CN116676241A - Chinese medicinal composition extract and bacterial culture medium - Google Patents
Chinese medicinal composition extract and bacterial culture medium Download PDFInfo
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- CN116676241A CN116676241A CN202310960209.1A CN202310960209A CN116676241A CN 116676241 A CN116676241 A CN 116676241A CN 202310960209 A CN202310960209 A CN 202310960209A CN 116676241 A CN116676241 A CN 116676241A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a traditional Chinese medicine composition extracting solution and a bacterial culture medium, which relate to the technical field of medical treatment and have the technical scheme that: an extract of a Chinese medicinal composition for preparing a bacterial culture medium; extracting the traditional Chinese medicine composition extract by adopting a water leaching method; the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass: 280-320 parts of aloe, 180-230 parts of barley seedling, 90-100 parts of Chinese yam, 80-110 parts of coix seed, 190-200 parts of mulberry leaf and 80-130 parts of earthworm. The aim of improving the bacterial detection efficiency is achieved.
Description
Technical Field
The invention relates to a bacterial culture technology, in particular to a traditional Chinese medicine composition extracting solution and a bacterial culture medium.
Background
The bacteria detection is a conventional medical detection means, and a human body specimen is required to be placed in a special culture medium for culture, so that the bacterial colony is increased in value to conveniently detect the bacterial type. A common aerobic or facultative anaerobic culture medium is trypticase soytone. When the culture medium is manufactured, raw materials are required to be mixed after being treated respectively, raw materials are not easy to obtain, the manufacturing steps are complicated, and the nutrient substances provided by each component are single. For example, peptone used in the prior art culture medium is used for providing amino acids required for culture, and the preparation process includes the steps of gelatinization, saccharification (fermentation), enzymolysis, filtration, sterilization and the like.
Disclosure of Invention
In a first aspect, the present invention provides an extract of a Chinese medicinal composition for use in the preparation of a bacterial culture medium; extracting the traditional Chinese medicine composition extract by adopting a water leaching method; the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
280-320 parts of aloe, 180-230 parts of barley seedling, 90-100 parts of Chinese yam, 80-110 parts of coix seed, 190-200 parts of mulberry leaf and 80-130 parts of earthworm.
Aloe: contains 75 elements and almost completely coincides with the substances required by bacterial cells
Barley seedling: is the single plant substance with the most abundant and balanced nutrient content, and is the optimal substance for nourishing cells and repairing cell wounds
Chinese yam: amino acids, e.g. lysine, glycine, tyrosine, vitamins, e.g. riboflavin, niacin, thiamine, vitamin C, proteins and polysaccharides, trace elements zinc, barium, copper, iron
Semen Coicis: contains polysaccharide, fat, amino acid and vitamin B1
Mulberry leaf: contains 17 amino acids and multiple vitamins
Earthworm: contains polypeptide proteins, riboflavin, vitamins C, DNA, RNA, total reducing sugar, 17 kinds of amino acids and 11 kinds of inorganic elements (including zinc, copper, iron, selenium, chromium, manganese, calcium, potassium, silicon, arsenic and lead)
Compared with the prior art, the Chinese medicine has the advantages that the nutrient components and the mineral matters in the Chinese medicine are more abundant, and compared with the raw materials used in the culture medium in the prior art, the nutrient components are abundant, so that the bacterial proliferation is facilitated. The nutrient components of the traditional Chinese medicine meet the production requirements of low-consumption and environment-friendly culture medium. And the standards of the traditional Chinese medicines are unified during processing, so that the nutrient components of the traditional Chinese medicines in different producing areas are nearly unified, and the production needs of a standardized culture medium are met.
Further, the traditional Chinese medicine composition extracting solution comprises the following production steps: pulverizing the Chinese medicinal materials; weighing traditional Chinese medicines with corresponding quality according to a formula of the traditional Chinese medicine composition, and mixing; adding 1-2 times of water into the traditional Chinese medicine composition for soaking; sending the soaked traditional Chinese medicine composition into an extraction device for extraction to obtain an extracting solution; and performing ultrafiltration sterilization on the extracting solution.
Further, the Chinese medicinal composition is soaked for at least 8 hours; the extraction temperature is less than 60 ℃.
