CN113832069A - Clostridium butyricum and application thereof, medicine, food, health product, veterinary drug, feed additive and microecological preparation - Google Patents
Clostridium butyricum and application thereof, medicine, food, health product, veterinary drug, feed additive and microecological preparation Download PDFInfo
- Publication number
- CN113832069A CN113832069A CN202111200171.5A CN202111200171A CN113832069A CN 113832069 A CN113832069 A CN 113832069A CN 202111200171 A CN202111200171 A CN 202111200171A CN 113832069 A CN113832069 A CN 113832069A
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- China
- Prior art keywords
- clostridium butyricum
- choline
- dopamine
- gamma
- neurotransmitter
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Abstract
The invention relates to clostridium butyricum and application thereof, a medicine, a food, a health product, a veterinary medicine, a feed additive and a microecological preparation, and belongs to the technical field of microorganisms. The Clostridium butyricum 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. The content of gamma-aminobutyric acid, dopamine, glutamic acid and choline in the clostridium butyricum fermentation liquor after the fermentation condition is optimized respectively reaches 4.78g/L, 5.82g/L, 135.9g/L and 3.78g/L, and the clostridium butyricum can be applied to production of 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, a medicine, a food, a health product, a veterinary medicine, a feed additive and a microecological preparation.
Background
Gamma-aminobutyric acid is an important neurotransmitter in the central nervous system, is a naturally-occurring non-protein amino acid, has extremely important physiological functions, can promote the activation of the brain, strengthen the brain, improve intelligence, resist epilepsy, promote sleep, beautify and moisten the skin, delay the aging function of the brain, supplement inhibitory neurotransmitters of a human body, and has good effect of reducing blood pressure. And secondly, the medicine can promote the improvement and protection of kidney functions. Inhibiting fatty liver and obesity, and activating liver function. The daily supplement of trace gamma-aminobutyric acid is beneficial to relieving the cardio-cerebral blood pressure, promoting the balance of amino acid metabolism in a human body and regulating the immune function.
Glutamate is an excitatory neurotransmitter and can only oxidize glutamate but not other amino acids in brain tissues, so that glutamate can be used as an energy substance of the brain tissues to improve and maintain the brain functions. Glutamic acid is used as supplement of nerve center and cerebral cortex, and has certain curative effect on treating concussion or nerve injury, epilepsy and children with mental retardation. Glutamic acid can promote the peristalsis of the digestive tract. Dopamine plays an important role in motor control, and parkinson's disease is caused by severe dopamine depletion due to dopaminergic neuronal degeneration. It is generally believed that the mesolimbic dopamine system and the mesocortical dopamine system play a role in learning and memory. Dopaminergic neurons in monkey a10 region have been reported to be involved in learning transient changes in impulsive activity during primary attention and motivation in cognitive behavior. The fact that cognitive function will decline with age is well documented in both humans and primates. In recent years, direct evidence is provided by research, obvious correlation among dopamine function reduction, cognitive function reduction and brain aging is proved, comparative research is carried out on volunteers of the same age, and the result shows that the dopamine level and the cognitive function are in positive correlation. Studies have shown depressed mood, depression, low pleasure, low mood, and low dopamine levels.
Choline is a constituent of all biological membranes and a precursor of acetylcholine in cholinergic neurons. Choline can promote brain development and improve memory, ensure information transmission, regulate and control apoptosis, prevent cholesterol deposition on the inner wall of blood vessel, remove partial deposition, and improve fat absorption and utilization, thereby preventing cardiovascular diseases.
Neurotransmitter substances such as gamma-aminobutyric acid, glutamic acid, dopamine, choline and the like can be obtained by conversion under the catalytic action of various enzymes in vivo, but under the conditions of age increase, stress state, intestinal microecological disorder, diseases or high mental stress, the synthesis of the neurotransmitter substances (the gamma-aminobutyric acid, the glutamic acid, the dopamine, the choline and the like) in vivo is remarkably reduced, so that the phenomenon of deficiency occurs. The addition of exogenous neurotransmitter can improve the condition of insufficient synthesis of endogenous neurotransmitter and promote the health of organism. It is also possible to produce neurotransmitter substances in the intestinal tract for direct absorption and utilization by supplementing the functional probiotics. Therefore, it has been a hot point of research through the development of foods, health products, feeds or microecological additives rich in neurotransmitter substances (gamma-aminobutyric acid, dopamine, glutamic acid, choline, etc.).
