CN111521631A - Method for evaluating quality consistency of donkey-hide gelatin - Google Patents
Method for evaluating quality consistency of donkey-hide gelatin Download PDFInfo
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Abstract
The invention discloses a method for evaluating the quality consistency of donkey-hide gelatin, which takes intestinal microorganisms as a model to analyze the associated biological thermal activity of a donkey-hide gelatin product, thereby establishing a donkey-hide gelatin quality biological evaluation method based on micro calorimetry analysis, realizing the donkey-hide gelatin quality evaluation purpose of the associated biological activity and providing technical support for evaluating the quality consistency of the donkey-hide gelatin. The method specifically comprises the following steps: firstly, establishing a stable and proper escherichia coli thermal spectrum curve model, carrying out similarity evaluation on an escherichia coli biological thermal activity spectrum according to an included angle cosine vector method, determining an available model and carrying out methodology investigation on the available model; secondly, determining a dose-effect curve and screening effective evaluation parameters by searching the administration concentration of the donkey-hide gelatin and investigating the overall influence of the donkey-hide gelatin on the biological thermal activity of the escherichia coli, and further determining the detection concentrations of different batches and carrying out methodology investigation on the detection concentrations; finally, fixing the detection conditions and determining the evaluation index, namely obtaining the method for evaluating the donkey-hide gelatin consistency.
Description
Technical Field
The invention relates to a detection technology of donkey-hide gelatin consistency, in particular to a method for evaluating donkey-hide gelatin quality consistency.
Background
The donkey-hide gelatin is solid gelatin prepared by decocting and concentrating dried or fresh skin of Equus asinm L, which is an equine animal, has sweet and mild taste, enters lung, liver and kidney channels, has the effects of enriching blood and nourishing yin, moistening dryness and stopping bleeding, and is widely applied in clinic. In recent years, with the expansion of market demand of donkey-hide gelatin and the increasing of donkey-hide gelatin and related products, the quality control of donkey-hide gelatin products by different manufacturers and different production places is difficult to unify; the clinical curative effect of the donkey-hide gelatin is seriously influenced by the frequent occurrence of false, false and genuine phenomena and secondary and good symptoms, and hidden dangers are buried when safe and reasonable medication of the donkey-hide gelatin is carried out. Therefore, establishing an effective quality evaluation method, especially an evaluation method aiming at the quality consistency of the donkey-hide gelatin, is an important problem to be solved urgently for the quality control of the donkey-hide gelatin.
The existing quality evaluation method mainly tends to identify the authenticity of the donkey-hide gelatin and determine the content of amino acid chemical components, but the donkey-hide gelatin as a traditional Chinese medicine has complex components and uncertain pharmacodynamic substances, and only uses partial chemical components as measurement indexes and is incomplete, so the establishment of the quality evaluation method related to the donkey-hide gelatin efficacy has indispensable significance for the quality evaluation of the donkey-hide gelatin. Biological evaluation is an important development direction of the quality control of the traditional Chinese medicine at present, and is particularly suitable for the traditional Chinese medicine with unclear or difficult-to-measure pharmacodynamic substances. The biological thermal activity fingerprint is a biological evaluation method for representing the overall quality characteristics of the traditional Chinese medicine, thermodynamic information is obtained through a micro calorimetric technology, so that a group of biological characteristic information or spectrums expressed by the action of the medicine on a biological system are comprehensively evaluated, and the biological thermal activity fingerprint has a good application prospect in the quality consistency evaluation of the traditional Chinese medicine. Therefore, on the basis of biological effect, the establishment of the biological heat activity fingerprint spectrum of the donkey-hide gelatin expresses and describes the effect of the donkey-hide gelatin on organisms to obtain the biological heat activity spectrum with the characteristics of the donkey-hide gelatin, and perhaps is a feasible way to establish a quality evaluation and control method of donkey-hide gelatin products.
However, most of the data analysis performed by the currently used thermal activity fingerprint is to evaluate the safety and effectiveness of the drug by using the detection time of the microorganism, the maximum heating power (Pi) and time (Ti) of each growth period, the growth index (ki), IC50, etc. as main indexes, the evaluation indexes are relatively single and one-sided, and it is difficult to comprehensively and rapidly evaluate the overall influence level of the sample to the microorganism model.
In conclusion, the method for detecting the quality of the donkey-hide gelatin, which can comprehensively and quantitatively evaluate the associated biological activity, is established, and has important significance for solving the problem that the existing evaluation means is difficult to reflect the consistency of the overall quality of the donkey-hide gelatin associated with the biological overall activity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for evaluating the quality consistency of donkey-hide gelatin, which can solve the problem that the existing evaluation means can not reflect the whole quality consistency of donkey-hide gelatin by the biological whole activity.
In order to achieve the aim, the invention provides a method for analyzing the associated biological thermal activity of the donkey-hide gelatin product for the first time by taking intestinal microorganisms as a biological model, exploring and establishing a donkey-hide gelatin quality biological evaluation method based on micro-calorimetry analysis, realizing the donkey-hide gelatin quality evaluation aim of the associated biological activity and providing technical support for evaluating the donkey-hide gelatin quality consistency. For the data analysis mode of the method, effective biological thermodynamic parameters obtained by origin9.0 are used for data processing, a calculation principal component analysis prediction calculation formula is obtained by combining a growth and metabolism thermogram curve of Escherichia coli according to the data distribution state in Principal Component Analysis (PCA), and characteristic values t1 and t 2 are obtained to evaluate the consistency of the characteristic values.
The method for evaluating the quality consistency of the donkey-hide gelatin provided by the invention comprises the following steps:
firstly, selecting more than two donkey-hide gelatin samples, dissolving the donkey-hide gelatin samples by culture medium solutions respectively to prepare donkey-hide gelatin sample solutions with the same mass concentration and dose-effect relationship, inoculating bacterial liquid into each donkey-hide gelatin sample solution, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of each donkey-hide gelatin sample; preparing a model sample containing the bacterial liquid with the concentration of the donkey-hide gelatin sample being 0mg/ml, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of each model sample;
further, extracting the biological thermodynamic parameters of each curve;
then, after the above-mentioned biological thermodynamic parameters of the described donkey-hide gelatin sample are normalized, and undergone the process of principal component analysis and calculation to obtain the characteristic values t1 and t 2 of biological thermal activity spectrum of every donkey-hide gelatin sample;
finally, combining with biological thermal activity spectrogram, using characteristic values t1 and t 2 as evaluation indexes to compare between multiple colla Corii Asini samples, and evaluating consistency of different colla Corii Asini samples.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the mass concentration of the donkey-hide gelatin sample solution with the dose-effect relationship is 0.625-20.0mg/ml, and optionally, the mass concentration of the donkey-hide gelatin sample solution is 5.0 mg/ml.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the specific step of inoculating the bacterial liquid into the donkey-hide gelatin sample solution is that the bacterial liquid is inoculated into a culture medium to form the bacterial liquid, then the bacterial liquid is diluted, and finally the bacterial liquid and the donkey-hide gelatin sample solution are mixed.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the strain is selected from escherichia coli (ATCC 25922).
