CN113832069B - Clostridium butyricum and application thereof - Google Patents
Clostridium butyricum and application thereof Download PDFInfo
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- CN113832069B CN113832069B CN202111200171.5A CN202111200171A CN113832069B CN 113832069 B CN113832069 B CN 113832069B CN 202111200171 A CN202111200171 A CN 202111200171A CN 113832069 B CN113832069 B CN 113832069B
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- clostridium butyricum
- choline
- dopamine
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Abstract
The invention relates to clostridium butyricum and application, medicine, food, health care product, veterinary medicine, feed additive and microecological preparation thereof, and belongs to the technical field of microorganisms. The clostridium butyricum of the invention is classified and named as clostridium butyricum (Clostridium butyricum) F06, and the preservation number is CCTCC NO: m2019962. The clostridium butyricum can be applied to producing neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline, acetylcholine, L-tryptophan, tyrosine, taurine, acetylcholine, tyramine and the like. After fermentation conditions are optimized, the contents of gamma-aminobutyric acid, dopamine, glutamic acid and choline in clostridium butyricum fermentation liquid respectively reach 4.78g/L,5.82g/L,135.9g/L and 3.78g/L, and clostridium butyricum can be applied to producing neurotransmitter substances and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to clostridium butyricum and application thereof.
Background
Gamma-aminobutyric acid is an important neurotransmitter in the central nervous system, is a naturally occurring non-protein constituent amino acid, has extremely important physiological functions, can promote the activation of brain, strengthen brain and improve intelligence, resist epilepsy, promote sleep, beautify and moisten skin, delay the aging function of brain, supplement human body inhibitory neurotransmitter, and has good blood pressure reducing effect. Secondly, it can also promote renal function improvement and protection. Inhibit fatty liver and obesity, and activate liver function. The daily supplementation of trace gamma-aminobutyric acid is beneficial to relieving heart and brain blood pressure, promoting the balance of amino acid metabolism in human body and regulating immune function.
Glutamate is an excitatory neurotransmitter, and brain tissue can only oxidize glutamate, but not other amino acids, so glutamate can be used as an energy substance for brain tissue to improve and maintain brain function. Glutamic acid is used as a supplement to the nerve center and cerebral cortex. Glutamate can promote peristalsis of the digestive tract. Dopamine plays an important role in motor control, and parkinson's disease is caused by severe dopamine reduction caused by dopaminergic neuronal degeneration. The limbic dopamine system and the mesocortical dopamine system are widely believed to play a role in learning and memory. It has been reported that the dopaminergic neurons of the monkey a10 region are involved in the learning of transient changes in basic attention and impulsive activity during motivation in cognitive behavior. The fact that cognitive function will decline with age is well documented in both humans and primates. In recent years, studies have provided direct evidence that demonstrates the obvious correlation between reduced dopamine function, reduced cognitive function and brain aging, and comparative studies have been performed on volunteers of the same age, which have shown that dopamine levels are positively correlated with cognitive function. Studies have shown that mood depression, low pleasure, low mood, and low dopamine levels.
Choline is a precursor of acetylcholine in all biological membrane components and cholinergic neurons. The choline can promote brain development and improve memory capacity, ensure information transmission, regulate apoptosis, prevent cholesterol deposition on the inner wall of blood vessel and remove partial sediment, and improve fat absorption and utilization, so that the choline has the effect of preventing cardiovascular diseases.
Neurotransmitters such as gamma-aminobutyric acid, glutamic acid, dopamine and choline can be converted and obtained under the catalytic action of various enzymes in the body, but under the conditions of aging, stress state, intestinal microecological disorder, diseases or high mental stress, the synthesis of neurotransmitters (gamma-aminobutyric acid, glutamic acid, dopamine and choline and the like) in the body is obviously reduced, so that the phenomenon of deficiency occurs. Exogenous neurotransmitter addition can improve the condition of insufficient synthesis of endogenous neurotransmitter, and promote body health. It is also possible to produce neurotransmitters in the intestine for direct absorption and utilization by supplementing the functional probiotics. Therefore, development of foods, health products, feeds or microecological additives rich in neurotransmitters (gamma-aminobutyric acid, dopamine, glutamic acid, choline, etc.) has become a hot spot of research.
