CN109097433A - Content of microorganisms detection method and application - Google Patents
Content of microorganisms detection method and application Download PDFInfo
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Abstract
本发明提供了一种微生物含量检测方法及应用，涉及微生物含量检测技术领域，检测方法包括如下步骤：首先利用生物热力学和生物动力学的关系构建微生物检出时间和微生物浓度的标准曲线；然后再测定未知浓度微生物溶液的微生物检出时间，根据微生物的检出时间，计算得到未知浓度微生物溶液中的微生物含量，改善现有平板菌落计数法检查周期长，操作繁琐，无法快速得到检测结果的技术问题，本发明提供的微生物含量检测方法，工艺简单，操作方便，检测周期短，精度高，在15h内即可检测出微生物含量，能够提高检查效率，节约时间和成本，为后续实时、在线、快速检测提供了重要支撑，也为食品和/或药品中微生物含量检测提供了一种新途径。
The invention provides a microbial content detection method and its application, and relates to the technical field of microbial content detection. The detection method includes the following steps: firstly, using the relationship between biothermodynamics and biokinetics to construct a standard curve of microbial detection time and microbial concentration; and then Measure the microbial detection time of the microbial solution with unknown concentration, calculate the microbial content in the microbial solution with unknown concentration according to the microbial detection time, improve the existing plate colony counting method, which has a long inspection period, cumbersome operation, and cannot quickly obtain the detection result. Problem, the microbial content detection method provided by the present invention has simple process, convenient operation, short detection cycle, high precision, can detect microbial content within 15 hours, can improve inspection efficiency, save time and cost, and provide real-time, online, Rapid detection provides important support and also provides a new way for the detection of microbial content in food and/or pharmaceuticals.
Description
Technical field
The present invention relates to content of microorganisms detection technique fields, more particularly, to a kind of content of microorganisms detection method and answer
With.
Background technique
Content of microorganisms detects the essential items for inspection for being to ensure that drug quality and drug safety, and promotes drug quality and comment control
Level reduces the important means that clinical adverse occurs.Currently, pharmaceutical factory generally uses the method for plate culture count to detect drug
In content of microorganisms, but the method for plate culture count inspection cycle is long (reaching 3-5 days), cumbersome, can not quickly be examined
The continuous production requirement as a result, especially for the following enterprise is surveyed, even more emphasizes that real-time online detects, therefore, develops micro- life
The research and development of object method for quickly detecting contents has important practical significance.
In view of this, the present invention is specifically proposed.
Summary of the invention
One of the objects of the present invention is to provide a kind of content of microorganisms detection methods, to improve existing plate count
The technical issues of method inspection cycle is long, cumbersome, can not quickly obtain testing result.
Content of microorganisms detection method provided by the invention, includes the following steps:
(a) mark of biothermodynamics and biokinetic relationship building microorganism detection time and microorganism concn is utilized
Directrix curve；
Construct the standard curve of microorganism detection time and microorganism concn；
(b) the microorganism detection time for measuring unknown concentration microbial solution is calculated according to the detection time of microorganism
To the content of microorganisms in unknown concentration microbial solution.
Further, in step (a), the standard curve of microorganism detection time and microorganism concn is constructed, including such as
Lower step:
(a1) it provides the microbial solution of various concentration and blank solution is cultivated respectively；
(a2) various concentration microbial solution and blank solution are measured respectively in the thermal power of different incubation times, when micro- life
When the thermal power of object solution is greater than the power of blank solution, then determine that microorganism detects, and detect using this time as microorganism
Time；
(a3) it according to the relationship of microorganism detection time and microorganism concn, constructs microorganism detection time and microorganism is dense
Scale directrix curve.
Further, in step (b), the microorganism detection time of unknown concentration microbial solution is measured, including as follows
Step:
(b1) unknown concentration microbial solution is provided to be cultivated respectively；
(b2) unknown concentration microbial solution is measured in the thermal power of different incubation times, when unknown concentration microbial solution
Thermal power when being greater than the power of blank solution, then determine that microorganism detects, and it is molten using this time as unknown concentration microorganism
The detection time of liquid.