In the method for evaluating the quality consistency of the donkey-hide gelatin, a culture medium selected from the bacterial liquid is an LB broth culture medium, and the relative OD value of the bacterial liquid is 0.3.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the bacterial liquid is diluted to 1 × 10 by equal proportion5And (3) multiplying, wherein the volume ratio of the diluted bacterium solution to the donkey-hide gelatin sample solution to be detected is 1:100, so that the donkey-hide gelatin sample solution and the Escherichia coli in the model sample have the same proportional dilution times.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the biological thermodynamic parameters are extracted by adopting origin9.0 software;
in the method for evaluating the quality consistency of the donkey-hide gelatin, the biological thermodynamic parameters comprise a first half-peak width F of a long-term life1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2As a parameter participating in principal component analysis.
In the method for evaluating the quality consistency of the donkey-hide gelatin, the principal component analysis of the biothermodynamic parameters is carried out in Simca-p14 software;
in the method for evaluating the quality consistency of the donkey-hide gelatin, the principal component analysis and prediction calculation formula is as follows:
Y＝{0.91,0.89,0.92,1.10,0.74,0.94,0.84,0.87,0.82,1.22}
P1＝{0.36,0.38,0.34,-0.29,0.58,0.18,0.20,0.03,0.26,-0.20}
P2＝{-0.16,-0.15,-0.14,0.08,-0.22,0.20,0.41,0.56,0.25,-0.56}
X＝{F1,LHW1,RHW1,Q1,AP1(％),LHW2,RHW2,F2,PE,EX2}
x is the biological thermodynamic parameter after normalization treatment, wherein the normalization treatment is the ratio of the biological thermodynamic parameter of the donkey-hide gelatin sample extracted by the origin9.0 software to the biological thermodynamic parameter of the model sample with the donkey-hide gelatin sample concentration of 0 mg/ml;
in the method for evaluating the quality consistency of the donkey-hide gelatin, the model sample and the donkey-hide gelatin sample have the same biological thermodynamic parameter acquisition process.
On the other hand, the invention provides a method for establishing a method for evaluating the quality consistency of donkey-hide gelatin, which comprises the following steps:
1) preparing initial bacterial liquid: the culture medium selected from the bacterial liquid is selected from LB broth culture medium, and the strain is selected from Escherichia coli (ATCC 25922);
2) and (3) investigating the concentration of the test bacterial liquid: taking the initial bacterial liquid prepared in the step 1), diluting a series of concentrations in an equal ratio, sealing respectively, shaking up, placing in a micro calorimeter for detection, and recording the energy change in the growth and metabolism process of the escherichia coli in a sealed state through the micro calorimeter to obtain a heat production power-time (P-t) curve; selecting a curve which has a stable plateau period, smooth line shape and obvious peak shape as a model group;
3) preparing test bacterial liquid: selecting the concentration of the test bacterial liquid as the specific dilution multiple of the initial bacterial liquid prepared in the step 1) according to the screening result of the step 2), so that the equal-ratio dilution multiple of the Escherichia coli in the mixed test bacterial liquid and the colla corii asini sample solution is the same as that of the model group;
4) preparation of test samples: preparing a donkey-hide gelatin sample solution with dose-effect relationship, weighing a donkey-hide gelatin sample, adding the donkey-hide gelatin sample into a culture medium, setting the final concentration of the donkey-hide gelatin sample to be more than two groups of 0.625mg/ml, 1.25mg/ml, 2.50mg/ml, 5.0mg/ml, 10.0mg/ml and 20.0mg/ml, namely an administration group, sterilizing the administration group and a model group of the donkey-hide gelatin sample with the concentration of 0mg/ml by high-pressure steam, and placing the administration group and the model group in a super clean bench to room temperature;
5) determining the administration concentration of the donkey-hide gelatin: adding the equi-specific diluted test bacterial liquid prepared in the step 3) into the donkey-hide gelatin samples with various concentrations prepared in the step 4), so that the equi-specific dilution multiple of the Escherichia coli in the test bacterial liquid is the same as that of the model group after the test bacterial liquid and the donkey-hide gelatin sample solution are mixed; sealing, shaking, detecting in a microcalorimeter, and recording heat generation power-time (P-t) curves of each group at each concentration;
6) screening evaluation parameters: analyzing heat production power-time (P-t) curve data of each group of concentrations by adopting origin9.0 software to obtain a biological thermodynamic parameter, and carrying out normalization treatment on the biological thermodynamic parameter;
the normalization processing is the ratio of the biomechanical parameters of the donkey-hide gelatin sample extracted by origin9.0 software to the biomechanical parameters of the donkey-hide gelatin sample with the concentration of 0mg/ml, and the data after normalization processing can reduce the error among groups, so that the method has universality.
Carrying out principal component analysis on the normalized biothermodynamic parameters by using Simca-P software; then selecting a donkey-hide gelatin sample with the concentration which has the maximum change of the principal component and a model group to respectively carry out partial least squares-differential analysis (OPLS-DA), and screening parameters which meet the requirements that VIP values are greater than 1, | P (corr) | is greater than or equal to 0.5 in the partial least squares-differential analysis and have a dose-effect relationship as evaluation parameters;
7) construction of evaluation indexes: characterizing the evaluation parameters screened in the step 6) in main component analysis, and selecting the concentration of an administration group with the largest influence on growth and metabolism of escherichia coli as the administration concentration for evaluating the quality consistency of the donkey-hide gelatin if the difference between the administration group and a model group presents a better dose-effect relationship along with the increase of the concentration of the donkey-hide gelatin;
meanwhile, the dose-effect relationship shown in the principal component analysis is used as a prediction blueprint, the evaluation parameters screened out in the step 6) are used as a fixed parameter combination and are converted into a formula, and the characteristic values t1 and t 2 are obtained, namely the consistency evaluation index of the donkey-hide gelatin quality.