The health food has been developed from rice germ in japan, has remarkable effects in improving insomnia, depression, climacteric syndrome, etc., and is widely added to various foods such as beverages, jams, and pastries. However, the rice germ has low content, and the rice germ can reach high content only by separation and concentration, thereby restricting the application of the rice germ. Therefore, the development of beneficial microorganisms by biotechnology and the production of foods, health products or feeds rich in gamma-aminobutyric acid and additives thereof by the beneficial microorganisms are key technologies for solving the problem. The application numbers CN201910502771.3 and CN201711386926.9 both disclose a streptococcus thermophilus with high gamma-aminobutyric acid yield. Application number CN201910502750.1 discloses a lactococcus lactis subspecies lactis with high yield of gamma-aminobutyric acid. The application number CN201910274454.0 discloses lactobacillus rhamnosus with high yield of gamma-aminobutyric acid. The application number CN201610964033.7 discloses a lactobacillus plantarum producing gamma-aminobutyric acid. Application number CN201611003216.9 discloses a strain of Lactobacillus pentosus producing gamma-aminobutyric acid. Application number CN201710149052.9 discloses rhizopus oryzae with high yield of gamma-aminobutyric acid. Application number CN201910773775.5 discloses a screening method of single bacteria of dopamine-promoting components. The application number CN201910257075.0 discloses a recombinant engineering bacterium for producing dopamine by catalyzing substrate dopa. Application number CN201911397692.7 discloses a strain of Escherichia coli for efficiently producing glutamic acid. The application numbers CN202010147933.9, CN201310361804.X and CN202010227468.X respectively disclose a recombinant corynebacterium glutamicum for efficiently producing L-glutamic acid. Most of the microorganisms mentioned above can be used as food or feed and additives therefor. However, these microorganisms produce a relatively single neurotransmitter, and only one neurotransmitter is produced. If these microorganisms are used in co-culture to produce a plurality of neurotransmitters, antagonism may exist between these microorganisms, thereby failing to achieve the effect of producing a plurality of neurotransmitters. In addition, some microorganisms (lactic acid bacteria, etc.) have poor in vitro viability, are intolerant to gastric acid and bile acid, and cannot simultaneously produce neurotransmitters such as gamma-aminobutyric acid, glutamic acid, dopamine, choline, etc. in the intestinal tract, thus limiting the application thereof. Thus, it is a new challenge to obtain probiotics that are more active and are capable of producing multiple neurotransmitters simultaneously.
Disclosure of Invention
The invention aims to provide clostridium butyricum capable of producing various neurotransmitters, and the functional probiotics metabolite is rich in not only gamma-aminobutyric acid, but also various neurotransmitters such as dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), tyrosine, taurine, acetylcholine, tyramine and the like.
It is a further object of the present invention to provide the use of clostridium butyricum as described above, including in particular in the production of neurotransmitters, in the preparation of pharmaceuticals, in the preparation of veterinary drugs, feed additives or in the preparation of micro-ecological preparations.
A third object of the present invention is to provide a pharmaceutical, veterinary, feed additive or microecological preparation comprising the clostridium butyricum described above.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides clostridium butyricum, which is classified and named as clostridium butyricum (Clostridium butyricum) F06, and has a preservation number of CCTCC NO: M2019962F06. The clostridium butyricum belongs to the family of bacillus, genus clostridium and species clostridium butyricum.
The clostridium butyricum is a strain which is used for screening neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like from intestinal contents of healthy carps by an anaerobic culture method by applying an improved RCM culture medium, and the functional probiotics metabolite is rich in not only gamma-aminobutyric acid, but also dopamine, glutamic acid, choline and L-tryptophan (5-hydroxytryptamine precursor), tyrosine, taurine, acetylcholine, tyramine and the like. Clostridium butyricum cctccc NO: m2019962, the colony is round and cloudy white. The microscopic thallus is in a short rod shape, gram positive, spore and spore oval. The 16S rDNA electrophoresis and sequencing analysis show that the homology of clostridium butyricum F06 and the 16S rDNA of clostridium butyricum (CP016332.1CP013352.1LN828942.1LN828931.1AB687551.1NR113244.1X68177.1) reaches 100 percent.
Clostridium butyricum, also known as Clostridium butyricum, clostridium butyricum. Clostridium butyricum belongs to the family Bacillus, genus Clostridium, G + The endophytic spore can resist gastric acid and bile acid and can resist adverse environment. The clostridium butyricum can be widely applied to the fields of medicines, veterinary medicines, feed additives, microecologics and the like.
