CN114214255A - Formula and preparation method of antibacterial peptide strain culture medium - Google Patents
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Abstract
The invention discloses a formula and a preparation method of an antibacterial peptide strain culture medium, relates to the technical field of antibacterial peptides, and aims to solve the problems that when the antibacterial peptide strain provided by the background technology is subjected to expanded culture, the titer of the antibacterial peptide strain after culture is low and the practicability is low due to the problems of the culture medium raw materials, the following scheme is provided, and the antibacterial peptide strain culture medium comprises the following raw materials in parts by mass: 5-13 parts of glucose, 10-15 parts of yeast extract, 10-20 parts of peptone, 10-20 parts of beef extract, 3-5 parts of sodium chloride, 3-5 parts of monopotassium phosphate, 3-5 parts of dipotassium hydrogen phosphate and the balance of distilled water. According to the invention, the yeast extract, the peptone, the beef extract and the glucose are used as the raw materials of the culture medium, the raw materials are easy to obtain and low in cost, and meanwhile, sufficient nutrient substances required by fermentation can be provided for the antibacterial peptide strain, the pH value of the culture medium is adjusted to be alkalescent, so that the growth of the antibacterial peptide is facilitated, the titer of the antibacterial peptide strain cultured by fermentation is improved, the germination rate is high, and the practicability is improved.
Description
Technical Field
The invention relates to the technical field of antibacterial peptide, in particular to a formula of an antibacterial peptide strain culture medium and a preparation method thereof.
Background
The antibacterial peptide antigen refers to a basic polypeptide substance with antibacterial activity generated by induction in an insect body, the molecular weight is about 2000-7000, the basic polypeptide substance is composed of 20-60 amino acid residues, and most of the active polypeptides have the characteristics of strong basicity, thermal stability, broad-spectrum antibacterial property and the like.
The antibacterial peptides can be classified into various types according to their structures, and they can be classified into 5 types: a single-chain alpha-helix without cysteine residues, or a peptide consisting of two alpha-helices joined by a random coil; antimicrobial peptides that are rich in certain amino acid residues but do not contain cysteine residues; an antimicrobial polypeptide comprising 1 disulfide bond; an antimicrobial peptide having 2 or more than 2 disulfide bonds and having a beta-sheet structure; peptides with antibacterial activity derived from other larger polypeptides of known function.
According to the source classification of antimicrobial peptides, they can be classified into 6 types: an insect antimicrobial peptide; a mammalian antimicrobial peptide; the amphibian antibacterial peptide has multiple functions, a large amount of skin active peptide existing in skin secretion has various biological activities, most of polypeptide substances have certain antimicrobial activity, and are extremely old and effective natural defense substances in evolution and often classified as the antibacterial peptide; at present, more than 49 antibacterial peptides are separated from fish; plant antimicrobial peptides, which are structurally similar to insect and mammalian defensins, are also present in plants and are called plant defensins, most of which have good activity against plant pathogens, some of which are toxic to gram-positive bacteria, gram-negative bacteria, fungi, yeasts and mammalian cells, and some of which are also called bacteriocins, including cationic peptides and neutral peptides, which are secreted by gram-positive bacteria and gram-negative bacteria.
At present, when the conventional antibacterial peptide strain culture medium is used for carrying out amplification culture on the antibacterial peptide strain, the titer of the antibacterial peptide strain after culture is low and the practicability is low due to the problems of the culture medium raw materials.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides an antibacterial peptide strain culture medium formula and a preparation method thereof.
The invention provides an antibacterial peptide strain culture medium formula which comprises the following raw materials in parts by weight: 5-13 parts of glucose, 10-15 parts of yeast extract, 10-20 parts of peptone, 10-20 parts of beef extract, 3-5 parts of sodium chloride, 3-5 parts of monopotassium phosphate, 3-5 parts of dipotassium hydrogen phosphate and the balance of distilled water.