In the establishing method of the method for evaluating the quality consistency of the donkey-hide gelatin, the OD600 value of the initial bacterial liquid is 0.3; the specific operation process is that the strain is inoculated into a culture medium to form initial bacterial liquid, and the initial bacterial liquid is shaken under the condition of 37 ℃ until the relative value of OD600 is 0.3.
In the establishing method of the method for evaluating the quality consistency of the donkey-hide gelatin, the series of concentrations of the equal-ratio dilution in the step 2) are equal-ratio dilution 1 × 101、1×102、1×103、1×104、1×105、1×106、1×107Seven concentrations.
In the method for establishing the method for evaluating the quality consistency of donkey-hide gelatin provided by the invention, the concentrations with the maximum change of the principal components are t 2: 2.50mg/ml and t 1: 20.0 mg/ml.
In the establishing method of the method for evaluating the quality consistency of the donkey-hide gelatin, the evaluation parameters screened in the step 6) are as follows: first life-time half-peak width F1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2。
Compared with the prior art, the method comprises the steps of generation of an evaluation system, screening of evaluation parameters, construction of evaluation indexes, scientificity and reasonability of application combinations of the evaluation indexes and the like. (independent technical scheme + technical purpose and corresponding technical effect).
The method related to biological thermodynamics and the index screening means have important significance for detecting and evaluating the quality consistency of the donkey-hide gelatin related to biological activity, can be used as an evaluation mode for evaluating the consistency of donkey-hide gelatin related products and other animal gelatin medicines or traditional Chinese medicines with unclear material bases, and can be used for exploring by replacing other biological models.
The invention can achieve the following technical effects:
1. the detection of different donkey-hide gelatin samples shows that the donkey-hide gelatin has the function of intervening the growth and metabolism of escherichia coli in intestinal tracts.
Regular dose-effect changes of donkey-hide gelatin with different mass concentrations on growth and metabolism of escherichia coli can be observed in the same batch of donkey-hide gelatin.
2. The detection of a plurality of donkey-hide gelatin samples of the same manufacturer shows that the donkey-hide gelatin products of the same manufacturer are relatively similar, which indicates that the method can be used for inspecting the quality consistency of the donkey-hide gelatin of the same manufacturer.
3. The detection of donkey-hide gelatin samples of different manufacturers shows that the donkey-hide gelatin samples of different manufacturers have certain differences in thermal activity maps, which indicates that the method can be used for inspecting the quality consistency of commercially available donkey-hide gelatin and can also be used for distinguishing part of different manufacturers.
4. Through the evaluation and investigation of the quality consistency of the finished product of the donkey-hide gelatin decoction pieces, aiming at the traditional Chinese medicine of donkey-hide gelatin which has wide raw material source, complicated production process, unclear material base and difficult quality control, compared with the existing relevant physical and chemical analysis means of donkey-hide gelatin, the method can correlate the biological activity, thereby more truly reflecting the effect of the donkey-hide gelatin on organisms and even human bodies; compared with the existing donkey-hide gelatin related pharmacological experiments, the method has the advantages of short time, less material consumption, high model stability and repeatability and the like, and is more suitable for the famous and precious traditional Chinese medicines such as donkey-hide gelatin.
5. The method can also be popularized and applied to other animal glue products and traditional Chinese medicines with unclear material bases.
6. The PCA principal component analysis model is established by adopting multi-parameter combination, so that the information of the biological thermal activity spectrum can be more comprehensively and specifically represented; the extracted data is normalized, so that errors inevitably from different groups in the experimental process can be eliminated to the greatest extent, and the method is more universal.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.
FIG. 1 shows the establishment of E.coli model and a methodology study.
Wherein A is a standard graph of a P-t curve of Escherichia coli; b is a P-t curve of the escherichia coli under different dilution times; c is an in-day repetition pattern of the E.coli P-t curve, and D is an in-day repetition pattern of the E.coli P-t curve. E and F are comparisons of the similarity of the in-day and the inter-day repeats, respectively, of the E.coli P-t curve.
FIG. 2 is a graph showing the thermodynamic changes of different concentrations of E.coli affecting the growth and metabolism of E.coli.
FIG. 3 is a graph showing the change of 10 normalized thermodynamic parameters on a P-t curve of different concentrations of E-gelatin affecting the growth and metabolism of Escherichia coli, including F1、LHW1、RHW1、Q1、AP1(％)、LHW2、RHW2、F2、PEAnd Ex2(n＝3)。
Compared to the control group,: p <0.05, x: p < 0.01.
FIG. 4 is a PCA analysis of 10 thermodynamic parameters on a P-t curve of 0mg/ml-20mg/ml E.coli affecting growth and metabolism.
Wherein, A is a score plot; b is a loading plot; c is the value of the principal component t1 in PCA; d is the value of the principal component t 2 in PCA.
FIG. 5 is a graph showing the effect of colla Corii Asini on growth and metabolism of Escherichia coli.
Wherein A, B, C respectively correspond to group A, group B and group C, and 12 batches of colla Corii Asini affects the P-t curve of growth and metabolism of Escherichia coli; d is a principal component analysis score chart of 12 batches of donkey-hide gelatin PCA; e and F are numerical graphs of characteristic values t1 and t 2 of A, B and C groups of samples respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.
The steps illustrated in the flow charts of the figures may be performed in a computer system such as a set of computer-executable instructions. Also, while a logical order is shown in the flow diagrams, in some cases, the steps shown or described may be performed in an order different than here.
In the embodiment of the invention, the method for evaluating the quality consistency of the donkey-hide gelatin comprises the following steps:
firstly, selecting more than two donkey-hide gelatin samples, dissolving the donkey-hide gelatin samples by culture medium solutions respectively to prepare donkey-hide gelatin sample solutions with the same mass concentration and dose-effect relationship, inoculating bacterial liquid into each donkey-hide gelatin sample solution, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of each donkey-hide gelatin sample; preparing a model sample containing the bacterial liquid with the concentration of the donkey-hide gelatin sample being 0mg/ml, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of each model sample;
further, extracting the biological thermodynamic parameters of each curve;
then, after the above-mentioned biological thermodynamic parameters of the described donkey-hide gelatin sample are normalized, and undergone the process of principal component analysis and calculation to obtain the characteristic values t1 and t 2 of biological thermal activity spectrum of every donkey-hide gelatin sample;
finally, combining with biological thermal activity spectrogram, using characteristic values t1 and t 2 as evaluation indexes to compare between multiple colla Corii Asini samples, and evaluating consistency of different colla Corii Asini samples.