The clostridium butyricum is preserved, and the preserved strain is: clostridium butyricum (Clostridium butyricum) F06, accession number: cctccc NO: m2019962, date of preservation: 11 months and 20 days in 2019, preservation unit: china Center for Type Culture Collection (CCTCC), preservation address: university of Chinese Wuhan, wuhan (university of Wuhan, hubei province, wuhan and Wuhan university of mountain Lopa nationality).
It is understood that neurotransmitters are collectively referred to herein as neurotransmitters and neurotransmitter precursors.
The invention provides the use of the clostridium butyricum described above for the production of neurotransmitters, which are neurotransmitters and/or neurotransmitter precursors.
In particular, the clostridium butyricum is used for producing neurotransmitter substances, wherein the neurotransmitter substances comprise one or any combination of gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, taurine and tyramine.
Further, the use of clostridium butyricum as described above for the production of neurotransmitters, including gamma-aminobutyric acid, dopamine, glutamate, choline, L-tryptophan and L-tyrosine. Still further, the gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan, L-tyrosine and tyramine.
Further, the neurotransmitters include gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine, and taurine.
Further, the neurotransmitters include gamma-aminobutyric acid, dopamine, glutamic acid and choline.
Specifically, the preservation number is CCTCC NO: clostridium butyricum of M2019962 was inoculated into the medium for fermentative production of neurotransmitters.
Furthermore, the invention optimizes the culture medium components and fermentation conditions for producing neurotransmitters by clostridium butyricum fermentation.
Preferably, the culture medium includes a seed medium and a fermentation medium. Wherein, the seed culture medium comprises the following components with the following concentration (g/L): glucose 25, soybean meal 10, corn steep liquor 30, sodium glutamate 10, potassium dihydrogen phosphate 1.5, magnesium sulfate 0.2, ammonium sulfate 0.2, disodium hydrogen phosphate 0.5 and calcium carbonate 0.3. The fermentation medium comprises the following components in the following concentration (g/L): glucose 140, soybean meal 15, corn steep liquor 40, magnesium sulfate 0.5, ammonium sulfate 0.2, monopotassium phosphate 0.5, disodium hydrogen phosphate 0.5 and calcium carbonate 0.3.
Preferably, the fermentation conditions are: the pH is 6.8-7.4, the temperature is 22-45 ℃, the inoculation amount is 1-10%, and the culture time is 12-36 h.
Further preferred, optimal fermentation conditions: the pH was 7.2, the temperature was 35 ℃, the inoculum size was 4%, and the incubation time was 24h.
The invention provides application of clostridium butyricum in preparation of medicines.
The invention provides a method for preparing the composite material, which comprises the following steps of: m2019962 Clostridium butyricum F06.
The invention provides application of clostridium butyricum F06 in preparation of veterinary drugs, feed additives or microecological preparations.
The invention provides a method for preparing the composite material, which comprises the following steps of: veterinary drug of clostridium butyricum F06 of M2019962.
The invention provides a method for preparing the composite material, which comprises the following steps of: clostridium butyricum F06 feed additive M2019962.
The invention provides a method for preparing the composite material, which comprises the following steps of: a probiotic of clostridium butyricum F06 of M2019962.
The invention has the beneficial effects that:
the invention uses an improved RCM culture medium, and screens out a strain with high yield of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like from intestinal contents of healthy carps by an anaerobic culture method: clostridium butyricum F06 (CCTCC No: M2019962). Compared with lactic acid bacteria for producing single neurotransmitter substances, the clostridium butyricum provided by the invention has the following characteristics: 1) Is bacillus, has strong stress resistance and can resist the actions of gastric acid and bile acid; 2) Anaerobic bacteria grow and reproduce in colon or cecum, and do not contend with host for nutrition; 3) Metabolites are rich in a variety of neurotransmitters including gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine, taurine, etc.; in addition, the clostridium butyricum provided by the invention is different from the conventional clostridium butyricum, and the metabolic product of the conventional clostridium butyricum is butyric acid, and the metabolic product of clostridium butyricum provided by the invention not only comprises butyric acid, but also is rich in neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine, taurine and the like. Experiments prove that after fermentation conditions of the strain are optimized, the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in fermentation liquor of the strain reaches 4.78g/L,5.82g/L,135.9g/L and 3.78g/L respectively, and the strain can be widely applied to the fields of feed additives, veterinary medicines, whole field probiotics and the like and has wide application prospects.