Health food developed from rice germ in Japan has remarkable effects in improving insomnia, depression and climacteric syndrome, and is widely added to various foods such as beverage, jam and cake. 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 utilizing biotechnology and the production of foods, health-care products or feeds and additives thereof rich in gamma-aminobutyric acid by utilizing the beneficial microorganisms are key technologies for solving the problems. Application numbers CN201910502771.3 and CN201711386926.9 both disclose a streptococcus thermophilus strain for high yield of gamma-aminobutyric acid. Application number CN201910502750.1 discloses a lactococcus lactis subspecies lactis capable of producing gamma-aminobutyric acid in high yield. Application number CN201910274454.0 discloses a lactobacillus rhamnosus strain for high yield of gamma-aminobutyric acid. Application number CN201610964033.7 discloses a lactobacillus plantarum for producing gamma-aminobutyric acid. Application number CN201611003216.9 discloses a lactobacillus pentosus strain producing gamma-aminobutyric acid. Application number CN201710149052.9 discloses rhizopus oryzae strain capable of producing gamma-aminobutyric acid in high yield. Application No. CN201910773775.5 discloses a screening method of single bacterium for promoting dopamine component. Application number CN201910257075.0 discloses a recombinant engineering bacterium for producing dopamine by catalyzing substrate dopa. Application number CN201911397692.7 discloses an escherichia coli for efficiently producing glutamic acid. Application numbers CN202010147933.9, CN201310361804.X and CN202010227468.X respectively disclose a recombinant corynebacterium glutamicum strain 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 single neurotransmitter, and only one neurotransmitter can be produced. If these microorganisms are used in combination culture to produce various neurotransmitters, antagonism between these microorganisms may be possible, and thus the production of various neurotransmitter substances is not achieved. In addition, some microorganisms (lactic acid bacteria and the like) have poor survival ability in vitro, are not resistant to gastric acid and bile acid, and cannot simultaneously produce neurotransmitter substances such as gamma-aminobutyric acid, glutamic acid, dopamine, choline and the like in intestinal tracts, so that the application of the microorganisms is limited. Therefore, it becomes a new challenge to obtain probiotics that are more active and capable of producing multiple neurotransmitter substances simultaneously.
Disclosure of Invention
The invention aims to provide a clostridium butyricum capable of producing various neurotransmitter substances, and the functional probiotic metabolite is rich in gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), tyrosine, taurine, acetylcholine, tyramine and other neurotransmitter substances.
The invention also aims to provide the application of the clostridium butyricum, and particularly comprises the application in producing neurotransmitter substances, the application in preparing medicines, foods or health products, and the application in preparing veterinary medicines, feed additives or microecologics.
The third object of the present invention is to provide a pharmaceutical product, food, health product, veterinary drug, feed additive or microecological preparation comprising the above clostridium butyricum.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides Clostridium butyricum, which is classified and named as Clostridium butyricum (Clostridium butyricum) F06, and the preservation number is CCTCC NO: M2019962F 06. The Clostridium butyricum belongs to the genus Clostridium, Clostridium butyricum, and Bacillus.
The clostridium butyricum is a strain which is prepared by applying an improved RCM culture medium and screening a high-yield neurotransmitter substance such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like from intestinal contents of healthy carps through an anaerobic culture method, and the functional probiotic metabolite is rich in gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), tyrosine, taurine, acetylcholine, tyramine and other neurotransmitter substances. Clostridium butyricum CCTCC NO: m2019962, the colony is round and cloudy white. The thalli is short rod-shaped, gram-positive, spore and spore oval in microscopic examination. The 16S rDNA electrophoresis and sequencing analysis show that the 16S rDNA homology of clostridium butyricum F06 and clostridium butyricum (CP016332.1CP013352.1LN828942.1LN828931.1AB687551.1NR113244.1X68177.1) reaches 100%.