In a second aspect, the present invention provides a bacterial culture medium comprising agar and the above-mentioned extract of a Chinese medicinal composition; the agar dosage is 0.5-2% of the mass of the traditional Chinese medicine composition extract.
Further, the preparation method of the culture medium comprises the following steps: taking the extract of the traditional Chinese medicine composition, and adding 0.5-2% of agar powder by mass percent; heating to dissolve agar powder, sterilizing, pouring into sterile plate, cooling, and solidifying to obtain culture medium.
Furthermore, the bacterial culture medium also comprises sterile animal blood, and the dosage of the sterile animal blood is 5-10% of the mass of the traditional Chinese medicine composition extract.
Further, the preparation method of the culture medium comprises the following steps: taking the traditional Chinese medicine composition extract, and adding 0.5-2% by mass of agar powder and/or 5-10% by mass of sterile animal blood; adding sterile animal blood at 80deg.C, packaging, cooling, solidifying to obtain chocolate plate, adding sterile animal blood at 45deg.C, packaging, cooling, solidifying to obtain blood plate culture medium.
Further, the bacterial culture medium is used for culturing anaerobic bacteria.
1. The extract of the traditional Chinese medicine composition is used as a nutrient component for culturing bacteria, and the bacteria detection time is superior to that of the prior art.
2. The extract of the traditional Chinese medicine composition is used as the main nutrition component of the culture medium, so that the nutrition component is more comprehensive, and the bacterial culture is facilitated.
3. The Chinese medicinal composition is extracted by water immersion, the extraction step is simple, the operation is convenient, and nutrient substances required by the culture medium can be obtained rapidly.
4. The Chinese medicinal composition has the advantages of easy acquisition of medicinal materials, low cost, less energy consumption of the Chinese medicinal preparation process, no waste emission, stable nutrient components of the Chinese medicinal materials, relatively stable formula of the Chinese medicinal composition, meeting the requirements of culture technology and good applicability.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail in the following examples.
First, the present embodiment provides a method for preparing an extract of a Chinese medicinal composition by water extraction, comprising the steps of:
step 1: after finishing the traditional Chinese medicine raw materials, cleaning the raw materials. The floating, sinking and pollutant attached to the traditional Chinese medicine can be washed away by washing, so that the content of the effective substances in the traditional Chinese medicine can be controlled conveniently during weighing.
The following cleaning modes can be selected according to the types and characteristics of the traditional Chinese medicines:
washing method: soaking the Chinese medicinal materials in clear water, and then lightly rubbing with hands or washing with a tap to remove impurities and pollutants on the surface. The cleaning agent can be used for thoroughly cleaning by replacing clean water for multiple times, and particularly for traditional Chinese medicinal materials with soil or relatively viscosity.
Soaking method: the Chinese medicinal materials are soaked in clear water for 15 minutes according to the characteristics and pollution degree of the Chinese medicinal materials. In the soaking process, the Chinese medicinal materials are stirred or gently rubbed periodically to promote the dissolution and falling of pollutants. And achieves the effect of swelling the medicinal materials by absorbing water.
Mortar grinding method: is suitable for hard Chinese medicinal materials such as rhizome or hard fruit. The Chinese medicinal materials are put into mortar, a small amount of water is added, and the mortar and pestle are used for grinding, so that impurities and pollutants are separated from the surfaces of the Chinese medicinal materials.
Rinsing method: for some smaller herbs, such as petals or leaves, a rinsing process may be used. Placing the Chinese medicinal materials into a fine mesh bag, soaking in water, slightly shaking to float impurities to water surface, and pouring out water.
Step 2: after pulverizing the raw materials, free water is removed by centrifugation. The traditional Chinese medicine raw materials are crushed, so that the surface area of the traditional Chinese medicine is improved, and the subsequent extraction is facilitated. According to the different traditional Chinese medicinal materials, the crushed particle size is controlled to be between 0.5 and 10 millimeters, so that the subsequent filtering operation is convenient. Free water is removed by centrifugation, and the purpose is to avoid the influence of water on the measurement of traditional Chinese medicine.
An alternative comminution method is as follows:
roller crushing method: is beneficial to leaching and infiltration processes and avoids dust pollution to the environment.