In a specific embodiment of the present invention, the mass concentration of the donkey-hide gelatin sample solution with dose-effect relationship is 0.625-20.0mg/ml, and optionally, the mass concentration of the donkey-hide gelatin sample solution is 5.0 mg/ml.
In the specific embodiment of the invention, the specific step of inoculating the bacterial liquid into the donkey-hide gelatin sample solution is to inoculate the bacterial liquid into the culture medium to form the bacterial liquid, dilute the bacterial liquid, and finally mix the bacterial liquid with the donkey-hide gelatin sample solution.
In a particular embodiment of the invention, the bacterial species is selected from escherichia coli (ATCC 25922).
In a specific embodiment of the invention, the culture medium selected from the bacterial liquid is selected from LB broth culture medium, and the relative OD value of the bacterial liquid is 0.3.
In the specific embodiment of the invention, the bacterial liquid is diluted to 1 × 10 by diluting the bacterial liquid in equal proportion5The volume ratio of the diluted bacterium solution to the donkey-hide gelatin sample solution to be detected is 1:100, so that the donkey-hide gelatin sampleThe product solution has the same geometric dilution factor as the Escherichia coli in the model sample.
In a particular embodiment of the invention, the biothermodynamic parameters are extracted by using origine 9.0 software;
in a particular embodiment of the invention, the biomechanical parameters comprise a first half-peak width in life F1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2As a parameter participating in principal component analysis.
In a particular embodiment of the invention, the principal component analysis of the biothermodynamic parameters is carried out in the Simca-p14 software;
in a specific embodiment of the present invention, the principal component analysis prediction calculation formula is:
Y＝{0.91,0.89,0.92,1.10,0.74,0.94,0.84,0.87,0.82,1.22}
P1＝{0.36,0.38,0.34,-0.29,0.58,0.18,0.20,0.03,0.26,-0.20}
P2＝{-0.16,-0.15,-0.14,0.08,-0.22,0.20,0.41,0.56,0.25,-0.56}
X＝{F1,LHW1,RHW1,Q1,AP1(％),LHW2,RHW2,F2,PE,EX2}
x is the biological thermodynamic parameter after normalization treatment, wherein the normalization treatment is the ratio of the biological thermodynamic parameter of the donkey-hide gelatin sample extracted by the origin9.0 software to the biological thermodynamic parameter of the model sample with the donkey-hide gelatin sample concentration of 0 mg/ml;
in a specific embodiment of the present invention, the process of obtaining the biothermodynamic parameters of the model sample is the same as that of the donkey-hide gelatin sample.
In the specific implementation mode of the invention, the invention also relates to an establishment method of the method for evaluating the quality consistency of the donkey-hide gelatin, which specifically comprises the following steps:
1) preparing initial bacterial liquid: the culture medium selected from the bacterial liquid is selected from LB broth culture medium, and the strain is selected from Escherichia coli (ATCC 25922);
2) and (3) investigating the concentration of the test bacterial liquid: taking the initial bacterial liquid prepared in the step 1), diluting a series of concentrations in an equal ratio, sealing respectively, shaking up, placing in a micro calorimeter for detection, and recording the energy change in the growth and metabolism process of the escherichia coli in a sealed state through the micro calorimeter to obtain a heat production power-time (P-t) curve; selecting a curve which has a stable plateau period, smooth line shape and obvious peak shape as a model group;
3) preparing test bacterial liquid: selecting the concentration of the test bacterial liquid as the specific dilution multiple of the initial bacterial liquid prepared in the step 1) according to the screening result of the step 2), so that the equal-ratio dilution multiple of the Escherichia coli in the mixed test bacterial liquid and the colla corii asini sample solution is the same as that of the model group;
4) preparation of test samples: preparing a donkey-hide gelatin sample solution with dose-effect relationship, weighing a donkey-hide gelatin sample, adding the donkey-hide gelatin sample into a culture medium, setting the final concentration of the donkey-hide gelatin sample to be more than two groups of 0.625mg/ml, 1.25mg/ml, 2.50mg/ml, 5.0mg/ml, 10.0mg/ml and 20.0mg/ml, namely an administration group, sterilizing the administration group and a model group of the donkey-hide gelatin sample with the concentration of 0mg/ml by high-pressure steam, and placing the administration group and the model group in a super clean bench to room temperature;
5) determining the administration concentration of the donkey-hide gelatin: adding the equi-specific diluted test bacterial liquid prepared in the step 3) into the donkey-hide gelatin samples with various concentrations prepared in the step 4), so that the equi-specific dilution multiple of the Escherichia coli in the test bacterial liquid is the same as that of the model group after the test bacterial liquid and the donkey-hide gelatin sample solution are mixed; sealing, shaking, detecting in a microcalorimeter, and recording heat generation power-time (P-t) curves of each group at each concentration;
6) screening evaluation parameters: analyzing heat production power-time (P-t) curve data of each group of concentrations by adopting origin9.0 software to obtain a biological thermodynamic parameter, and carrying out normalization treatment on the biological thermodynamic parameter;
the normalization processing is the ratio of the biomechanical parameters of the donkey-hide gelatin sample extracted by origin9.0 software to the biomechanical parameters of the donkey-hide gelatin sample with the concentration of 0mg/ml, and the data after normalization processing can reduce the error among groups, so that the method has universality.