Clostridium butyricum, also known as butyric acid bacteria, Clostridium butyricum, butyric acid bacteria. Clostridium butyricum belongs to the family Bacillaceae, genus Clostridium, G+And the endospores can resist gastric acid and bile acid and resist adverse environment. The clostridium butyricum can be widely applied to the fields of medicines, health products, foods, veterinary medicines, feed additives, microecologics and the like.
The invention preserves the clostridium butyricum, and the preserved strains are as follows: clostridium butyricum (Clostridium butyricum) F06, accession no: CCTCC NO: m2019962, deposit date: 20/11/2019, depository: china Center for Type Culture Collection (CCTCC), preservation address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
It is understood that the neurotransmitter is a generic term for neurotransmitters and neurotransmitter precursors in the present invention.
The invention provides the use of clostridium butyricum as described above in the production of neurotransmitter substances which are neurotransmitters and/or neurotransmitter precursors.
Specifically, 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 the above clostridium butyricum for producing neurotransmitter substances, wherein the neurotransmitter substances comprise gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan and L-tyrosine. Further, the gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan, L-tyrosine and tyramine.
Further, the neurotransmitter substances comprise gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine and taurine.
Further, the neurotransmitter substances include gamma-aminobutyric acid, dopamine, glutamic acid and choline.
Specifically, the preservation number is CCTCC NO: the clostridium butyricum of M2019962 is inoculated into a culture medium to ferment and produce neurotransmitter substances.
Furthermore, the invention optimizes the culture medium components and fermentation conditions for producing the neurotransmitter substances by fermenting clostridium butyricum.
Preferably, the culture medium includes a seed culture medium and a fermentation culture medium. Wherein, the seed culture medium comprises the following components in concentration (g/L): 25 parts of glucose, 10 parts of bean pulp, 30 parts of corn steep liquor, 10 parts of sodium glutamate, 1.5 parts of monopotassium phosphate, 0.2 part of magnesium sulfate, 0.2 part of ammonium sulfate, 0.5 part of disodium hydrogen phosphate and 0.3 part of calcium carbonate. The fermentation medium comprises the following components in concentration (g/L): glucose 140, soybean meal 15, corn steep liquor 40, magnesium sulfate 0.5, ammonium sulfate 0.2, potassium dihydrogen 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 preferably, the optimal fermentation conditions are: the pH was 7.2, the temperature was 35 ℃, the inoculum size was 4%, and the cultivation time was 24 h.
The invention provides application of clostridium butyricum in preparation of medicines, foods or health-care products.
The invention provides a novel culture medium containing the CCTCC NO: a pharmaceutical, food or health product of clostridium butyricum F06 of M2019962.
The invention provides application of clostridium butyricum F06 in preparation of veterinary drugs, feed additives or microecologics.
The invention provides a novel culture medium containing the CCTCC NO: a veterinary drug of clostridium butyricum F06 of M2019962.
The invention provides a novel culture medium containing the CCTCC NO: clostridium butyricum F06 feed supplement of M2019962.
The invention provides a novel culture medium containing the CCTCC NO: a microecological formulation of clostridium butyricum F06 of M2019962.
The invention has the following beneficial effects:
the invention uses an improved RCM culture medium to screen out a strain capable of highly producing neurotransmitter substances 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 producing single neurotransmitter substances, the clostridium butyricum provided by the invention has the following characteristics: 1) the bacillus is high in stress resistance and can resist the action of gastric acid and bile acid; 2) is anaerobic bacteria, grows and breeds in colon or caecum, and does not compete with the host for nutrition; 3) the metabolite is rich in various neurotransmitter substances, including gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine, taurine and the like; in addition, the clostridium butyricum provided by the invention is also different from the conventional clostridium butyricum, the metabolite of the conventional clostridium butyricum is butyric acid, and the metabolite of the clostridium butyricum provided by the invention not only contains butyric acid, but also is rich in neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline, L-tryptophan (5-hydroxytryptamine precursor), L-tyrosine, taurine and the like. Experiments prove that after the fermentation conditions of the strain are optimized, the contents of neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in the fermentation liquor respectively reach 4.78g/L, 5.82g/L, 135.9g/L and 3.78g/L, and the strain can be widely applied to the fields of medicines, health-care foods, feed additives, veterinary medicines, whole-field probiotics and the like and has wide application prospects.