Further, when the thermal power of microbial solution and the thermal power difference of blank solution are not less than 10 μ W, determine
Microorganism detection.
Further, the cultivation temperature of microbial solution is 25-35 DEG C, preferably 30 DEG C.
Further, in step (a1), respectively provide concentration be 10cfu/mL, 100cfu/mL, 1000cfu/mL,
The microbial solution of 10000cfu/mL and 100000cfu/mL.
Further, using the thermal power of thermal activities micro-calorimeter measurement microbial solution
Further, using nutrient broth medium culture microbial solution.
Further, microorganism is in staphylococcus aureus, escherichia coli, Candida albicans and aspergillus niger spore
At least one.
The second object of the present invention is to provide the detection method of content of microorganisms in drug and/or microbe content in food
Application in detection.
Content of microorganisms detection method provided by the invention is detected using biothermodynamics with biokinetic relationship micro-
Biological content, simple process is easy to operate, and detection cycle is short, and precision is high, can be detected out content of microorganisms in 15h, can
It improves and checks efficiency, save time and cost, provide a kind of new way for content of microorganisms detection in food and/or drug.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the thermal power spectrogram that the microbial solution for the various concentration that embodiment 1 provides changes with incubation time；
Fig. 2 is the standard curve of embodiment 1 the microorganism detection time provided and microbial solution concentration；
Fig. 3 be verify example 2 in embodiment 1-3 provide Radix Notoginseng powder detection liquor and blank solution difference incubation time and
The curve of thermal power.
Specific embodiment
Technical solution of the present invention will be clearly and completely described below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, shall fall within the protection scope of the present invention.
According to an aspect of the present invention, the present invention provides a kind of content of microorganisms detection method, include the following steps:
(a) mark of biothermodynamics and biokinetic relationship building microorganism detection time and microorganism concn is utilized
Directrix curve；
(b) the microorganism detection time for measuring unknown concentration microbial solution is calculated according to the detection time of microorganism
To the content of microorganisms in unknown concentration microbial solution.
Content of microorganisms detection method provided by the invention is detected using biothermodynamics with biokinetic relationship micro-
Biological content, simple process is easy to operate, and detection cycle is short, and precision is high, can be detected out content of microorganisms in 15h,
Content of microorganisms can be quickly measured, provides a kind of new way for the content of microorganisms detection in food and/or drug.
In the preferred embodiment of the present invention, it in step (a), constructs microorganism detection time and microorganism is dense
The standard curve of degree, includes the following steps:
(a1) it provides the microbial solution of various concentration and blank solution is cultivated respectively；
(a2) various concentration microbial solution and blank solution are measured respectively in the thermal power of different incubation times, when micro- life
When the thermal power of object solution is greater than the power of blank solution, then determine that microorganism detects, and detect using this time as microorganism
Time；
(a3) it according to the relationship of microorganism detection time and microorganism concn, constructs microorganism detection time and microorganism is dense
Scale directrix curve.
In the preferred embodiment of the present invention, by measurement various concentration microbial solution and blank solution in different cultures
The thermal power of time draws the spectrogram of thermal power and incubation time, then compare the thermal power of certain concentration microbial solution with
The thermal power of blank solution, when certain concentration microbial solution thermal power be greater than blank solution thermal power, then determine the training
Supporting the time is microorganism detection time, then draws microorganism inspection according to the detection time of certain concentration microorganism and certain concentration
Time and microorganism concn standard curve out.
In the preferred embodiment of the present invention, in step (b), the microorganism inspection of measurement unknown concentration microorganism
Time out includes the following steps:
(b1) unknown concentration microbial solution is provided to be cultivated respectively；
(b2) unknown concentration microbial solution is measured in the thermal power of different incubation times, when unknown concentration microbial solution
Thermal power when being greater than the power of blank solution, then determine that microorganism detects, and it is molten using this time as unknown concentration microorganism
The detection time of liquid.