And (3) manually grinding: the herbs were placed in a mortar and manually ground using a pestle, and repeated force rotation and ramming until the desired degree of comminution was achieved. The method is suitable for coarse crushing of a small amount of Chinese medicinal materials, but requires a certain strength and endurance.
Mechanical grinding: grinding is performed using a mechanical device such as a pulverizer, a grinder, or a food processor. And (3) putting the Chinese medicinal materials into equipment, operating according to an operation guideline of the equipment, and selecting proper setting parameters and grinding time until the required grinding degree is reached.
Stirring and grinding: the Chinese medicinal materials are placed in a closed container, a proper amount of grinding medium (such as ball milling medium) is added, and stirring and grinding are carried out by a stirring device. The method is suitable for coarse grinding of large-scale Chinese medicinal materials, and can achieve a certain grinding degree rapidly.
Step 3: the pretreated traditional Chinese medicine is weighed according to the formula of the traditional Chinese medicine composition, and the traditional Chinese medicine is mixed to obtain the raw materials for extracting the traditional Chinese medicine extract.
Step 4: adding pure water with the volume 1.5 times of the raw materials, and soaking for 1 hour. The raw materials can fully absorb water through soaking, so that the precipitation of effective components is facilitated.
Step 5: pasteurizing the mixture of step 4.
Step 6: after sterilization, the soaking is continued for 8 hours.
Step 7: the mixture is sent into an extraction device, and the Chinese medicinal composition is extracted by the extraction device to obtain Chinese medicinal composition extract. The extraction temperature is less than 60 ℃. The low-temperature extraction can prevent the effective components of the Chinese medicinal materials from being destroyed, and simultaneously avoid generating a large amount of ineffective components at high temperature.
Step 8: performing ultrafiltration sterilization on the traditional Chinese medicine composition extract to obtain a sterile traditional Chinese medicine composition extract; the precipitate and the residue are removed at the same time in this step.
Next, the present embodiment provides a method for preparing a culture medium by using the extract of the above-mentioned Chinese medicinal composition. The common culture medium and the preparation method are as follows:
common agar plate medium: and taking the traditional Chinese medicine composition extract, and adding 0.5-2% of agar powder by mass fraction, namely adding 5-20 g of agar powder into each kilogram of traditional Chinese medicine composition extract. Heating to dissolve agar powder, sterilizing, pouring the dissolved mixed solution into a sterile plate, cooling and solidifying to obtain the common agar plate culture medium.
Chocolate or blood agar plate medium: and (3) taking the extract of the traditional Chinese medicine composition, adding 0.5-2% of agar powder by mass percent for dissolving, sterilizing, and adding 5-10% of animal blood by mass percent. Namely, 5 to 20 grams of agar powder is added into each kilogram of the extract of the traditional Chinese medicine composition, 50 to 100 grams of sterile animal blood is added into the extract at 80 ℃ or 45 ℃ after dissolution and sterilization, and the extract is poured into a sterile plate, and the chocolate agar plate culture medium or blood agar plate culture medium is obtained after cooling and solidification. The animal blood is at least one of bovine blood, sheep blood and chicken blood, and the treatment mode is a routine treatment mode of a person skilled in the art.
Selective agar plates: adding inhibitor into the extractive solution of the Chinese medicinal composition, and preparing into tablet. A selective agar plate medium or a selective blood agar plate medium can be obtained.
Liquid medium: the liquid culture medium can be obtained by placing the sterile Chinese medicinal composition extract into a sterile culture bottle and sterilizing.
This example also provides an aerobic/facultative anaerobic culture medium. The method described in this example was used to extract the extract of the Chinese medicinal composition and prepare the culture medium using the extract of the Chinese medicinal composition.
The traditional Chinese medicine composition for preparing the aerobic bacteria/facultative anaerobic bacteria culture medium comprises the following traditional Chinese medicines in parts by mass:
200-250 parts of pine needles, 210-240 parts of whole oyster, 240-280 parts of reed rhizome, 100-150 parts of gorgon euryale seed and 150-200 parts of acanthopanax.