Drawings
FIG. 1 is a graph of Clostridium butyricum F06 colonies;
FIG. 2 is a staining microscopic image of Clostridium butyricum F06;
FIG. 3 is a graph showing the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in clostridium butyricum F06 fermentation broth at different time points;
FIG. 4 is a graph showing the content of gamma-aminobutyric acid, dopamine, glutamic acid, choline and other neurotransmitters in clostridium butyricum F06 fermentation broth at different pH, temperature and inoculum size;
FIG. 5 shows the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid and choline in clostridium butyricum F06 fermentation broth at different temperatures;
FIG. 6 is a graph showing the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in clostridium butyricum F06 fermentation broth at different inoculum sizes;
FIG. 7 shows the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid and choline in fermentation broth after optimization of clostridium butyricum F06 fermentation conditions.
Detailed Description
The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples were all conventionally commercially available unless otherwise specified.
Example 1 screening and identification of Clostridium butyricum F06
1. Screening of clostridium butyricum for the production of various neurotransmitters:
the media involved in the screening were as follows:
1) Modified RCM liquid screening media, 1000mL, contained: 10g of tryptone; 10g of beef extract; 3g of yeast extract; glucose 5g; 1g of soluble starch; 5g of sodium chloride; 3g of sodium acetate; 0.5g of L-cysteine;
3g of sodium glutamate; 0.02g of polymyxin B; 6% yeast extract powder; 1.74% ferrous sulfate; 0.37% dipotassium hydrogen phosphate; 0.2% sodium chloride; 0.024% magnesium sulfate.
2) Modified RCM solid screening media: 1000mL of modified RCM broth was added with 15g agar.
3) The modified enhanced clostridium medium, 1000mL, contains: 10g of protein; 10g of beef extract; 3g of yeast powder;
glucose 5g; 1g of soluble starch; 5g of sodium chloride; 0.5g of L-cysteine; 3g of sodium glutamate; 15g of agar;
pH7.0-7.2。
4) Improved enhanced clostridium solid media: 1000mL of modified enhanced Clostridium medium was added with 15g of agar.
5) Seed fermentation medium, 1000mL contains: glucose 25g; 10g of soybean meal; 30g of corn steep liquor; 10g of sodium glutamate;
1.5g of monopotassium phosphate; 0.2g of magnesium sulfate; 0.2g of ammonium sulfate; disodium hydrogen phosphate 0.5g; 0.3g of calcium carbonate.
All media were sterilized at high temperature and pressure (121 ℃, 1X 105 kPa).
The specific screening steps are as follows:
1) The intestinal contents of healthy carp in the Henan province area of yellow river are weighed 5g, and 1:4, adding the strain into sterile physiological saline, and placing the strain in a water bath at 70 ℃ for 15 minutes for preliminary screening to kill part of non-spore bacteria;
2) Then 5mL of culture solution is extracted and transferred into 100mL of modified RCM liquid culture medium, and anaerobic culture is carried out for 48h under the conditions of 37 ℃ and 180 rpm;
3) Extracting 5mL of the culture solution into 100mL of modified reinforced clostridium liquid culture medium, and selectively enriching and culturing for 48h under the anaerobic condition at 37 ℃;
4) Extracting 100uL of culture solution for gradient dilution, then selecting proper dilution gradients to be respectively coated in modified reinforced clostridium solid medium plates, placing in an anaerobic incubator, and culturing for 48 hours at 37 ℃;
5) Selecting a gram-positive strain with colony morphology, culture characteristics, biochemical characteristics and microscopic morphology which all meet the culture characteristics of clostridium butyricum for subsequent experiments;
6) And (3) fermenting and culturing the strain conforming to the culturing characteristics of clostridium butyricum for 24 hours by a seed fermentation medium, centrifuging a fermentation liquid (4 ℃,12000r/min,10 min) to obtain a supernatant, measuring the content of gamma-aminobutyric acid, dopamine, choline and other neurotransmitters in the fermentation supernatant by adopting a high performance liquid chromatography, screening out the strain with the strongest neurotransmitter production capacity, and storing.
