitrogen source is 1.5:2.5:

1.

8. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The buffer system consists of potassium dihydrogen phosphate and disodium hydrogen phosphate, and the pH value of the recovery medium is 7.4 The color change indicator is 2% bromothymol blue solution; The substrate is 4-MUG.

9. The ultra-rapid ethylene oxide biological indicator according to claim 1, characterized in that: The formula of the recovery medium is specifically: Glucose 10g / L, starch 5g / L, ascorbic acid 4g / L, cottonseed oil 3g / L, L-asparagine 1.5g / L, L-cysteine ​​0.5g / L, calcium 2,6-pyridinedicarboxylate 0.5g / L, yeast extract 3g / L, soy peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 0.2245g / L, disodium hydrogen phosphate 1.9734g / L, 2% bromothymol blue solution 1.6ml / L, substrate 4-MUG 0.8735g / L, solvent is water.

10. A method for preparing the ultra-rapid ethylene oxide biological indicator according to any one of claims 1 to 9, characterized in that: The following steps are involved: The water temperature is raised to 70°C to 80°C, starch is added under stirring, and the temperature is lowered to 20°C to 30°C after dissolution; glucose, ascorbic acid, cottonseed oil, composite germination agent, composite nitrogen source and substrate are then added and stirred continuously; a buffer system and a color change indicator are then added for ultrasonic treatment to obtain a recovery medium; The above recovery culture medium is sealed in an ampoule bottle and combined with biological indicator spores to obtain an ultra-rapid ethylene oxide biological indicator.

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