Pine needles: contains multiple amino acids, carotene, vitamins B1, B2, B6, C, E, K
Whole oyster: contains multiple sugar, taurine, microelements and vitamins
Reed rhizome: contains proteins, vitamins B1, B2, C, asparagine, polysaccharide and water-soluble monosaccharides
Gorgon fruit: comprises starch, protein, fat, saccharide, thiamine, riboflavin, nicotinic acid, carotene, vitamin B1, B2, C, glucoside, trace elements such as phosphorus, ferrum, barium, aluminum, and copper
Radix Et caulis Acanthopanacis Senticosi: contains seed glycoside, polysaccharide, fatty acid, chlorogenic acid, and antioxidant.
The embodiment also provides an anaerobic bacteria culture medium, the method of the embodiment is adopted to extract the traditional Chinese medicine composition extract, and the culture medium is prepared by using the traditional Chinese medicine composition extract.
The traditional Chinese medicine composition for preparing the anaerobic bacteria culture medium comprises the following traditional Chinese medicines in parts by mass:
280-320 parts of aloe, 180-230 parts of barley seedling, 90-100 parts of Chinese yam, 80-110 parts of coix seed, 190-200 parts of mulberry leaf and 80-130 parts of earthworm.
Aloe: contains 75 elements and almost completely coincides with the substances required by bacterial cells
Barley seedling: is the single plant substance with the most abundant and balanced nutrient content, and is the optimal substance for nourishing cells and repairing cell wounds
Chinese yam: amino acids, e.g. lysine, glycine, tyrosine, vitamins, e.g. riboflavin, niacin, thiamine, vitamin C, proteins and polysaccharides, trace elements zinc, barium, copper, iron
Semen Coicis: contains polysaccharide, fat, amino acid and vitamin B1
Mulberry leaf: contains 17 amino acids and multiple vitamins
Earthworm: contains polypeptide proteins, riboflavin, vitamins C, DNA, RNA, total reducing sugar, 17 kinds of amino acids and 11 kinds of inorganic elements (including zinc, copper, iron, selenium, chromium, manganese, calcium, potassium, silicon, arsenic and lead)
In order to verify the culture medium prepared by the extract of the traditional Chinese medicine composition provided by the invention, the culture medium is verified by adopting the following method.
1. Culture medium verification basis
Control medium: the ingredients are used for culturing fungi and aerobic bacteria according to a 1101 aseptic inspection method in the source of Chinese pharmacopoeia 2020 edition, and a tryptone liquid culture medium is adopted in comparative example 1; comparative example 2 thioglycolate fluid medium was used for anaerobic cultivation. The same morphology medium as that of the comparative example was prepared according to the relevant requirements.
The verification method comprises the following steps: CNAS-GL028 section 4.4.1 of the clinical microbiological test program verification guide
2. Culture medium verification using instrument
Full-automatic bacteria culture instrument
3. Culture medium validation use of test strains
Aerobic/facultative anaerobic strains: pseudomonas aeruginosa, acinetobacter baumannii, streptococcus agalactiae, escherichia coli, streptococcus pyogenes, staphylococcus aureus, enterococcus faecalis, salmonella and Klebsiella pneumoniae
Anaerobic strain: clostridium perfringens and bacteroides fragilis
Fungus strain: candida albicans, candida krusei and candida parapsilosis
Causticizing oxygen strain: haemophilus influenzae, streptococcus pneumoniae
4. Culture medium Performance validation parameters
Detecting strains: aerobic/facultative anaerobic gram-positive bacteria, aerobic/facultative anaerobic gram-negative bacteria, fastidious bacteria, fungi, obligate anaerobic bacteria, and the like.
Detection time: positive was found within 72 hours.
Verification indexes: positive detection rate = instrument number of detected vials/total number of experimental vials
Results and evaluation criteria: according to CNAS-GL028, the 3 day period should be sufficient to detect at least 95% of clinically relevant bacteria, and must be capable of detecting causticizing bacteria, fungi, anaerobic bacteria, etc.
Examples 1 to 4 are aerobic/facultative anaerobic culture mediums prepared from extract of Chinese medicinal composition;
examples 5 to 8 are anaerobic bacteria culture mediums prepared from extract of Chinese medicinal composition;
example 1:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
240 parts of pine needles, 210 parts of whole oyster, 250 parts of reed rhizome, 150 parts of gorgon euryale seed and 150 parts of acanthopanax.
Example 2:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
200 parts of pine needles, 220 parts of whole oyster, 240 parts of reed rhizome, 100 parts of gorgon euryale seed and 160 parts of acanthopanax.
