Standard number: GB/T 38569-2020
Standard name: Raman spectroscopy for quality evaluation of industrial microorganism strains
English name: Performance evaluation of industrial microorganism strain—Raman spectroscopy ||
tt||Standard format: PDF
Release time: 2020-03-31
Implementation time: 2020-03-31
Standard size: 9.76M
Standard introduction: 1 Scope
This standard specifies the method for evaluating the quality of industrial microorganism strains by Raman spectroscopy.
This standard is applicable to the quality consistency evaluation of bacteria, fungi, microalgae, etc. used in industrial fermentation relative to reference strains.
2 Normative reference documents
The following documents are indispensable for the application of this document. For all referenced documents with dates, only the versions with dates apply to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document.
GB/T6682 Specifications and test methods for water used in analytical laboratories
This standard specifies the method for evaluating the quality of industrial microbial strains using Raman spectroscopy. This standard is applicable to the quality consistency evaluation of bacteria, fungi, microalgae, etc. used in industrial fermentation relative to reference strains.

ICS07.080
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National Standard of the People's Republic of China
GB/T38569—2020
Quality Evaluation of Industrial Microorganism Strain
Raman Spectroscopy
Performanceevaluation of industrial microorganism strain-Ramanspectroscopy
Published on 2020-03-31
State Administration for Market Regulation
National Administration of Standardization
Implementation on 2020-03-31
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. The drafting units of this standard are: Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences, and China National Institute of Standardization. The main drafters of this standard are: Ma Bo, Ji Yuetong, He Rihui, Li Xunrong, Xu Jian, and Ma Aijin. iirKAa~cJouakAa
GB/T38569—2020
1 Scope
Quality evaluation of industrial microbial strains
Raman spectroscopy
This standard specifies the method for evaluating the quality of industrial microbial strains by Raman spectroscopy. firKAa~cJouaKA
GB/T38569—2020
This standard applies to the quality consistency evaluation of bacteria, fungi, microalgae, etc. for industrial fermentation relative to reference strains. Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water for analytical laboratories Terms and definitions
The following terms and definitions apply to this document. 3.1
Performance evaluation of industrial microorganism strain Raman spectroscopy is used to carry out consistency evaluation of the quality of industrial microorganism strains. 3.2
Reference strain
Reference strain
An identified and preserved strain with clear fermentation performance used for fermentation production. Principle
The superposition information of Raman spectra of internal compounds of cells can reflect the comprehensive phenotype of the cell. By comparing the similarity of single-cell Raman spectra of the strain to be evaluated with that of the reference strain, it can be determined whether the strain to be evaluated has fermentation performance comparable to that of the reference strain. Reagents or materials
Unless otherwise specified, only analytically pure reagents are used, and water is secondary water as specified in GB/T6682. 5.1 Sterile culture medium
Prepare according to the type of bacteria, or select corresponding commercial culture medium. 2 Slides
Calcium fluoride slides (25mm×75mm). 6
Instruments
The instruments used in the test are:
GB/T38569—2020
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Micro-Raman spectrometer: the spectral resolution is not less than 5cm-, and the Raman shift wavenumber range includes at least 400cm-1~1850cm
b) Incubator:
Shaker;
d) Centrifuge.
7 Determination steps
Strain activation
Prepare the corresponding solid plates and liquid culture media for industrial microorganisms according to the production process. Use a sterile inoculation loop to dip the seed culture liquid of the reference strain and the strain to be evaluated, streak on the solid plate, and select the corresponding temperature and other conditions according to the production process parameters for cultivation until a single colony grows: Pick 3 reference strains and 3 single colonies of the strain to be evaluated, inoculate them into test tubes containing 5mL of liquid culture medium, select the corresponding temperature and speed according to the production process parameters, and cultivate them to the logarithmic phase. 7.2
Strain cultivation
Use a micropipette to transfer 200L of the activated reference strain and the strain to be evaluated. Inoculate them into 3 containers containing 20mL of liquid culture medium, select the corresponding temperature and speed according to the production process parameters, and cultivate them to the stable phase. 7.3
Sample pretreatment
7.3.1 Transfer 1ml of the reference strain and the strain to be evaluated after cultivation into sterile tubes. 7.3.2 Select the appropriate speed for centrifugation, discard the supernatant, add 1mL of sterile double distilled water, and mix the bacteria. 7.3.3 Repeat 7.3.2 twice, transfer 10μL of bacterial solution to a sterile tube, add 990L of sterile double distilled water, mix well, transfer 2pL of diluted bacterial solution, spot on a clean calcium fluoride slide, and air-dry at room temperature under a sterile environment. 7.4 Measurement
Place the air-dried calcium fluoride slide on the stage of the micro-Raman spectrometer, select a single cell in the transmission bright field mode, 7.4.1
Switch to the spectrum acquisition mode, and measure the Raman spectrum signal. 7.4.2 Randomly select at least 20 single cells for each sample as sample signals. 7.4.3 Collect signals from 3 cell-free areas in the spotting area, and calculate the average value as the background signal. 7.5 Data Processing
7.5.1 The single-cell Raman spectroscopy raw data were subjected to background signal subtraction (sample signal minus background signal), baseline correction (>5th order function) and area-based normalization.
7.5.2 Calculate the signal-to-noise ratio, where the signal intensity corresponding to the sharp Raman peak at 1001cm- is used. Data with a signal-to-noise ratio less than 3 are discarded. The number of data is supplemented so that the evaluated strain group and the reference strain group have the same number of data. 8 Result calculation and analysis
Result calculation
The cosine distance is calculated according to formula (1):
Where:
coso(a,b)
Xb,）
The angle between the Raman spectrum vectors of single cell α and single cell 6b; cos(ab)—the cosine distance between the Raman spectra of single cell a and single cell b: a
—the intensity corresponding to the Raman shift i of single cell a; the intensity corresponding to the Raman shift of single cell 6.
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GB/T38569-—2020
Calculate the mean and standard deviation of the cosine distance, and plot with the number of cells as the horizontal axis and the coefficient of variation as the vertical axis. If the curve reaches a platform, it means that the number of cells is sufficient. If the curve does not reach a platform, increase the number of single-cell Raman spectra collected until saturation. The similarity is calculated according to formula (2):
Where:
[n(n-1)]/4
Similarity between the strain to be evaluated and the reference strain; The average value of the Raman spectrum cosine distance between each cell of the strain to be evaluated and each cell of the reference strain: - The average value of the Raman spectrum cosine distance between each cell in the strain group to be evaluated: The number of single cells in the strain group to be evaluated.
The calculation result is expressed as the arithmetic mean of the parallel determination values, retaining three significant figures. 8.2
Result Analysis
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The smaller the R value, the more similar the strain to be evaluated is to the reference strain. When the R values ​​of three independent experiments obtained under repeatability conditions are all less than 0.05, it is determined that the fermentation performance of the strain to be evaluated is consistent with that of the reference strain.
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