2. Identification of clostridium butyricum
1) Morphological and physiological Biochemical characterization
According to the experimental method in the common bacteria system identification manual, the strain with the strongest neurotransmitter substance capability of producing gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like is identified in morphological characteristics and physiological and biochemical characteristics.
2) 16S rDNA sequence analysis
The total DNA of the genome was extracted, and the 16S rRNA fragment of the strain was amplified using the 16S rRNA universal primers 27F,5 '-AGAGTTTGATCCTGGGCTCAG-3' and 1492R,5 '-TACGGCTACCTTGTTACGAC-3'.
The PCR reaction system (25. Mu.L) was: 2 XTaq Mix 12.5. Mu.L, upstream primer 27F (10. Mu. Mol/L) 1. Mu.L, downstream primer 1492R (10. Mu. Mol/L) 1. Mu.L, bacterial DNA 2.5. Mu.L, and sterile water 8. Mu.L.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 50s,28 cycles; extending at 72℃for 10min.
The PCR product was subjected to 2% TAE agarose gel electrophoresis, and the size of the PCR product was detected, and then the 16S rDNA gene product was submitted to Shanghai Bioengineering Co. The sequencing results were BLAST against GenBank databases in NCBI and the phylogenetic tree was constructed to analyze sequence homology.
3. Results and analysis
The 12 strains which meet the culture characteristics of clostridium butyricum are screened out, seed fermentation broths of the 12 strains are respectively detected for the capability of producing neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid and choline, and the result shows that the 6 strain has the strongest capability of producing neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid and choline. Thus strain 06 was selected for subsequent experiments and designated clostridium butyricum F06. Clostridium butyricum cctccc NO: m2019962, colony (FIG. 1) was round and cloudy white. Microscopic examination (FIG. 2) shows that the thallus is in a short rod shape, gram positive, spore and oval.
The 16S rDNA gene sequence of the strain F06 is obtained through PCR amplification, sequencing is carried out on NCBI by using Blast comparison, related sequences are obtained from a GenBank database and are subjected to phylogenetic analysis, and the sequencing result is shown in SEQ ID NO.1. The result shows that the homology of the strain F06 and the 16S rDNA of clostridium butyricum (CP016332.1CP013352.1LN828942.1LN828931.1AB687551.1NR113244.1X68177.1) reaches 100 percent, and the strain F06 is judged to be clostridium and named clostridium butyricum F06 by combining physiological and biochemical characteristics.
The above results show that clostridium butyricum F06 can produce neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in high yield, so that the clostridium butyricum F06 is preserved in China Center for Type Culture Collection (CCTCC) for 11 months and 20 days in 2019, with the preservation number: CCTCCNO: m2019962, deposit address: university of Chinese Wuhan, wuhan (university of Wuhan, hubei province, wuhan and Wuhan university of mountain Lopa nationality).
EXAMPLE 2 use of Clostridium butyricum F06 for the production of neurotransmitters
This example provides the use of clostridium butyricum of example 1 for the production of neurotransmitters, including gamma-aminobutyric acid, dopamine, glutamate and choline.
1. Clostridium butyricum F06 growth curve assay
Clostridium butyricum F06, which is the strain with the strongest neurotransmitter-producing ability and stored in the laboratory, is inoculated into a modified RCM solid medium, subjected to stationary culture at 37 ℃ under anaerobic conditions for 24 hours, and then the activated strain is inoculated into the modified enhanced Clostridium culture medium at 37 ℃ and 180rpm, and cultured overnight under anaerobic conditions as a seed solution.
6 test tubes containing 3.9mL of RCM medium were taken and the incubation times were marked with markers, namely 0, 4, 8, 12, 24, 48h. 100. Mu.L of seed solution was accurately aspirated each time using a pipette, and inoculated into 6 numbered tubes, respectively. Anaerobic stationary culture at 37 deg.C after inoculation, taking out test tubes numbered as corresponding time at 0, 4, 8, 12, 24 and 48 hr respectively, and immediately storing in refrigerator.
Finally, the non-inoculated RCM liquid culture medium is used as a blank control, and the 600nm wavelength is selected for photoelectric turbidimetry to determine the growth curve of clostridium butyricum F06. And detecting the content of neurotransmitters such as gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu) and Choline (Choline) at each time point by using high performance liquid chromatography.