Then the thermal power for measuring unknown concentration microbial solution difference incubation time again trains it with blank solution difference
The thermal power for supporting the time compares, when unknown concentration microorganism thermal power is greater than the thermal power of blank solution, then when determining this
Between be unknown concentration microbial solution microorganism detection time, then according to the mark of microorganism detection time and microorganism concn
The content of microorganisms in unknown concentration microbial solution is calculated in directrix curve.
In the preferred embodiment of the present invention, when the power difference of the thermal power of microbial solution and blank solution
When not less than 10 μ W, microorganism detection is determined.
Since there are certain detection errors for thermal power measurement, it will test error and be set as 10 μ W, be more advantageous to just
Really determine microorganism detection time.
In the preferred embodiment of the present invention, bent in the standard of building microorganism detection time and microorganism concn
The thermal power of the microbial solution of various concentration is measured when line and measures the thermal power of unknown concentration microbial solution by 10 μ W
It is set as detection error, to guarantee the accuracy of testing result.
In the preferred embodiment of the present invention, the cultivation temperature of microbial solution is 25-35 DEG C.
In the preferred embodiment of the present invention, the typical but non-limiting cultivation temperature of microbial solution is for example
25,26,27,28,29,30,31,32,33,34 or 35 DEG C.
By the way that the cultivation temperature of microbial solution is set as 25-35 DEG C, it is more advantageous to the growth of microorganism.
In the preferred embodiment of the present invention, in step (a1), respectively provide concentration be 10cfu/mL,
The microbial solution of 100cfu/mL, 1000cfu/mL, 10000cfu/mL and 100000cfu/mL.
By the way that when constructing the standard curve of microorganism detection time and microorganism concn, the concentration of microbial solution is divided
It is not set as 10cfu/mL, 100cfu/mL, 1000cfu/mL, 10000cfu/mL and 100000cfu/mL, to guarantee standard song
The accuracy of line.
In the preferred embodiment of the present invention, when instrument measurement microbial solution difference culture micro using thermal activities
Between thermal power.
By using the thermal power of thermal activities micro-calorimeter measurement microorganism difference incubation time, measurement result is more quasi-
Really, and microbial solution can be placed in the sense channel of the micro instrument of thermal activities and is cultivated, it is simpler and more direct more convenient.
In the preferred embodiment of the present invention, using nutrient broth medium culture microbial solution.
By using nutrient broth medium culture microbial solution, it is more advantageous to the life of microorganism in microbial solution
It is long, so as to effectively shorten detection cycle.
In the preferred embodiment of the present invention, microorganism is selected from staphylococcus aureus, escherichia coli, white
It is one or more of in candida albicans and aspergillus niger spore.
In present invention further optimization embodiment, microorganism is staphylococcus aureus, escherichia coli, white
When the mixture of candida albicans and aspergillus niger spore, the microbial total content being more advantageous in detection food and/or drug is more convenient
It is faster.
According to the second aspect of the invention, the present invention provides the detection method of mentioned microorganism content food and/
Or the application in drug in content of microorganisms detection.
Technical solution provided by the invention is further described below with reference to embodiment and comparative example.
Embodiment 1
The detection method for present embodiments providing content of microorganisms in a kind of Radix Notoginseng powder, includes the following steps:
(a) standard curve for constructing microorganism detection time and microorganism concn, specifically comprises the following steps:
(a1) various concentration microbial solution and blank solution are configured
(a11) prepare nutrient agar, nutrient broth medium and Rose Bengal Sodium agar medium
(1) preparation of nutrient agar includes the following steps: to weigh nutrient agar powder 33g, adds distilled water 1000mL,
Heating stirring dissolution, in 121 DEG C of high pressure 0.1Mpa sterilizing 30min, 48 DEG C of waters bath with thermostatic control heat preservations are spare.
(2) preparation of nutrient broth medium: nutrient meat powder 18g is weighed, distilled water 1000mL is added, heating stirring is molten
Solution, in 121 DEG C of high pressure sterilization 30min, 48 DEG C of water bath with thermostatic control heat preservations are spare.
(3) preparation of Rose Bengal Sodium agar medium: weighing Rose Bengal Sodium agar 31.5g, adds distilled water 1000mL, heating
Stirring and dissolving, in 121 DEG C of high pressure sterilization 30min, 48 DEG C of water bath with thermostatic control heat preservations are spare.