Preferably, the feed comprises the following raw materials in parts by mass: 5-9 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
Preferably, the feed comprises the following raw materials in parts by mass: 10-13 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
A preparation method of an antibacterial peptide strain culture medium comprises the following steps:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone, sodium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, respectively adding the yeast extract, the glucose, the peptone, the sodium chloride, the potassium dihydrogen phosphate and the dipotassium hydrogen phosphate into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to completely mix all the components, dissolving the components in distilled water, and naturally cooling to obtain a culture medium stock solution;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially placing the subpackaged culture dishes into a sterilizing pot, performing high-pressure steam sterilization, taking out the culture dishes while the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain the culture medium.
Preferably, the heating temperature in the step S3 is 80 to 90 ℃.
Preferably, the sterilization temperature in the step S7 is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
The invention has the beneficial effects that:
according to the invention, the yeast extract, the peptone, the beef extract and the glucose are used as the raw materials of the culture medium, the raw materials are easy to obtain and low in cost, and meanwhile, sufficient nutrient substances required by fermentation can be provided for the antibacterial peptide strain, the pH value of the culture medium is adjusted to be alkalescent, so that the growth of the antibacterial peptide is facilitated, the titer of the antibacterial peptide strain cultured by fermentation is improved, the germination rate is high, and the practicability is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1: the antibacterial peptide strain culture medium formula comprises the following raw materials in parts by weight: 6 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
A preparation method of an antibacterial peptide strain culture medium comprises the following steps:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone and sodium chloride, respectively adding the yeast extract, the glucose, the peptone and the sodium chloride into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to ensure that all the components are completely mixed and dissolved in distilled water, naturally cooling to obtain a culture medium stock solution, wherein the heating temperature is 80-90 ℃;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially putting the subpackaged culture dishes into a sterilizing pot, sterilizing by high-pressure steam, taking out the culture dishes when the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain a culture medium, wherein the sterilization temperature is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
Example 2: the antibacterial peptide strain culture medium formula comprises the following raw materials in parts by weight: 7 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
A preparation method of an antibacterial peptide strain culture medium comprises the following steps:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone and sodium chloride, respectively adding the yeast extract, the glucose, the peptone and the sodium chloride into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to ensure that all the components are completely mixed and dissolved in distilled water, naturally cooling to obtain a culture medium stock solution, wherein the heating temperature is 80-90 ℃;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially putting the subpackaged culture dishes into a sterilizing pot, sterilizing by high-pressure steam, taking out the culture dishes when the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain a culture medium, wherein the sterilization temperature is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
Example 3: the antibacterial peptide strain culture medium formula comprises the following raw materials in parts by weight: 11 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
A preparation method of an antibacterial peptide strain culture medium comprises the following steps:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone and sodium chloride, respectively adding the yeast extract, the glucose, the peptone and the sodium chloride into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to ensure that all the components are completely mixed and dissolved in distilled water, naturally cooling to obtain a culture medium stock solution, wherein the heating temperature is 80-90 ℃;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially putting the subpackaged culture dishes into a sterilizing pot, sterilizing by high-pressure steam, taking out the culture dishes when the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain a culture medium, wherein the sterilization temperature is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
Example 4: the antibacterial peptide strain culture medium formula comprises the following raw materials in parts by weight: 12 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
A preparation method of an antibacterial peptide strain culture medium comprises the following steps:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone and sodium chloride, respectively adding the yeast extract, the glucose, the peptone and the sodium chloride into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to ensure that all the components are completely mixed and dissolved in distilled water, naturally cooling to obtain a culture medium stock solution, wherein the heating temperature is 80-90 ℃;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially putting the subpackaged culture dishes into a sterilizing pot, sterilizing by high-pressure steam, taking out the culture dishes when the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain a culture medium, wherein the sterilization temperature is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
Potency assay
Taking two parts of the culture medium of example 1, example 2, example 3 and example 4 respectively, inoculating the antibacterial peptide strain when the culture medium is cooled to 35 ℃ after autoclaving, culturing for 24 h-48 h under the constant temperature condition of 30 ℃, observing and recording the germination rate, then perforating by using a perforator, wherein the aperture is 2.7mm, taking the culture medium with the outer diameter of 2.7mm, inoculating the Escherichia coli strain when the culture medium of another part of example 1, example 2, example 3 and example 4 is cooled to 55 ℃, culturing for 24 h-48 h under the constant temperature condition of 37 ℃, then perforating by using the perforator, putting the culture medium inoculated with the antibacterial peptide strain into a hole with the outer diameter of 2.7mm on the culture medium inoculated with the Escherichia coli strain, culturing for 24h under the condition of 37 ℃, measuring the diameter (mm) of a bacteriostatic ring, and calculating according to the following formula:
bactericidal potency (IU/mL) is 2XX1000, wherein X ═ 2.7mm diameter of zone of inhibition ÷ 2.1
The results are calculated and shown in Table 1.