As a result, as shown in FIG. 3, the number of colonies was measured at 24 hours, and the content of neurotransmitters such as gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu) and Choline (Choline) was maximized.
2. Determination of optimal pH, temperature and inoculum size of clostridium butyricum F06 fermentation medium
The initial pH of the seed culture medium was adjusted to 6.8, 7.0, 7.2, 7.4, 7.6 and 7.8 with 4mol/L HCl and 20% NaOH solution, respectively, and 2% seed solution was added thereto, and the culture was performed under anaerobic conditions at 37℃and 180rpm for 24 hours. The number of bacteria was measured by the hemocytometer method. 2% Clostridium butyricum F06 bacteria solution was inoculated into 100mL of modified RCM medium, and cultured at constant temperature of 30℃at 37℃at 40℃and at 45℃at 180rpm, respectively, for 24 hours under anaerobic conditions. After inoculating clostridium butyricum F06 seed solution at 0.5%, 1%, 2%, 4%, 6% and 8%, culturing was performed under anaerobic conditions at 37 ℃,180rpm for 24 hours. Then, the colony count and the content of neurotransmitters such as gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu), choline (Choline) and the like under the conditions of pH, temperature and inoculum size are respectively measured by a blood cell count method and a high performance liquid chromatography method.
The results are shown in FIG. 4, FIG. 5 and FIG. 6, and the optimal culture conditions are as follows: the pH was 7.4, the temperature was 37℃and the inoculum size was 4%, and the number of colonies was measured at 24 hours for the culture, and the amounts of neurotransmitters such as gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu) and Choline (Choline) were the largest.
3. Optimization of Clostridium butyricum F06 Medium composition
Glucose, soybean meal, corn steep liquor are the main components affecting the culture medium. Thus, test design L16 (3 4 ) Orthogonal experiments, which determine the components of clostridium butyricum F06 medium, were designed as follows: glucose (100 g,120g,140 g), soybean meal (10 g,15g,20 g), corn steep liquor (20 g,30g,40 g). Then, the colony number and the content of neurotransmitters such as gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu) and Choline (Choline) under the conditions of various components are respectively measured by a blood cell count method and a high performance liquid chromatography method.
TABLE 1 results of the influence of the orthogonal Medium composition test on GABA content
Note that: the ia value reflects the effect of levels of each factor B, C of 1, 2, 3 on A1 level (A, B, C stands for glucose, soybean meal, corn meal, respectively). Similarly, IA reflects the effect of three levels of each factor B, C on A2 (A3) levels once each. When comparing the differences between IA, IIA, IIIA, the effect of B, C on IA, IIA, IIIA can be considered to be substantially identical. Thus, the difference between IA, IIA, IIIA can be seen as being due to factor A taking three different levels. According to this method, IB, IIB, IIIB and IC, IIC, IIIC can be calculated. The difference between the maximum and minimum values in each of columns I, II, III is then calculated and is the very difference, designated R. The range of each column reflects the magnitude of the effect of the selected level variation of the factor corresponding to that column on GABA content. The greater the value, the greater the extent to which the factor affects GABA content. Tables 2, 3 and 4 are the same.