(4) pH7.0 sterile NaCl-peptone buffer agent is prepared: weighing pH7.0 sterile NaCl-peptone buffer agent
Powder 14.31g, is added distilled water 1000mL, heating stirring dissolution, and it is spare to make dilution by 121 DEG C of high pressure sterilization 30min.
(a12) prepare various concentration microbial solution and blank solution
(1) be inoculated with escherichia coli, staphylococcus aureus fresh cultured object into nutrient broth medium or nutrition
On agar medium, culture 18~for 24 hours；It is inoculated on the fresh cultured object to Rose Bengal Sodium agar medium of Candida albicans, training
Support 24~48h；On the fresh cultured object of inoculated aspergillus niger spore to sterile improvement Martin's culture medium, 24~48h is cultivated.Above-mentioned training
Supporting object and every 1mL is made containing bacterium number with 0.9% aseptic sodium chloride solution is 106-108The bacteria suspension of cfu.
(2) take it is above-mentioned by sterilizing nutrient broth medium 2mL, take respectively above-mentioned staphylococcus aureus bacteria suspension,
Escherichia coli bacteria suspension, Candida albicans bacteria suspension and aspergillus niger spore bacteria suspension are according to staphylococcus aureus, large intestine angstrom
The clump count of uncommon bacterium, Candida albicans and aspergillus niger spore, which is respectively that 1:1:1:1 is uniformly mixed, obtains plastc ring.
(3) being diluted to plastc ring containing bacterium number with 0.9% sterilizing sodium chloride solution is 101、102、103、104、
105The bacteria suspension of cfu/mL takes above-mentioned each concentration bacteria suspension mL to mix respectively with 1mL nutrient broth medium, obtains different dense
Spend microbial solution；
(4) it takes 0.9% sterilizing sodium chloride solution 1mL and 1mL nutrient broth medium to mix, obtains blank solution.
(a2) measurement various concentration microbial solution and blank solution difference incubation time thermal power
(a21) above-mentioned various concentration microbial solution and blank solution be respectively put into isothermal biology thermal activities microcalorimetry
In the sense channel of instrument, keeping temperature is 30 DEG C, detects above-mentioned various concentration microbial solution and blank solution difference respectively and trains
The thermal power for supporting the time, obtains various concentration microbial solution and blank solution in the thermal power spectrogram of different incubation times.
As shown in Figure 1, wherein the microbial solution that 1-5 curve respectively represents various concentration in figure changes with incubation time
Thermal power curve, the 6 thermal power curves changed with incubation time for blank solution, it will be seen from figure 1 that same concentration is micro-
Biological solution is with the extension of incubation time, and thermal power significantly improves, and various concentration microbial solution is in identical culture
In time, content of microorganisms is higher, and thermal power is higher, and the thermal power of blank solution is commonly with the extension of incubation time,
Its thermal power does not have significant change, this explanation in microbial solution content of microorganisms increase, thermal power is higher.
(a22) building microorganism detection time and microorganism concn standard curve
(1) since there are certain errors for detection, it will test error and be set as 10 μ W, when the thermal power of microbial solution
When being not less than 10 μ W with the thermal power difference of blank solution, microorganism detection is determined, incubation time at this time is detection time.
According to the pass of content of microorganisms in the detection time of various concentration microbial solution and various concentration microbial solution
Its corresponding concentration of the detection time of microorganism is done dose-effect relationship analysis, constructs microorganism detection time and microorganism by system
The standard curve of solution concentration.
As shown in Fig. 2, obtaining equation y=after the linear recurrence of the logarithm of microorganism detection time and microorganism concn
3.213x+149.11, y are microorganism detection time, and x is the logarithm of microorganism concn, the complex phase relationship after linear regression
Number R2=0.998.