Drawings
FIG. 1 is a colony diagram of Clostridium butyricum F06;
FIG. 2 is a stained 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 in a Clostridium butyricum F06 fermentation broth at different time points;
FIG. 4 is a graph showing the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline 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, choline, etc. in the fermentation broth of Clostridium butyricum F06 at different temperatures;
FIG. 6 is a graph showing the content of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline in the fermentation broth of Clostridium butyricum F06 at different inoculation amounts;
FIG. 7 shows the contents of neurotransmitters such as gamma-aminobutyric acid, dopamine, glutamic acid, choline, etc. in the fermentation broth after the fermentation conditions of Clostridium butyricum F06 are optimized.
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the equipment and reagents used in the examples are, unless otherwise specified, conventionally available commercially.
Example 1 screening and identification of Clostridium butyricum F06
1. Screening of Clostridium butyricum for production of various neurotransmitter substances:
the media involved in the screening were as follows:
1) modified RCM liquid screening medium, 1000mL containing: 10g of tryptone; 10g of beef extract; 3g of yeast extract; 5g of glucose; 1g of soluble starch; 5g of sodium chloride; 3g of sodium acetate; 0.5g of L-cysteine; 3g of sodium glutamate; polymyxin B0.02 g; 6% of 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 of agar.
3) Modified enriched clostridia culture medium, 1000mL containing: 10g of protein; 10g of beef extract; 3g of yeast powder; 5g of glucose; 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) Modified enhanced clostridial solid media: 1000mL of modified enriched Clostridium culture medium was supplemented with 15g of agar.
5) 1000mL of seed fermentation medium contains: 25g of glucose; 10g of soybean meal; 30g of corn steep liquor; 10g of sodium glutamate; 1.5g of monopotassium phosphate; magnesium sulfate 0.2 g; 0.2g of ammonium sulfate; 0.5g of disodium hydrogen phosphate; 0.3g of calcium carbonate.
All media were sterilized by autoclaving (121 ℃, 1X 105 kPa).
The specific screening steps are as follows:
1) weighing 5g of intestinal contents of healthy carp in the watershed of yellow river Henan province, and mixing the intestinal contents in a ratio of 1: 4, adding the mixture into sterile physiological saline, placing the mixture into a water bath at 70 ℃ for 15 minutes for primary screening to kill part of non-spore bacteria;
2) then 5mL of culture solution is extracted and transferred into 100mL of improved 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 improved reinforced clostridium liquid culture medium, and selectively enriching and culturing for 48h under the anaerobic condition at 37 ℃;
4) extracting 100uL culture solution for gradient dilution, then selecting appropriate dilution gradient to respectively coat the dilution gradient on an improved reinforced clostridium solid culture medium plate, placing the plate in an anaerobic incubator, and culturing for 48h at 37 ℃;
5) selecting gram-positive strains of which the colony morphology, the culture characteristic, the biochemical characteristic and the microscopic morphology all accord with the culture characteristic of clostridium butyricum to carry out subsequent tests;
6) fermenting and culturing strains according with the culture characteristics of clostridium butyricum for 24 hours by using a seed fermentation culture medium, centrifuging (4 ℃,12000r/min,10min) fermentation liquor to obtain supernatant, measuring the content of neurotransmitter substances such as gamma-aminobutyric acid, dopamine, choline and the like in the fermentation supernatant by using a high performance liquid chromatography, screening the strains with the strongest capability of producing the neurotransmitter substances, and storing.
2. Identification of Clostridium butyricum
1) Morphological characteristics and physiological and biochemical characteristics identification
According to an experimental method in a 'common bacteria system identification manual', the strain with the strongest capacity of producing neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like is identified by morphological characteristics and physiological and biochemical characteristics.
2)16S rDNA sequence analysis
Total genomic DNA was extracted and 16S rRNA fragments of the strains were amplified using 16S rRNA universal primers 27F, 5'-AGAGTTTGATCCTGGCTCAG-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, sterile water 8. mu.L.
The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 50s, for 28 cycles; extension at 72 ℃ for 10 min.
And (3) carrying out electrophoresis on the PCR product by using 2% TAE agarose gel, detecting the size of the PCR product, and then carrying out sequencing on the 16S rDNA gene product by handing over to Shanghai biological engineering Co., Ltd. BLAST was performed on the sequencing results and the GenBank database in NCBI and an evolutionary tree was constructed to analyze sequence homology.