TABLE 1 Titer test results
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Germination Rate (%) | 96.5 | 97.3 | 98.9 | 98.9 |
| Potency (IU/mL) | 5773 | 5862 | 5963 | 5946 |
In conclusion, the culture medium containing 11 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate and 3 parts of dipotassium phosphate is the optimal culture medium of the invention.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. The formula of the antibacterial peptide strain culture medium is characterized by comprising the following raw materials in parts by weight: 5-13 parts of glucose, 10-15 parts of yeast extract, 10-20 parts of peptone, 10-20 parts of beef extract, 3-5 parts of sodium chloride, 3-5 parts of monopotassium phosphate, 3-5 parts of dipotassium hydrogen phosphate and the balance of distilled water.
2. The antibacterial peptide strain culture medium formula according to claim 1, characterized by comprising the following raw materials in parts by mass: 5-9 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
3. The antibacterial peptide strain culture medium formula according to claim 1, characterized by comprising the following raw materials in parts by mass: 10-13 parts of glucose, 10 parts of yeast extract, 15 parts of peptone, 15 parts of beef extract, 3 parts of sodium chloride, 3 parts of monopotassium phosphate, 3 parts of dipotassium phosphate and the balance of distilled water.
4. The method for preparing a culture medium for an antimicrobial peptide strain according to claim 1, comprising the steps of:
s1: putting a proper amount of distilled water into a 250ml beaker A, dipping beef extract for a plurality of times by using a glass rod, putting the beef extract on parchment paper for weighing, mixing the beef extract and the parchment paper after weighing, putting the mixture into a container, stirring by using the glass rod to dissolve the beef extract in the distilled water, and taking out the parchment paper by using a pair of tweezers;
s2: weighing a proper amount of yeast extract, glucose, peptone, sodium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, respectively adding the yeast extract, the glucose, the peptone, the sodium chloride, the potassium dihydrogen phosphate and the dipotassium hydrogen phosphate into a beaker A, and adding a proper amount of distilled water into the beaker A to obtain a mixed solution;
s3: placing the beaker A on a magnetic stirrer, heating and stirring simultaneously to completely mix all the components, dissolving the components in distilled water, and naturally cooling to obtain a culture medium stock solution;
s4: slowly dripping 5% sodium hydroxide solution into the culture medium stock solution, and adjusting the pH value of the culture medium stock solution to 7.5-7.7;
s5: taking a clean 250ml beaker B for containing the filtered clear solution of the mixed solution filtered by filter paper;
s6: taking a sufficient culture dish, uniformly filling the filtered liquid into the culture dish, and covering the culture dish with a dish cover;
s7: and sequentially placing the subpackaged culture dishes into a sterilizing pot, performing high-pressure steam sterilization, taking out the culture dishes while the culture dishes are hot after the sterilization is finished, and naturally cooling to obtain the culture medium.
5. The method for preparing a culture medium of antibacterial peptide species according to claim 4, wherein the heating temperature in step S3 is 80-90 ℃.
6. The method for preparing the antibacterial peptide strain culture medium according to claim 4, wherein the sterilization temperature in the step S7 is 100-120 ℃, the pressure is 1.5-1.8 Mpa, and the sterilization time is 40-60 min.
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