Carrying out principal component analysis on the normalized biothermodynamic parameters by using Simca-P software; then selecting a donkey-hide gelatin sample with the concentration which has the maximum change of the principal component and a model group to respectively carry out partial least squares-differential analysis (OPLS-DA), and screening parameters which meet the requirements that VIP values are greater than 1, | P (corr) | is greater than or equal to 0.5 in the partial least squares-differential analysis and have a dose-effect relationship as evaluation parameters;
7) construction of evaluation indexes: characterizing the evaluation parameters screened in the step 6) in main component analysis, and selecting the concentration of an administration group with the largest influence on growth and metabolism of escherichia coli as the administration concentration for evaluating the quality consistency of the donkey-hide gelatin if the difference between the administration group and a model group presents a better dose-effect relationship along with the increase of the concentration of the donkey-hide gelatin;
meanwhile, the dose-effect relationship shown in the principal component analysis is used as a prediction blueprint, the evaluation parameters screened out in the step 6) are used as a fixed parameter combination and are converted into a formula, and the characteristic values t1 and t 2 are obtained, namely the consistency evaluation index of the donkey-hide gelatin quality.
In the specific embodiment of the invention, the OD600 value of the initial bacterial liquid is 0.3; the specific operation process is that the strain is inoculated into a culture medium to form initial bacterial liquid, and the initial bacterial liquid is shaken under the condition of 37 ℃ until the relative value of OD600 is 0.3.
In one embodiment of the present invention, the geometric dilution in step 2) is a series ofColumn concentration of 1 × 10 diluted in equal ratio1、1×102、1×103、1×104、1×105、1×106、1×107Seven concentrations.
In a specific embodiment of the present invention, the concentrations at which the main components change most are t 2: 2.50mg/ml and t 1: 20.0 mg/ml.
In a specific embodiment of the present invention, the evaluation parameters selected in step 6) are: first life-time half-peak width F1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2。
The equipment used in the examples was: TAM Air milliwatt level thermal conductivity type isothermal micro calorimeter (thermo company, Sweden, provided with 8 independent channels, can simultaneously measure 8 samples; detection limit is 4 uW; precision is 20 uW; error is less than 23 uW; system temperature is controlled at 37 ℃); a vertical pressure steam sterilizer (Chongqing Yamaoto science and technology Co., Ltd.); an electronic balance model XS205DU (Metter Toledo Sartorious, switzerland); SW-CT-2FD double single-side purification bench (Suzhou purification plant); TH2-22 desk type constant temperature oscillator (Experimental facilities, Taicang, Jiangsu), SynergH2 full-function microplate detector (BioTek, USA).
The detection conditions used in the examples were: the detection is carried out at a constant temperature of 37 ℃.
The strains used in the examples were: escherichia coli (Escherichia coli ATCC25922, provided by China pharmaceutical biologicals institute); LB broth (purchased from tokyo boxing biotechnology, llc).
The analysis procedure in the examples also used biothermodynamic data analysis software V1.0 copyright accession number: 2011SR0955405
The raw materials used in the examples: the donkey-hide gelatin is purchased from 3 different manufacturers and 13 different batches, and the detailed information is shown in table 1.
Table 1: sample information table
Example 1
The preparation, conditions and used instruments and equipment of the donkey-hide gelatin quality consistency detection method are established according to the above description.
1. Preparation of initial bacterial liquid
Accurately weighing 10.0g LB broth culture medium powder, dissolving in 500ml distilled water, adjusting pH to 7.0-7.2, sterilizing with high pressure steam at 121 deg.C for 30min, cooling to room temperature, and storing in refrigerator at 4 deg.C. Inoculating Escherichia coli (ATCC25922) to a centrifugal tube containing 10ml of LB broth culture medium, shaking overnight, and detecting (wavelength is 600nm) relative OD value of the bacterial liquid to be 0.3 by an enzyme-linked immunosorbent assay (namely a microplate detector), thus obtaining the prepared original Escherichia coli liquid.
2. Establishing a growth and metabolism thermal activity spectrum curve of Escherichia coli
Diluting the Escherichia coli liquid prepared in step 1 at a constant temperature of 37 deg.C to 7 concentrations (1 × 10) at a ratio of 1:101、1×102、1×103、1×104、1×105、1×106、1×107Respectively placing 10ml of the obtained product in an ampoule bottle, sealing, shaking up, placing in a micro calorimeter for detection, recording the energy change in the growth and metabolism process of the Escherichia coli in a sealed state by the micro calorimeter, recording a heat production power-time (P-t) curve as shown in B in figure 1, when the curve returns to a base line again, ending the experiment, finally selecting a curve which meets the requirements of stable platform period, smooth linear shape and obvious peak shape as a model group through comprehensive investigation, namely diluting at equal ratio of 1 × 107The (P-t) curve of the fold, as shown in A in FIG. 1.
In order to ensure the stability and reliability of the experiment. The model curves were subjected to an intra-day reproducibility study (FIG. 1-C) and an inter-day reproducibility study (FIG. 1-D). The similarity of the curves is inspected by using a cosine vector method, and the similarity value is more than 0.9 (shown in figures 1-E and 1-F), so that the model is stable and reproducible, and can be used as an evaluation model to be applied to the method.
According to the growth metabolism maps of the Escherichia coli with different concentrations and the growth metabolism rule of the Escherichia coli, the thermal spectrum curve can be divided into 5 stages: a lag phase (A-B), a first growth phase (B-C), a stationary phase (C-D), a second growth phase (D-E), and a decline phase (E-F).
3. Preparation of test bacterial liquid
Selecting the concentration of the bacterial liquid as the initial bacterial liquid equal ratio dilution 1 × 10 according to the screening result of the step 25The mixed liquid is mixed with donkey-hide gelatin sample to make the concentration of Escherichia coli bacteria liquid of different concentration donkey-hide gelatin sample equal to the original bacteria liquid diluted 1 × 107Concentration of fold, consistent with model group.
4. Preparation of test samples
Accurately weighing colla Corii Asini samples of number S1 batch with required concentration, placing in penicillin bottles, adding 10ml culture medium respectively to make final concentration of colla Corii Asini in the liquid be 0.625mg/ml, 1.25mg/ml, 2.50mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml, sterilizing with high pressure steam at 121 deg.C for 30min, and placing in ultra-clean bench to room temperature.
5. Donkey-hide gelatin administration concentration detection
And (3) determining by using a microcalorimetry method under the condition of constant temperature of 37 ℃, thus obtaining the donkey-hide gelatin sample model group with the concentration of 0 mg/ml. And (3) adding 100 mu l of the escherichia coli liquid diluted in the step (3) into the newly-prepared 10ml test samples with different concentrations prepared in the step (4) to keep the bacterial liquid concentrations of the samples to be tested consistent, respectively sealing, shaking up, and placing in a micro calorimeter for detection. The heat generation power-time (P-t) curve was recorded for each concentration and the experiment was ended when the curve returned to baseline again.
