Method for evaluating raw material soy peptone of culture medium

Abstract

The invention belongs to the technical field of culture media, and particularly relates to a method for evaluating a culture medium raw material soy peptone, which comprises the steps of antibacterial substance detection, microbial growth promotion effect evaluation and soy peptone quality judgment according to a comprehensive evaluation standard. According to the method, the antibacterial substances in the soy peptone are accurately measured through a protein sequencing method, and the quality of the soy peptone can be comprehensively analyzed from multiple dimensions by combining growth data of various indication microorganisms, so that the limitation of a single evaluation index is avoided, the accuracy is greatly improved, the evaluation result is more accurate and reliable, and the method is suitable for large-scale popularization and application. The experiment and production efficiency is improved, the production cost is reduced, and the resource waste is reduced.

Description

Technical Field

[0001] The invention belongs to the field of culture medium, and in particular relates to a method for evaluating soy peptone as a raw material of the culture medium. Background Art

[0002] In modern biology and biotechnology, culture media serve as the foundation for the growth and reproduction of microorganisms and cells. Their quality directly impacts the accuracy and reliability of experimental results, as well as the quality and yield of biotechnology products. Soy peptone, as one of the key raw materials for culture media, plays a crucial role in the nutrient composition of these media.

[0003] Soy peptone is a product obtained through a series of processing steps, including hydrolysis and purification of soy protein. Rich in nutrients such as amino acids, peptides, vitamins, and trace elements, it provides microbial and cellular growth with nitrogen and carbon sources, as well as other essential nutrients. Due to its wide availability, relatively low cost, and nutrient-rich nature, soy peptone has found widespread application in various fields, including microbial culture, fermentation, and biopharmaceuticals.

[0004] However, there are significant differences in the quality of soy peptones from different manufacturers and batches. These variations may stem from a variety of factors, including the raw soybean variety, origin, and processing technology. For example, different soybean varieties can vary in protein content and composition, which in turn affects the nutritional profile of the peptone. Improper control of the degree of hydrolysis during processing can alter the composition and ratio of amino acids and peptides in the peptone, affecting its nutritional value and suitability for use in culture media.

[0005] The quality of soy peptone currently on the market varies widely. In practical applications, these differences can directly affect microbial growth rate, growth volume, and metabolite synthesis, leading to unstable culture medium performance.

[0006] Existing evaluation methods mostly focus on the determination of a single indicator, such as only detecting protein content or simply observing microbial growth, which cannot fully consider the overall quality of soy peptone. This single evaluation method is difficult to accurately determine whether it is suitable for the cultivation needs of specific microorganisms, which makes it difficult for researchers and manufacturers to choose the right soy peptone. This may affect the accuracy and repeatability of experimental results, and may also cause waste of resources and increased costs in the production process. Therefore, the development of a comprehensive, accurate and efficient evaluation method is imminent. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a method for evaluating soy peptone, a raw material for culture medium. The method uses high-performance liquid chromatography to accurately determine antibacterial substances. Combined with the growth data of multiple indicator microorganisms, it can comprehensively analyze the quality of soy peptone from multiple dimensions, avoiding the limitations of a single evaluation indicator, greatly improving the accuracy, making the evaluation results more accurate and reliable, improving experimental and production efficiency, reducing production costs, and reducing resource waste.

[0008] The present invention provides a method for evaluating soy peptone as a culture medium raw material, comprising: Step 1: Antibacterial substance detection; Step 2: Evaluation of microbial growth promotion effect; Step 3: Determine the quality of soy peptone based on comprehensive evaluation criteria.

[0009] In some embodiments, the antibacterial substance detection in step 1 is performed using protein sequencing.

[0010] In some embodiments, the protein sequencing method comprises: extracting proteins from a soy peptone sample, performing reduction alkylation, enzyme digestion, desalting, and then detecting using LC-MS / MS, performing qualitative protein matching and identification using software, and performing relative quantitative analysis of the same protein / peptide in different groups using peak area; In some embodiments, the microbial growth promotion effect evaluation in step 2 includes: Step 2-1: Weigh the soy peptone to be tested and prepare peptone aqueous solutions with mass concentrations of 0.25%, 0.5%, and 1% respectively; Step 2-2: Dispense the prepared peptone aqueous solution into 96-well ELISA plates, and then inoculate the corresponding indicator microorganisms into each well; Step 2-3: Place the ELISA plate in a 36°C constant temperature incubator for 24 hours, and use a microplate reader to measure the OD value of each well at a wavelength of 600nm; by comparing the changes in the OD600 values ​​of microorganisms cultured with different concentrations of peptone water, the growth-promoting ability of soy peptone was evaluated.