3. Results and analysis
And screening 12 strains meeting the culture characteristics of clostridium butyricum, and respectively detecting the capability of seed fermentation liquor of the 12 strains for producing neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like, wherein the result shows that the capability of the No. 6 strain for producing the neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like is strongest. Strain 06 was therefore selected for subsequent experiments and was designated clostridium butyricum F06. Clostridium butyricum CCTCC NO: m2019962, the colony (FIG. 1) is round and cloudy white. The thallus is short rod-shaped, gram-positive, spore-containing and spore-oval in microscopic examination (figure 2).
Obtaining a 16S rDNA gene sequence of the strain F06 through PCR amplification, obtaining a related sequence from a GenBank database by using Blast comparison on NCBI for sequencing, and performing phylogenetic analysis, wherein a sequencing result is shown in SEQ ID NO. 1. The result shows that the homology of the strain F06 and the 16S rDNA of the 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 results show that clostridium butyricum F06 can produce neurotransmitter substances such as gamma-aminobutyric acid, dopamine, glutamic acid, choline and the like in high yield, and therefore, the clostridium butyricum is preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 20 days in 2019, and the preservation number is as follows: CCTCC NO: m2019962, deposit address: wuhan university in Wuhan, Hubei (preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei province).
Example 2 use of Clostridium butyricum F06 for production of neurotransmitter substances
This example provides the use of clostridium butyricum of example 1 in the production of neurotransmitter substances, including gamma aminobutyric acid, dopamine, glutamate and choline.
1. Clostridium butyricum F06 growth curve assay
Inoculating a strain Clostridium butyricum F06 which is stored in a laboratory and has the strongest capability of producing neurotransmitter substances into an improved RCM solid culture medium, standing and culturing for 24 hours at 37 ℃ under an anaerobic condition, and then inoculating an activated strain into an improved reinforced Clostridium culture medium, culturing at 37 ℃ and 180rpm under an anaerobic condition overnight to serve as a seed solution.
6 tubes containing 3.9mL of RCM medium were taken and marked with a marker for incubation times of 0, 4, 8, 12, 24, 48 h. Using a pipette, 100. mu.L of seed solution was accurately aspirated each time and inoculated into 6 numbered test tubes. After inoculation, anaerobic static culture is carried out at 37 ℃, test tubes with the numbers of corresponding time are taken out in 0, 4, 8, 12, 24 and 48 hours respectively, and the test tubes are immediately stored in a refrigerator.
And finally, using an unseeded RCM liquid culture medium as a blank control, and selecting a wavelength of 600nm to perform photoelectric turbidimetry so as to determine the growth curve of the clostridium butyricum F06. And detecting the content of neurotransmitter substances such as gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu), Choline (Choline) and the like at each time point by using a high performance liquid chromatography.
As shown in FIG. 3, the number of colonies and the contents of neurotransmitters such as gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu) and Choline (Choline) were the greatest when the culture time was 24 hours.
2. Determination of optimal pH, temperature and inoculation amount of clostridium butyricum F06 fermentation medium
Adjusting the initial pH of the seed culture medium to 6.8, 7.0, 7.2, 7.4, 7.6 and 7.8 respectively by using 4mol/L HCl and 20% NaOH solution, inoculating 2% seed solution, culturing at 37 ℃ and 180rpm under anaerobic condition for 24 h. The number of bacteria was measured by a hemacytometer. Inoculating 2% Clostridium butyricum F06 bacterial liquid into 100mL improved RCM culture medium, and culturing at 30 deg.C, 37 deg.C, 40 deg.C, 45 deg.C, 180rpm under anaerobic condition for 24 h. After inoculating the seed solution of Clostridium butyricum F06 with the inoculum size of 0.5%, 1%, 2%, 4%, 6% and 8%, culturing at 37 ℃ and 180rpm under anaerobic condition for 24 h. 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 various pH, temperature and inoculation amount conditions were measured by using a blood cell count method and a high performance liquid chromatography, respectively.