To ensure the reliability of the experiment, the experiment was repeated three times.
6. Screening evaluation parameters:
However, under the condition of limited culture, the microbial population composition has the characteristic of heterogeneity, and belongs to a nonlinear and multivariable complex dynamic process. The parameters obtained in the experimental method have adverse factors such as information overlapping and low quality, so in order to further optimize the evaluation index and discover the regularity information, the obtained parameters need to be subjected to mathematical comprehensive treatment, so that the evaluation index with more regularity is obtained. Therefore, Principal Component Analysis (PCA) was performed on all normalized parameters using Simca-p software, as shown in FIG. 2, panel B.
Selected in the principal component t [1]]，t[2]The 20.0mg/ml and 2.5mg/ml groups with the largest upper variation were subjected to partial least squares-differential analysis (OPLS-DA) with the model group (0mg/ml group), respectively, to screen VIP values in the OPLS-DA analysis>1, | p (corr) | ≧ 0.5 and has a parameter of dose-effect relationship, as shown in C in fig. 2 and D in fig. 2, the union of them is taken as an evaluation parameter. Obtaining 10 biomechanical parameters of F1、LHW1、RHW1、Q1、AP1(％)、LHW2、RHW2、F2、PEAnd EX2The values are shown in Table 2. The trend and dose-effect relationship in PCA is shown in figure 3.
TABLE 2 Biothermodynamic principal parameter information
7. Determination of the evaluation dose
And (3) characterizing the 10 evaluation parameters screened in the step 6 in Principal Component Analysis (PCA), and obtaining a PCA graph (A in figure 4) and a loading graph (B in figure 4) which are consistent with the principal component analysis of all the parameters. The biological thermal activity spectrum of the screened donkey-hide gelatin with 0-20mg/ml administration concentration has obvious dose-effect relationship in the main components t1 and t 2 as shown in figure 4C and D.
By combining the direct mapping (A in figure 2), principal component analysis (B in figure 2) and (A in figure 4) and the significance p value and the principal components t1 and t 2 value of each index parameter administration group and model group, it can be found that the difference between the administration group and the model group presents a certain dose-effect relationship along with the increase of the administration concentration of the donkey-hide gelatin between 0mg/ml and 20mg/ml, and the change is maximum at 5.0mg/ml, thereby influencing a plurality of indexes. Thus, a 5mg/ml donkey-hide gelatin solution was selected as the dosing concentration to evaluate consistency.
8. Methodology investigation
In order to ensure the stability of the escherichia coli model and the applicability of the detection dose after the administration of the donkey-hide gelatin, the research carries out methodology investigation on the effect of the donkey-hide gelatin solution on the escherichia coli model when the concentration is 5.0mg/ml, selects the same donkey-hide gelatin sample S1, and carries out repeated tests according to the steps 5 and 6 to obtain 10 evaluation parameters. The results show that the model and 10 main parameters in the thermal spectrum curve after donkey-hide gelatin administration have good reproducibility. This method is suggested to be stable and this dose can be used for evaluation.
9. Model construction
Using the principal component analysis algorithm related to the dosage obtained in the step 7) as a model, and combining the 10 screened parameters as fixed parameters to obtain a calculation formula of evaluation index principal components t1 and t 2 values taking the dose-effect relationship as a blueprint, namely a principal component analysis prediction calculation formula.
Y＝{0.91,0.89,0.92,1.10,0.74,0.94,0.84,0.87,0.82,1.22}
P1＝{0.36,0.38,0.34,-0.29,0.58,0.18,0.20,0.03,0.26,-0.20}
P2＝{-0.16,-0.15,-0.14,0.08,-0.22,0.20,0.41,0.56,0.25,-0.56}
X＝{F1,LHW1,RHW1,Q1,AP1(％),LHW2,RHW2,F2,PE,EX2}
And X is the biological thermodynamic parameter after normalization treatment, wherein the normalization treatment is the ratio of the biological thermodynamic parameter of the donkey-hide gelatin sample extracted by the origin9.0 software to the biological thermodynamic parameter of the model group with the concentration of the donkey-hide gelatin sample being 0 mg/ml.
The principal components t1 and t 2 can be used to comprehensively evaluate the information in the biological heat activity spectrum, so that it can be used as characteristic index to evaluate the consistency of donkey-hide gelatin quality. The smaller the SD value of the evaluation indexes t1 and t 2 in the multi-batch test, the better the consistency is proved.
Example 2
Donkey-hide gelatin product consistency detection based on biological thermal activity fingerprint spectrum
By way of example 1, it was determined that the detection of donkey-hide gelatin was carried out in 3 factories (A, B and group C), 12 batches (samples No. S2-S13) at a mass concentration of donkey-hide gelatin of 5 mg/ml. The specific detection method is as follows:
1. preparation of initial bacterial liquid
Accurately weighing 10.0g LB broth culture medium powder, dissolving in 500ml distilled water, adjusting pH to 7.0-7.2, sterilizing with high pressure steam at 121 deg.C for 30min, cooling to room temperature, and storing in refrigerator at 4 deg.C. Inoculating Escherichia coli (ATCC25922) to a centrifugal tube containing 10ml of LB broth culture medium, shaking overnight, and detecting (wavelength is 600nm) relative OD value of the bacterial liquid to be 0.3 by an enzyme-linked immunosorbent assay (namely a microplate detector), thus obtaining the prepared original Escherichia coli liquid.
2. Preparation of test samples
Accurately weighing each donkey-hide gelatin sample with the serial numbers of S2-S13 and a model sample with the donkey-hide gelatin sample concentration of 0mg/ml into each penicillin bottle, respectively adding 10ml of culture medium to ensure that the final concentration of the donkey-hide gelatin in the liquid is 5.0mg/ml, the donkey-hide gelatin concentration of 0mg/ml in the model sample only contains 10ml of culture medium, sterilizing by high-pressure steam at 121 ℃ for 30min, and placing the model sample in a super clean bench to be at room temperature.