[0011] In some embodiments, the indicator microorganisms include Escherichia coli ATCC25922, Escherichia coli CMCC44102, Staphylococcus aureus ATCC 25923, Staphylococcus aureus CMCC 26003, Staphylococcus aureus ATCC6538, Shigella flexneri CMCC51572, Bacillus subtilis CMCC63501, Enterobacter aerogenes ATCC13048, Micrococcus luteus CMCC 28001, Bacillus clausii CMCC 7316, beta-hemolytic Streptococcus pyogenes CMCC32210, Streptococcus pyogenes CICC 10373, Pseudomonas aeruginosa CMCC10104, Listeria monocytogenes ATCC19115, Salmonella paratyphi B CMCC50094, and Salmonella typhimurium ATCC14028.

[0012] In some embodiments, in step 2-2, 200 μL of peptone aqueous solution is dispensed into each well, and the inoculation amount of the indicator microorganism is 1×10 6 CFU / mL.

[0013] In some embodiments, the comprehensive evaluation criteria in step 3 include: the comprehensive evaluation criteria in step 3 include: if the content of antibacterial substances in soy peptone is lower than 0.01 mg / g, the soy peptone is suitable for microbial growth; the higher the OD600 value of the indicator microorganism, the better the effect of soy peptone in promoting microbial growth.

[0014] In some embodiments, the quality of soy peptone is determined. If the content of antibacterial substances is suitable for microbial growth and the OD600 value is high, the soy peptone is determined to have a strong growth-promoting ability; if the content of antibacterial substances is suitable for microbial growth and the OD600 value is intermediate, the soy peptone is determined to have a medium growth-promoting ability; if the content of antibacterial substances is not suitable for microbial growth or the OD600 value is low, the soy peptone is determined to have a weak growth-promoting ability and needs to be compounded with other nitrogen sources to promote growth.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: Accuracy is greatly improved: Protein sequencing is used to accurately measure antibacterial substances. Combined with the growth data of multiple indicator microorganisms, the quality of soy peptone can be comprehensively analyzed from multiple dimensions, avoiding the limitations of a single evaluation indicator. Compared with traditional methods, the evaluation results are more accurate and reliable.

[0016] Improve experimental and production efficiency: Help researchers quickly screen soy peptone that meets experimental requirements, reduce experimental errors and the number of repeated experiments caused by raw material quality issues, and improve research efficiency. In the production field, it can enable companies to accurately select the appropriate soy peptone, optimize production processes, improve product quality and production efficiency, and reduce production costs.

[0017] Reduced resource waste: This effectively avoids microbial culture failures caused by poor soy peptone quality, reducing the waste of raw materials, culture media, laboratory equipment, and manpower, aligning with the concept of sustainable development. This technology has significant application value in all aspects of microbial culture research and production, driving the development and progress of the entire industry. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific embodiments: To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0019] Example 1 Method for evaluating soy peptone as a culture medium raw material The method for evaluating soy peptone as a culture medium raw material provided by the present invention comprises: 1. Determination of antibacterial substances: Detection of antibacterial substances in soy peptone by protein sequencing.

[0020] Sequencing was performed on four soy peptone samples from two groups (two with good bacterial growth and two with poor growth). Proteins were extracted, reductively alkylated, enzymatically digested, and desalted before detection by LC-MS / MS. Software was used for qualitative protein matching and identification, and peak area analysis was used for relative quantitative analysis of identical proteins / peptides in different groups. A total of 217 proteins and 472 peptides were identified.

[0021] Protein sequencing results showed that the glycinin protein content in the two groups of soy peptones was different. The possible antibacterial mechanism of glycinin was analyzed as follows: Destroy bacterial cell membranes: Soybean globulin glycine basic peptides can interact with bacterial cell membranes. They can insert into the phospholipid bilayer of bacterial cell membranes, resulting in increased permeability of the cell membrane.

[0022] Inhibit bacterial cell wall synthesis: Cell wall synthesis is a key step in bacterial growth. Soybean globulin glycine basic peptide may interfere with the activity of enzymes related to cell wall synthesis.