As shown in FIGS. 4, 5 and 6, the optimum culture conditions were as follows: the pH value is 7.4, the temperature is 37 ℃, the inoculation amount is 4%, and the colony count and the content of neurotransmitters such as gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu), Choline (Choline) and the like are the largest when the culture time is 24 hours.
3. Optimization of clostridium butyricum F06 medium components
Glucose, soybean meal, corn steep liquor are the main components affecting the culture medium. Thus, design of experiment L16 (3)4) Orthogonal experiments were performed to determine the composition of clostridium butyricum F06 medium, and the experimental design was as follows: glucose (100g, 120g, 140g), soybean meal (10g, 15g, 20g), corn steep liquor (20g, 30g, 40 g). Then, the colony count and the contents of neurotransmitters such as gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu), Choline (Choline) and the like under various component conditions were measured by using a blood cell count method and a high performance liquid chromatography, respectively.
TABLE 1 results of the Effect of the orthogonal assay of the Medium composition on GABA content
Note: the ia values reflect the effect of levels of B, C at 1, 2, and 3 times per factor on the a1 levels (A, B, C represents glucose, soybean meal, corn meal, respectively). Similarly, IA reflects the effect of B, C on the level of A2(A3) once at each of the three levels of factor. When comparing differences between ia, iia, iiia, B, C was found to have substantially the same effect on ia, iia, iiia. Thus, the differences between IA, IIA and IIIA can be considered to be due to the 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 column I, II, III is then calculated and this difference is the range, denoted R. The range of each column reflects the magnitude of the effect of the level shift selected by the factor corresponding to that column on GABA content. The larger the value, the greater the degree of influence of this factor on the GABA content. Tables 2, 3 and 4 are the same.
TABLE 2 results of the Effect of the orthogonal assay of the Medium composition on Glu content
TABLE 3 results of the Effect of the orthogonal test of the Medium composition on the DA content
| Numbering | 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 Effect of the orthogonal assay of Medium composition on Choline content
As shown in tables 1 to 4, the medium components of Clostridium butyricum F06 were glucose, 140 g; 15g of soybean meal; 20g of corn flour; the culture conditions were pH 7.4, temperature 37 ℃, inoculum size 4%, and the maximum gamma-aminobutyric acid (GABA) content was measured at 24h culture time (Table 1). The component of clostridium butyricum F06 in the culture medium is glucose, 140 g; 20g of soybean meal; 30g of corn flour; the culture conditions were pH 7.4, temperature 37 ℃, inoculum size 4%, and maximum glutamic acid (Glu) content was measured at 24h of culture time (Table 2). The component of clostridium butyricum F06 in the culture medium is glucose, 130 g; 10g of soybean meal; 30g of corn flour; the conditions of the culture were pH 7.4, temperature 37 ℃, inoculum size 4%, and maximum Dopamine (DA) content measured at 24h of culture time (Table 3). The component of clostridium butyricum F06 in the culture medium is glucose, 140 g; 10g of soybean meal; 40g of corn flour; the culture conditions were pH 7.4, temperature 37 ℃, inoculum size 4%, and maximum Choline (Choline) content measured at 24 h. Since the above results indicate that the combination under laboratory conditions has not been subjected to production practice, further experiments are needed to verify the colony count of clostridium butyricum F06 and the content of related neurotransmitter substances under the conditions of the combination. From tables 1 to 4, it can be seen that glucose is the main component affecting the GABA content, glucose is the main component affecting the Glu content, soybean meal is the main component affecting the DA content, and corn starch is the main component affecting the Choline content. Therefore, in consideration of the test result and the production cost, the component of the clostridium butyricum F06 in the culture medium is glucose, 140 g; 15g of soybean meal; 40g of corn flour; the culture conditions are that the pH is 7.4, the temperature is 37 ℃, the inoculum size is 4 percent, and the content of the neurotransmitter substances in the fermentation liquor is detected when the culture time is 24 hours. As can be seen from FIG. 7, the contents of gamma-aminobutyric acid, glutamic acid, dopamine and choline in the Clostridium butyricum fermentation broth measured under the conditions reach 4.78g, 135.9g, 5.82g and 3.78g/L respectively.