TABLE 2 results of the influence of the orthogonal Medium composition experiments on Glu content
TABLE 3 results of the influence of the orthogonal Medium composition test on DA content
Numbering device | Glucose (g) | Bean pulp (g) | Corn flour (g) | DA content (g) |
1 | 100 | 10 | 20 | 3.89 |
2 | 100 | 15 | 30 | 4.38 |
3 | 100 | 20 | 40 | 5.14 |
4 | 120 | 10 | 30 | 5.24 |
5 | 120 | 15 | 40 | 3.86 |
6 | 120 | 20 | 20 | 4.26 |
7 | 140 | 10 | 40 | 4.32 |
8 | 140 | 15 | 20 | 5.08 |
9 | 140 | 20 | 30 | 3.69 |
Ⅰ | 13.41 | 12.07 | 13.23 | |
Ⅱ | 13.36 | 13.32 | 13.31 | |
Ⅲ | 13.09 | 13.09 | 13.32 | |
R | 0.32 | 1.02 | 0.09 |
TABLE 4 results of the influence of orthogonal Medium composition experiments on Choline content
The results are shown in tables 1 to 4, and clostridium butyricum F06 contained 140g of glucose in the medium; bean pulp, 15g; corn flour, 20g; the culture conditions were pH 7.4, the temperature was 37℃and the inoculum size was 4%, and the maximum gamma-aminobutyric acid (GABA) content was measured at 24 hours of the culture (Table 1). Clostridium butyricum F06 in the medium composition of glucose, 140g; bean pulp, 20g; corn flour, 30g; the culture conditions were pH 7.4, the temperature was 37℃and the inoculum size was 4%, and the maximum glutamic acid (Glu) content was measured at 24 hours of culture (Table 2). Clostridium butyricum F06 in the medium composition of glucose, 130g; 10g of soybean meal; corn flour, 30g; the cultivation conditions were pH 7.4, the temperature was 37℃and the inoculum size was 4%, and the maximum Dopamine (DA) content was measured for 24 hours (Table 3). Clostridium butyricum F06 in the medium composition of glucose, 140g; 10g of soybean meal; corn flour, 40g; the cultivation conditions were pH 7.4, temperature 37℃and inoculum size 4%, and the maximum Choline (Choline) content was measured at 24 hours. Since the above results are that the combination under laboratory conditions has not been subjected to production practice, further experiments have been carried out to verify the colony count of clostridium butyricum F06 and the content of the related neurotransmitters under the combination conditions. From tables 1 to 4, it is understood that the main component affecting GABA content is glucose, the main component affecting Glu content is glucose, the main component affecting DA content is soybean meal, and the main component affecting Choline content is corn starch. Therefore, in consideration of test results and production cost, clostridium butyricum F06 is selected to be 140g of glucose in the culture medium; bean pulp, 15g; corn flour, 40g; the culture condition is that the pH is 7.4, the temperature is 37 ℃, the inoculation amount is 4 percent, and the neurotransmitter content in the fermentation broth is detected when the culture time is 24 hours. As can be seen from FIG. 7, the content of gamma-aminobutyric acid, glutamic acid, dopamine and choline in clostridium butyricum fermentation broth measured under the conditions reached 4.78g,135.9g,5.82g and 3.78g/L, respectively.
EXAMPLE 3 Clostridium butyricum F06 simulated gut fermentation product metabonomics analysis
This example the specific test method for the analysis of the metabonomics of the simulated gut fermentation products of clostridium butyricum F06 provided in example 1 is as follows:
after inoculation of the fermentation broth with clostridium butyricum F06, samples were taken at 2, 4, 6, 8, 12, 24 hours, numbered A, B, C, D, E, F in sequence. Then, non-targeted metabonomics analysis based on ultra-high performance liquid chromatography-Q-TOF MS is carried out to obtain primary mass spectrum and secondary mass spectrum data, and XCMS is adopted to carry out peak extraction and metabolite identification on the data.
In the test results, A, B, C, D (2, 4, 6, 8 hours) samples, i.e. during clostridium butyricum F06 breeding season: the transmitters related to the nervous system signal transmission, such as gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, tyramine, taurine and the like are obviously rich. The test results are shown in Table 5.
TABLE 5 results of metabonomic analysis of clostridium butyricum F06 simulated digestive tract fermentation products
Note that: mzmed is the median of m/z, the intermediate number of mass to charge ratios. rtmed is median of retention time, the median of the retention times, which together with mzmed is characteristic of this substance. POS a, B, C, D, E, F represent the analysis results in positive ion mode at 2, 4, 6, 8, 12, 24 hours, respectively (POS is an abbreviation for positive, i.e. positive ion or positive).
Example 4
This example is the use of clostridium butyricum as described in example 1 in the preparation of a medicament.
In practical application, the medicines comprise the components with the preservation number of CCTCC NO: clostridium butyricum of M2019962.
And (3) setting the preservation number as CCTCC NO: clostridium butyricum of M2019962 fed mice, the test was set to 4 groups (negative control group, low dose 5000CFU, medium dose 10000CFU and high dose 50000CFU, respectively), each group being repeated 10 times. After 1 week of continuous feeding, the heart was sampled and plasma was isolated. Based on LC-MS/GC-MS technology, targeted metabonomics analysis is carried out, and the feeding preservation number is CCTCC NO: m2019962 Clostridium butyricum mice contained higher levels of gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu), choline (Choline), L-tryptophan, L-tyrosine, taurine, acetylcholine and tyramine in the plasma than mice without the addition of the bacteria.