[0023] Affecting bacterial metabolic processes: Peptides may interfere with metabolic pathways within bacterial cells. They can bind to certain key enzymes within bacterial cells, changing enzyme activity and thus affecting bacterial energy metabolism, protein synthesis, or nucleic acid synthesis.

[0024] Differences in antibacterial effects against different bacteria: Soybean globulin glycine basic peptide may have a more pronounced antibacterial effect against Gram-positive bacteria. This is because the cell wall of Gram-positive bacteria is primarily composed of a thick peptidoglycan layer, making it easier for peptides to interact with the peptidoglycan and insert into the cell membrane. Gram-negative bacteria, on the other hand, have an outer membrane structure that hinders the entry of peptides.

[0025] 2. Evaluation of microbial growth promotion effect: Accurately weigh different batches of soy peptone to be tested and prepare peptone water with mass concentrations of 0.25%, 0.5%, and 1%, respectively.

[0026] The microorganisms listed in Table 1 were selected as indicator microorganisms, including Escherichia coli ATCC 25922, Escherichia coli CMCC44102, Staphylococcus aureus ATCC 25923, Staphylococcus aureus CMCC 26003, Staphylococcus aureus ATCC6538, Shigella flexneri CMCC51572, Bacillus subtilis CMCC63501, Enterobacter aerogenes ATCC13048, Micrococcus luteus CMCC 28001, Bacillus clausii CMCC 7316, beta-hemolytic Streptococcus pyogenes CMCC32210, Streptococcus pyogenes CICC 10373, Pseudomonas aeruginosa CMCC10104, Listeria monocytogenes ATCC19115, Salmonella paratyphi B CMCC50094, Salmonella typhimurium ATCC14028, etc.

[0027] The prepared peptone water was dispensed into 96-well ELISA plates, 200 μL per well, and then the corresponding indicator microorganisms were inoculated into each well at a volume of 1×10 6 CFU / mL.

[0028] The ELISA plate was placed in a 36°C constant temperature incubator for 24 h, and the OD value of each well was measured at a wavelength of 600 nm using a microplate reader.

[0029] The growth-promoting ability of soy peptone was evaluated by comparing the changes in OD600 values ​​of microorganisms cultured in peptone water with different concentrations.

[0030] The OD values ​​of microorganisms cultured in different batches of soytone were finally measured and shown in Table 1: Table 1. OD values ​​of microorganisms ;

[0031] 3. Quality assessment: Establish comprehensive assessment standards.

[0032] Soy peptone is graded according to the content of antibacterial substances and the OD value of indicator microorganisms. If the content of antibacterial substances in soy peptone is less than 0.01 mg / g, it is suitable for bacterial growth; for Escherichia coli, the OD600 value reaches above 0.8, and for Staphylococcus aureus, the OD600 value reaches above 0.7, the soy peptone is judged to have a high-quality effect on promoting microbial growth. For example, the soy peptones of batches 160985 and 115350 shown in Table 1 are both high-quality soy peptones; the OD600 value is between 0.5-0.8 (Escherichia coli) or 0.4- The values ​​between 0.7 (Staphylococcus aureus) and 0.7 (Staphylococcus aureus) are moderate, such as the soytone of batches 163065, 20240412, and 20240409 shown in Table 1; those below 0.5 (Escherichia coli) or 0.4 (Staphylococcus aureus), such as the soytone of batches 20240407 and 158186, require the addition of other nitrogen sources to promote microbial growth.

[0033] Based on the results of antibacterial substance determination and microbial growth promotion effect evaluation, if the antibacterial substance content is suitable for microbial growth and the OD600 value is high, the soy peptone is judged to have a strong growth-promoting ability; if the antibacterial substance content is suitable for microbial growth and the OD600 value is medium, the soy peptone is judged to have a medium growth-promoting ability; if the antibacterial substance content is not suitable for microbial growth or the OD600 value is low, the soy peptone is judged to have a weak growth-promoting ability and needs to be compounded with other nitrogen sources to promote growth.