(b) content of microorganisms in Radix Notoginseng powder is detected
(b1) Radix Notoginseng powder 10g is taken, Radix Notoginseng powder is diluted to Radix Notoginseng with 100mLpH7.0 sterile NaCl-peptone buffer agent
Powder test liquid takes Radix Notoginseng powder test liquid 1mL, is diluted with pH7.0 sterile NaCl-peptone buffer agent, obtains Radix Notoginseng powder detection
Liquid；
(b2) Radix Notoginseng powder detection liquid is placed in the sense channel of isothermal biology thermal activities micro-calorimeter, holding temperature is
30 DEG C, the thermal power of above-mentioned Radix Notoginseng powder detection liquor difference incubation time is detected, when obtaining Radix Notoginseng powder detection liquid difference culture
Between thermal power curve, the thermal power curve of the curve and blank solution is compared, when Radix Notoginseng powder detection liquid thermal power
When being not less than 10 μ W with the thermal power difference of blank solution, microorganism detection in Radix Notoginseng powder detection liquid is determined, when culture at this time
Between be Radix Notoginseng powder detection liquid microorganism detection time.
(c) the microorganism detection time of Radix Notoginseng powder detection liquid is substituted into the standard of microorganism detection time and microorganism concn
Curve, so that the microorganism concn in Radix Notoginseng powder detection be calculated.
In the present embodiment, the microorganism detection time of Radix Notoginseng powder detection liquid and microorganism concn numerical value are as shown in table 1.
Embodiment 2
A kind of detection method of content of microorganisms in Radix Notoginseng powder is present embodiments provided, the present embodiment and embodiment 1 are not
Be with place, used Radix Notoginseng powder detection liquid carries out non-thorough sterilization treatment, other steps and raw material with 1 phase of embodiment
Together.
Embodiment 3
A kind of detection method of content of microorganisms in Radix Notoginseng powder is present embodiments provided, the present embodiment and embodiment 1 are not
Be with place, used Radix Notoginseng powder detection liquid carries out thorough sterilization treatment, other steps and raw material with 1 phase of embodiment
Together.
Verify example 1
The Radix Notoginseng powder provided in embodiment 1-3 is detected into liquid and blank solution according to plate bacterium as defined in Chinese Pharmacopoeia respectively
It falls counting method and carries out limit test of microbe, the flat-plate bacterial colony for respectively obtaining the Radix Notoginseng powder detection liquid of embodiment 1-3 offer calculates
The microorganism concn in liquid is detected in method detection time and Radix Notoginseng powder, the results are shown in Table 1.
The Radix Notoginseng powder detection liquid and blank solution determination data table that 1 embodiment 1-3 of table is provided
Note: 1, the Radix Notoginseng powder detection liquid and blank solution that each embodiment provides are repeated three times, and are averaged；
2, p < 0.05 * compared with classic flat-plate method；
3, "-" indicates no data or is not detected.
As it can be seen from table 1 the Radix Notoginseng powder detection liquid that embodiment 1-3 is provided is carried out using detection method provided by the invention
The accuracy of detection, detection is suitable with flat-plate bacterial colony method, and detection cycle is shorter, and practicability is stronger, and sensitivity is higher (to be less than
10cfu/mL), the detection efficiency that can more effectively improve content of microorganisms in food and/or drug, saves time and cost, tool
Have broad application prospects.
Verify example 2
The Radix Notoginseng powder provided in embodiment 1-3 is detected into liquid respectively and blank solution is placed in the micro amount of isothermal biology thermal activities
In the sense channel of hot instrument, keeping temperature is 30 DEG C, when detecting above-mentioned Radix Notoginseng powder detection liquor and blank solution difference culture
Between thermal power, as a result as shown in Figure 3.
From figure 3, it can be seen that in identical incubation time, the thermal power for the Radix Notoginseng powder detection liquid that embodiment 1-3 is provided
For embodiment 1 > embodiment, 2 > embodiment, 3 > blank solution, this matches with actual conditions, this illustrates micro- life provided by the invention
Object detection method of content has good sensitivity and applicability.
Content of microorganisms detection method provided in this embodiment is also applied for the test of single culture content, works as test sample
When middle single culture content, detecting step is identical as the method that embodiment 1 provides, the difference is that, using single culture
Bacteria suspension replaces the mixed bacteria bacteria suspension in embodiment 1, and details are not described herein.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
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