Example 3 Clostridium butyricum F06 metabolomics analysis of simulated gut fermentation products
This example describes a specific assay method for performing metabolomics analysis of simulated gut fermentation products for clostridium butyricum F06 provided in example 1 as follows:
after the fermentation liquor is inoculated with clostridium butyricum F06, samples are respectively taken at 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours, and the serial number of the samples is A, B, C, D, E, F. And then, acquiring primary mass spectrum data and secondary mass spectrum data based on non-targeted metabonomics analysis of ultra performance liquid chromatography-Q-TOF MS, and performing peak extraction and metabolite identification on the data by using XCMS.
In the results of the test, it was found that in the A, B, C, D (2, 4, 6, 8 hours) sample, that is, in the c.butyricum F06 reproductive stage: transmitters related to signal conduction of the nervous system, 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 Clostridium butyricum F06 results of metabonomics analysis of simulated gut fermentation products
Note: mzmed is mean of m/z, the median of the mass to charge ratios. rtmed is the mean of retention time, the median of the retention times, which together with mzmed is the characteristic of the substance. POS a, B, C, D, E, F represent the results of analyses in positive ion mode (POS is the abbreviation for positive ion or positive) at 2, 4, 6, 8, 12, 24 hour sampling, respectively.
Example 4
This example is the use of the clostridium butyricum of example 1 for the preparation of a medicament, food or health care product.
In practical application, the medicines, foods and health care products all comprise the components with the preservation number of CCTCC NO: clostridium butyricum of M2019962.
And (3) the preservation number is CCTCC NO: the M2019962 Clostridium butyricum was fed to mice and the test was set up as 4 groups (negative control, low 5000CFU, medium 10000CFU and high 50000CFU, respectively) with 10 replicates per group. After 1 week of continuous feeding, blood was collected from the heart and plasma was separated. The targeted metabonomics analysis is carried out based on the LC-MS/GC-MS technology, and the feeding preservation number is CCTCC NO: the content levels of gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu), Choline (Choline), L-tryptophan, L-tyrosine, taurine, acetylcholine and tyramine in the plasma of the mice with the Clostridium butyricum M2019962 are higher than those of the mice without the Clostridium butyricum.
Example 5
This example is the use of clostridium butyricum according to example 1 for the preparation of a veterinary drug, a feed additive or a probiotic.
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) the preservation number is CCTCC NO: m2019962 clostridium butyricum F06 is fed to broilers and pigs, targeted metabonomics analysis detects the content levels of gamma-aminobutyric acid (GABA), Dopamine (DA), glutamic acid (Glu), Choline (Choline), L-tryptophan, L-tyrosine, taurine, acetylcholine and tyramine in blood, and the feeding preservation number is CCTCC NO: the content of the clostridium butyricum F06 of M2019962 in the blood of the chicken and the pig is higher than that of the chicken and the pig which are not fed with the clostridium butyricum.
<110> Henan university of agriculture
Clostridium butyricum with effects of inhibiting growth of microorganisms and promoting growth of microorganisms, and application, medicine, food, health product, veterinary drug, feed additive and microecological preparation thereof
<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 (9)
1. The Clostridium butyricum is classified and named Clostridium butyricum (Clostridium butyricum) F06 with the preservation number of CCTCC NO: m2019962.
2. Use of clostridium butyricum according to claim 1 for the production of neurotransmitter substances.
3. The use of clostridium butyricum according to claim 2, for producing a neurotransmitter substance, wherein the neurotransmitter substance comprises one or any combination of gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, taurine and tyramine.
4. The use of clostridium butyricum according to claim 3, for the production of neurotransmitter substances, wherein said neurotransmitter substances include gamma-aminobutyric acid, dopamine, glutamic acid, acetylcholine, choline, L-tryptophan, L-tyrosine, tyramine.
5. The use of clostridium butyricum of claim 3 for the production of neurotransmitter substances, wherein the neurotransmitter substances comprise gamma-aminobutyric acid, dopamine, glutamic acid and choline.
6. Use of clostridium butyricum according to claim 1 for the preparation of a medicament, food or health product.
7. A pharmaceutical, food or health product comprising clostridium butyricum of claim 1.
8. Use of clostridium butyricum according to claim 1 for the preparation of a veterinary drug, a feed additive or a probiotic.
9. A veterinary drug, feed additive or probiotic comprising clostridium butyricum of claim 1.
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