Example 5
This example is the use of clostridium butyricum as described in example 1 in the preparation of veterinary drugs, feed additives or microecologics.
In practical application, the veterinary drug, the feed additive and the microecological preparation all comprise the components with the preservation number of CCTCC NO: clostridium butyricum F06 of M2019962.
And (3) setting the preservation number as CCTCC NO: clostridium butyricum F06 of M2019962 is fed to broilers and pigs, and targeted metabonomics analysis detects the content level of gamma-aminobutyric acid (GABA), dopamine (DA), glutamic acid (Glu), choline (Choline), L-tryptophan, L-tyrosine, taurine, acetylcholine and tyramine in blood, and finds that the feeding and preserving number is cctccc NO: both chickens and pigs of clostridium butyricum F06 of M2019962 had higher blood levels than were not fed with this bacterium.
<110> Henan agricultural university
<120> Clostridium butyricum and its application, medicine, food, health product, veterinary drug, feed additive, and microecological preparation
<160> 1
<170> PatentIn version 3.5
<211> 1415
<212> DNA
<213> Clostridium butyricum
<221> 16s rDNA
<400> 1
gagtttgatc ctggctcagg acgaacgctg gcggcgtgct taacacatgc aagtcgagcg 60
atgaagctcc ttcgggagtg gattagcggc ggacgggtga gtaacacgtg ggtaacctgc 120
ctcatagagg ggaatagcct ttcgaaagga agattaatac cgcataagat tgtagtaccg 180
catggtacag caattaaagg agtaatccgc tatgagatgg acccgcgtcg cattagctag 240
ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc 300
cacattggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca 360
caatggggga aaccctgatg cagcaacgcc gcgtgagtga tgacggtctt cggattgtaa 420
agctctgtct ttagggacga taatgacggt acctaaggag gaagccacgg ctaactacgt 480
gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttactg ggcgtaaagg 540
gagcgtaggt ggatatttaa gtgggatgtg aaatacccgg gcttaacctg ggtgctgcat 600
tccaaactgg atatctagag tgcaggagag gaaaggagaa ttcctagtgt agcggtgaaa 660
tgcgtagaga ttaggaagaa taccagtggc gaaggcgcct ttctggactg taactgacac 720
tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780
cgatgaatac taggtgtagg ggttgtcatg acctctgtgc cgccgctaac gcattaagta 840
ttccgcctgg ggagtacggt cgcaagatta aaactcaaag gaattgacgg gggcccgcac 900
aagcagcgga gcatgtggtt taattcgaag caacgcgaag aaccttacct agacttgaca 960
tctcctgaat tactctgtaa tggaggaagc cacttcggtg gcaggaagac aggtggtgca 1020
tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080
tattgttagt tgctaccatt tagttgagca ctctagcgag actgcccggg ttaaccggga 1140
ggaaggtggg gatgacgtca aatcatcatg ccccttatgt ctagggctac acacgtgcta 1200
caatggtcgg tacaatgaga tgcaacctcg cgagagtgag caaaactata aaaccgatct 1260
cagttcggat tgtaggctga aactcgccta catgaagctg gagttgctag taatcgcgaa 1320
tcagaatgtc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag 1380
agttggcaat acccaaagtt cgtgagctaa ccgca 1415
Claims (8)
1. A clostridium butyricum is characterized in that the clostridium butyricum is classified and named clostridium butyricum @Clostridium butyricum) F06, the preservation number is CCTCC NO: m2019962.
2. The use of clostridium butyricum according to claim 1 for the production of neurotransmitters, wherein the neurotransmitters comprise one or any combination of gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, taurine, tyramine.
3. The use of clostridium butyricum according to claim 2 for the production of neurotransmitters, comprising gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, tyramine.
4. Use of clostridium butyricum according to claim 2 for the production of neurotransmitters, including gamma aminobutyric acid, dopamine, glutamate, choline.
5. Use of clostridium butyricum according to claim 1 for the preparation of a medicament.
6. A pharmaceutical product comprising clostridium butyricum as claimed in claim 1.
7. Use of clostridium butyricum according to claim 1 for the preparation of veterinary or microecological formulations.
8. A veterinary or probiotic formulation comprising clostridium butyricum as claimed in claim 1.
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