3. Establishing a heat production power-time (P-t) curve
Diluting the Escherichia coli liquid prepared in the step 1 to 10 in equal proportion5Doubling, taking 100 μ l of the solution and passing through a filter 105And (3) inoculating the diluted escherichia coli liquid in the equal ratio to each newly-configured donkey-hide gelatin test sample and model sample (0mg/ml donkey-hide gelatin sample) of S2-S13 prepared in the step 2, keeping the concentration of the bacterial liquid of the sample to be detected consistent, respectively sealing, shaking up, and placing in a micro calorimeter for detection. At a constant temperature of 37 ℃, a microcalorimetry measurement is adopted, and heat generation power-time (P-t) curves of the donkey-hide gelatin from S2 to S13 and heat generation power-time (P-t) curves of model samples are recorded, and when the curves return to a base line again, the experiment is ended.
4. Data analysis
Analyzing the heat generation power-time (P-t) curve data of the 12 samples obtained in the step 3 and the corresponding model samples by adopting origin9.0 software, and extracting F in the curve1、LHW1、RHW1、Q1、AP1(％)、LHW2、RHW2、F2、PEAnd EX2Performing normalization treatment on 10 biological thermodynamic parameters, namely selecting the biological thermodynamic parameters of the donkey-hide gelatin sample extracted by origin9.0 software and the biological thermodynamic of the model sample with the donkey-hide gelatin sample concentration of 0mg/mlThe ratio of the mathematical parameters and the normalized values are input into the principal component analysis prediction formula in example 1 to obtain the evaluation index t [1]]、t[2]。
5. And (3) consistency detection results:
according to the visual phenotype of the map (A, B, C in figure 5), the following results are obtained when the donkey-hide gelatin concentration of 5mg/mL is used as the detection dose:
(1) the three groups of donkey-hide gelatin have the function of intervening the growth and metabolism of Escherichia coli;
(2) the difference of the influence points of the three groups of donkey-hide gelatin samples on the growth and metabolism heat activity spectrum curve of the escherichia coli is large, and the influence points are mainly expressed on the maximum heating power and the total heating duration of the second growth period (namely an x axis and a y axis);
the influence of the samples S8-S10E-gelatin on the growth metabolism heat activity spectrum curve of the Escherichia coli in the group B is mainly to increase the heat production power of the second growth period of the Escherichia coli and shorten the heat production time of the Escherichia coli; the influence of the samples S11-S13 of the group C on the growth and metabolism heat activity spectrum curve of the Escherichia coli is mainly to prolong the heat production time of the Escherichia coli; the samples S2-S7 in the group A have small influence on the heat production power and the total heat production time of the second growth period of the Escherichia coli, as shown in A, B and C in figure 5, and the three can be obviously distinguished in an intuitive map.
Taking A in figure 4 which can represent dosage relation and weight bias direction as a blueprint, and screening 10 parameters F in the loading figure (B in figure 4)1、LHW1、RHW1、Q1、AP1(％)、LHW2、RHW2、F2PE and EX2The coordinates of the donkey-hide gelatin are fixed, and main component analysis is carried out on donkey-hide gelatin of 3 manufacturers and 12 batches to obtain D in the graph 5; the corresponding coordinate in the figure is an evaluation index t [1] obtained by a calculation formula]、t[2]The values of (a) are shown as E and F in fig. 5. As shown in D and E in FIG. 5, the difference between the effects of the three groups of E-gelatin samples on E.coli can be clearly characterized and distinguished.
In conclusion, the method for evaluating the quality consistency of the donkey-hide gelatin can be used for evaluating the consistency of products sold in markets and can distinguish donkey-hide gelatin products of different manufacturers; the standard deviation SD value is used as an index for measuring the consistency of the donkey-hide gelatin products, and the lower the SD value is, the better the overall consistency of the donkey-hide gelatin products is.
Claims (13)
1. A method for evaluating the quality consistency of donkey-hide gelatin comprises the following steps:
firstly, selecting more than two donkey-hide gelatin samples, dissolving the donkey-hide gelatin samples by culture medium solutions respectively to prepare donkey-hide gelatin sample solutions with the same mass concentration and dose-effect relationship, inoculating bacterial liquid into each donkey-hide gelatin sample solution, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of each donkey-hide gelatin sample; preparing a model sample containing the bacterial liquid with the concentration of the donkey-hide gelatin sample being 0mg/ml, and detecting by adopting a microcalorimetry method to obtain a corresponding biological thermal activity spectrum curve of the model sample;
further, extracting the biological thermodynamic parameters of each curve;
then, after the above-mentioned biological thermodynamic parameters of the described donkey-hide gelatin sample are normalized, and undergone the process of principal component analysis and calculation to obtain the characteristic values t1 and t 2 of biological thermal activity spectrum of every donkey-hide gelatin sample;
finally, combining with biological thermal activity spectrogram, using characteristic values t1 and t 2 as evaluation indexes to compare between multiple colla Corii Asini samples, and evaluating consistency of different colla Corii Asini samples.
2. The method for evaluating the quality consistency of donkey-hide gelatin according to claim 1, wherein the donkey-hide gelatin sample solution with dose-effect relationship has a mass concentration of 0.625-20.0mg/ml, optionally the donkey-hide gelatin sample solution has a mass concentration of 5.0 mg/ml.
3. The method for evaluating the quality consistency of the donkey-hide gelatin according to claim 1 or 2, wherein the step of inoculating the bacterial liquid into the donkey-hide gelatin sample solution comprises the steps of inoculating the bacterial liquid into a culture medium to form the bacterial liquid, diluting the bacterial liquid, and finally mixing the bacterial liquid with the donkey-hide gelatin sample solution.
4. The method for evaluating the quality consistency of donkey-hide gelatin according to claim 3, wherein the bacterial species is selected from Escherichia coli (ATCC 25922).
5. The method for evaluating the quality consistency of donkey-hide gelatin according to claim 3, wherein the culture medium selected from the bacterial liquid is LB broth, and the relative OD value of the bacterial liquid is 0.3.
6. The method for evaluating the quality consistency of donkey-hide gelatin according to claim 3, wherein the bacterial liquid is diluted to 1 × 10 in equal proportion5And (3) multiplying, wherein the volume ratio of the diluted bacterium solution to the donkey-hide gelatin sample solution to be detected is 1:100, so that the donkey-hide gelatin sample solution and the Escherichia coli in the model sample have the same proportional dilution times.
7. The method for evaluating the quality consistency of donkey-hide gelatin according to any one of claims 1 to 6, wherein the biothermodynamic parameters are extracted by using origin9.0 software,
the biomechanical parameters include a first half-peak width for life1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2As a parameter participating in principal component analysis.