Example 3:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
230 parts of pine needles, 230 parts of whole oyster, 270 parts of reed rhizome, 110 parts of gorgon euryale seed and 180 parts of acanthopanax.
Example 4:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
250 parts of pine needles, 240 parts of whole oyster, 280 parts of reed rhizome, 150 parts of gorgon euryale seed and 200 parts of acanthopanax.
Example 5:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
300 parts of aloe, 200 parts of barley seedling, 100 parts of Chinese yam, 100 parts of coix seed, 200 parts of mulberry leaf and 100 parts of earthworm.
Example 6:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
280 parts of aloe, 180 parts of barley seedling, 90 parts of Chinese yam, 80 parts of coix seed, 190 parts of mulberry leaf and 80 parts of earthworm.
Example 7:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
320 parts of aloe, 230 parts of barley seedling, 100 parts of Chinese yam, 110 parts of coix seed, 200 parts of mulberry leaf and 130 parts of earthworm.
Example 8:
the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
290 parts of aloe, 210 parts of barley seedling, 10 parts of Chinese yam, 90 parts of coix seed, 200 parts of mulberry leaf and 110 parts of earthworm.
Table 1 summary of validation results
Analysis of results: sixteen strains of the verification strain are covered with aerobic bacteria, fungi, facultative anaerobic bacteria, anaerobic bacteria and causticizing bacteria. Each strain was inoculated with each strain, and each strain was inoculated with the culture medium described in examples and comparative examples, and all the bacteria were detected.
Verification conclusion: the average detection time of the examples 1-8 is better than that of the comparative examples 1 and 3, and the clinical examination quality requirement is met.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (8)
1. The Chinese medicinal composition extracting solution is characterized in that the extracting solution of the Chinese medicinal composition is used for preparing a bacterial culture medium; extracting the traditional Chinese medicine composition extract by adopting a water leaching method; the traditional Chinese medicine composition comprises the following traditional Chinese medicines in parts by mass:
280-320 parts of aloe, 180-230 parts of barley seedling, 90-100 parts of Chinese yam, 80-110 parts of coix seed, 190-200 parts of mulberry leaf and 80-130 parts of earthworm.
2. The extract of a Chinese medicinal composition according to claim 1, wherein: the traditional Chinese medicine composition extracting solution comprises the following production steps:
pulverizing the Chinese medicinal materials;
weighing traditional Chinese medicines with corresponding quality according to a formula of the traditional Chinese medicine composition, and mixing;
adding 1-2 times of water into the traditional Chinese medicine composition for soaking;
sending the soaked traditional Chinese medicine composition into an extraction device for extraction to obtain an extracting solution;
and performing ultrafiltration sterilization on the extracting solution.
3. The extract of a Chinese medicinal composition according to claim 2, wherein: soaking the Chinese medicinal composition for at least 8 hours; the extraction temperature is less than 60 ℃.
4. A bacterial culture medium, characterized by: the bacterial culture medium comprises agar and the Chinese medicinal composition extract as claimed in claim 3; the agar dosage is 0.5-2% of the mass of the traditional Chinese medicine composition extract.
5. The bacterial culture medium according to claim 4, wherein the medium is prepared by the following method:
taking the extract of the traditional Chinese medicine composition, and adding 0.5-2% of agar powder by mass percent; heating to dissolve agar powder, sterilizing, pouring the dissolved mixed solution into a sterile plate, and cooling and solidifying to obtain a culture medium plate.
6. A bacterial culture according to claim 4, wherein: the bacterial culture medium plate also comprises a blood plate or a chocolate plate added with sterile animal blood, wherein the animal blood is 5-10% of the mass of the traditional Chinese medicine composition extract.
7. The bacterial culture medium according to claim 6, wherein the medium is prepared by the following method:
adding 0.5-2% of agar powder by mass percent into the extract of the traditional Chinese medicine composition to dissolve, sterilizing, cooling to 80 ℃ or 45 ℃, and adding 5-10% of sterile animal blood by mass percent; pouring the dissolved mixed solution into a sterile plate, and cooling and solidifying to obtain a nutrient medium plate.
8. A bacterial culture according to claim 4, wherein: the bacterial culture medium is used for culturing anaerobic bacteria.
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