[0034] The soy peptone evaluated is suitable for culture media commonly used in the art. Specific examples of culture media include but are not limited to: TSA medium (g / L): Tryptone 15.0 Soy Peptone 5.0 Sodium chloride 5.0 Agar 15.0 pH 7.3 ± 0.2 at 25°C; mTSB medium (g / L): Tryptone 17.0 Soy Peptone 3.0 Sodium chloride 5.0 Bile Salt No. 3 1.5 Dipotassium hydrogen phosphate 4.0 Glucose 2.5 pH 7.4 ± 0.2 at 25°C; Modified Tryptone Soy Broth Medium (g / L): Tryptone 17.0 Soy Peptone 3.0 Sodium chloride 5.0 Anhydrous potassium dihydrogen phosphate 2.5 Glucose 2.5 pH 7.3 ± 0.2 at 25°C; Magnesium chloride malachite green soytone enrichment solution (g / L): Soybean Peptone 4.5 Sodium chloride 7.2 Potassium dihydrogen phosphate 1.26 Dipotassium hydrogen phosphate 0.18 Anhydrous magnesium chloride 13.4 Malachite green 0.036 pH 5.2±0.2 at 25℃.

[0035] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

[0036] Although some specific embodiments of the present invention have been described in detail by way of example, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It should also be understood by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for evaluating soy peptone, characterized in that The method comprises: Step 1: Antibacterial substance detection; Step 2: Evaluation of microbial growth promotion effect; Step 3: Determine the quality of soy peptone based on comprehensive evaluation criteria.

2. The method for evaluating soy peptone according to claim 1, wherein The antibacterial substance detection in step 1 adopts protein sequencing method.

3. The method for evaluating soy peptone according to claim 2, wherein The protein sequencing method includes: extracting proteins from soy peptone samples, reducing and alkylating them, enzymatically digesting them, and detecting them using LC-MS / MS after desalting, using software to perform protein matching and qualitative identification, and using peak area to perform relative quantitative analysis of the same proteins / peptides in different groups.

4. The method for evaluating soy peptone according to claim 1, wherein The microbial growth promotion effect evaluation in step 2 includes: Step 2-1: Weigh the soy peptone to be tested and prepare peptone aqueous solutions with mass concentrations of 0.25%, 0.5%, and 1% respectively; Step 2-2: Dispense the prepared peptone aqueous solution into 96-well ELISA plates, and then inoculate the corresponding indicator microorganisms into each well; Step 2-3: Place the ELISA plate in a 36°C constant temperature incubator for 24 hours, and use a microplate reader to measure the OD value of each well at a wavelength of 600nm; by comparing the changes in the OD600 values ​​of microorganisms cultured with different concentrations of peptone water, the growth-promoting ability of soy peptone was evaluated.

5. The method for evaluating soy peptone according to claim 4, wherein The indicator microorganisms include Escherichia coli ATCC 25922, Escherichia coli CMCC44102, Staphylococcus aureus ATCC 25923, Staphylococcus aureus CMCC 26003, Staphylococcus aureus ATCC6538, Shigella flexneri CMCC 51572, Bacillus subtilis CMCC63501, Enterobacter aerogenes ATCC13048, Micrococcus luteus CMCC28001, Bacillus clausii CMCC 7316, and Streptococcus aureus. pyogenes) CMCC 32210, Streptococcus pyogenes CICC10373, Pseudomonas aeruginosa CMCC10104, Listeria monocytogenes ATCC19115, Salmonella paratyphi B CMCC50094, and Salmonella typhimurium ATCC14028.

6. The method for evaluating soy peptone according to claim 5, wherein: In step 2-2, 200 μL of peptone aqueous solution was dispensed into each well, and the inoculum size of the indicator microorganism was 1×10 6 CFU / mL.

7. The method for evaluating soy peptone according to any one of claims 1 to 6, wherein: The comprehensive evaluation criteria in step 3 include: if the content of antibacterial substances in soy peptone is lower than 0.01 mg / g, the soy peptone is suitable for microbial growth; the higher the OD600 value of the indicator microorganism, the better the effect of soy peptone in promoting microbial growth.

8. The method for evaluating soy peptone according to claim 7, wherein To determine the quality of soy peptone, if the antibacterial substance content is suitable for microbial growth and the OD600 value is high, the soy peptone is judged to have a strong growth-promoting ability; if the antibacterial substance content is suitable for microbial growth and the OD600 value is medium, the soy peptone is judged to have a medium growth-promoting ability; if the antibacterial substance content is not suitable for microbial growth or the OD600 value is low, the soy peptone is judged to have a weak growth-promoting ability and needs to be compounded with other nitrogen sources to promote growth.

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