8. The method for evaluating the quality consistency of donkey-hide gelatin according to any one of claims 1 to 6, wherein the principal component analysis of the biothermodynamic parameters is performed in Simca-p14 software;
the principal component analysis prediction calculation formula is as follows:
Y＝{0.91,0.89,0.92,1.10,0.74,0.94,0.84,0.87,0.82,1.22}
P1＝{0.36,0.38,0.34,-0.29,0.58,0.18,0.20,0.03,0.26,-0.20}
P2＝{-0.16,-0.15,-0.14,0.08,-0.22,0.20,0.41,0.56,0.25,-0.56}
X＝{F1,LHW1,RHW1,Q1,AP1(％),LHW2,RHW2,F2,PE,EX2}
x is the biological thermodynamic parameter after normalization treatment, wherein the normalization treatment is the ratio of the biological thermodynamic parameter of the donkey-hide gelatin sample extracted by the origin9.0 software to the biological thermodynamic parameter of the model sample with the donkey-hide gelatin sample concentration of 0 mg/ml;
the model sample and the donkey-hide gelatin sample have the same biological thermodynamic parameter acquisition process.
9. The method for establishing the method for evaluating the quality consistency of donkey-hide gelatin according to any one of claims 1 to 8, which comprises the following steps:
1) preparing initial bacterial liquid: the culture medium selected from the bacterial liquid is selected from LB broth culture medium, and the strain is selected from Escherichia coli (ATCC 25922);
2) and (3) investigating the concentration of the test bacterial liquid: taking the initial bacterial liquid prepared in the step 1), diluting a series of concentrations in an equal ratio, sealing respectively, shaking up, placing in a micro calorimeter for detection, and recording the energy change in the growth and metabolism process of the escherichia coli in a sealed state through the micro calorimeter to obtain a heat production power-time (P-t) curve; selecting a curve which has a stable plateau period, smooth line shape and obvious peak shape as a model group;
3) preparing test bacterial liquid: selecting the concentration of the test bacterial liquid as the specific dilution multiple of the initial bacterial liquid prepared in the step 1) according to the screening result of the step 2), so that the equal-ratio dilution multiple of the Escherichia coli in the mixed test bacterial liquid and the colla corii asini sample solution is the same as that of the model group;
4) preparation of test samples: preparing a donkey-hide gelatin sample solution with dose-effect relationship, weighing a donkey-hide gelatin sample, adding the donkey-hide gelatin sample into a culture medium, setting the final concentration of the donkey-hide gelatin sample to be more than two groups of 0.625mg/ml, 1.25mg/ml, 2.50mg/ml, 5.0mg/ml, 10.0mg/ml and 20.0mg/ml, namely an administration group, sterilizing the administration group and a model group of the donkey-hide gelatin sample with the concentration of 0mg/ml by high-pressure steam, and placing the administration group and the model group in a super clean bench to room temperature;
5) determining the administration concentration of the donkey-hide gelatin: adding the equi-specific diluted test bacterial liquid prepared in the step 3) into the donkey-hide gelatin samples with various concentrations prepared in the step 4), so that the equi-specific dilution multiple of the Escherichia coli in the test bacterial liquid is the same as that of the model group after the test bacterial liquid and the donkey-hide gelatin sample solution are mixed; sealing, shaking, detecting in a microcalorimeter, and recording heat generation power-time (P-t) curves of each group at each concentration;
6) screening evaluation parameters: analyzing heat production power-time (P-t) curve data of each group of concentrations by adopting origin9.0 software to obtain a biological thermodynamic parameter, and carrying out normalization treatment on the biological thermodynamic parameter;
the normalization treatment is the ratio of the biological thermodynamic parameters of the donkey-hide gelatin sample extracted by origin9.0 software to the biological thermodynamic parameters of the donkey-hide gelatin sample with the concentration of 0 mg/ml;
carrying out principal component analysis on the normalized biothermodynamic parameters by using Simca-P software; then selecting a donkey-hide gelatin sample with the concentration which has the maximum change of the principal components and a model group to respectively carry out partial least square method-differential analysis, and screening parameters which meet the requirements that the VIP value is greater than 1 and the P (corr) is greater than or equal to 0.5 in the partial least square method-differential analysis and have a dose-effect relationship as evaluation parameters;
7) construction of evaluation indexes: characterizing the evaluation parameters screened in the step 6) in main component analysis, and selecting the concentration of an administration group with the largest influence on growth and metabolism of escherichia coli as the administration concentration for evaluating the quality consistency of the donkey-hide gelatin if the difference between the administration group and a model group presents a better dose-effect relationship along with the increase of the concentration of the donkey-hide gelatin;
meanwhile, the dose-effect relationship shown in the principal component analysis is used as a prediction blueprint, the evaluation parameters screened out in the step 6) are used as a fixed parameter combination and are converted into a formula, and the characteristic values t1 and t 2 are obtained, namely the consistency evaluation index of the donkey-hide gelatin quality.
10. The method for establishing the method for evaluating the quality consistency of the donkey-hide gelatin according to claim 9, wherein the OD600 value of the initial bacterial liquid is 0.3; the specific operation process is that the strain is inoculated into a culture medium to form initial bacterial liquid, and the initial bacterial liquid is shaken under the condition of 37 ℃ until the relative value of OD600 is 0.3.
11. The method for establishing the method for evaluating the quality consistency of donkey-hide gelatin according to claim 9, wherein the series of concentrations of the equal-ratio dilution in the step 2) is equal-ratio dilution 1 × 101、1×102、1×103、1×104、1×105、1×106、1×107Seven concentrations.
12. The method of claim 9, wherein the concentrations of the main components that change the most are t 2: 2.50mg/ml and t 1: 20.0 mg/ml.
13. The method for establishing the method for evaluating the quality consistency of donkey-hide gelatin according to claim 9, wherein the evaluation parameters screened in the step 6) are as follows: first life-time half-peak width F1First life-span left half-peak width LHW1Right half peak width in first life cycle RHW1First long-term heat generation area Q1First life-time heating area/total heating area AP1(%), left half-peak width of the second growth phase LHW2Right half peak width RHW of second growth period2Half-peak width F of second growth period2Maximum heating power P in the second growth periodEAnd end time of second growth phase EX2。
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