CN117343879B - Lactobacillus plantarum 9A-11 and application thereof in preparation of autism improvement products - Google Patents
Lactobacillus plantarum 9A-11 and application thereof in preparation of autism improvement products Download PDFInfo
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- CN117343879B CN117343879B CN202311638991.1A CN202311638991A CN117343879B CN 117343879 B CN117343879 B CN 117343879B CN 202311638991 A CN202311638991 A CN 202311638991A CN 117343879 B CN117343879 B CN 117343879B
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Abstract
The invention belongs to the field of microorganisms, relates to application of lactobacillus, and in particular relates to lactobacillus plantarum 9A-11 and application thereof in preparation of products for improving autism. The lactobacillus plantarum 9A-11 is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023625. Proved by researches, the lactobacillus plantarum can regulate the balance of intestinal flora, promote digestion and absorption of human bodies, and simultaneously has the effects of conditioning and effectively relieving and treating anxiety, depression and improving sleep quality; meanwhile, the lactobacillus plantarum can generate endogenous sources in the intestinal canal reproduction and growth process, can continuously provide necessary amounts of beta-alanine, valine and docosahexaenoic acid, and is beneficial to preventing, improving and treating the occurrence of autism patients.
Description
Technical Field
The invention belongs to the field of microorganisms, relates to application of lactobacillus, and in particular relates to lactobacillus plantarumLactobacillus plantarum) 9A-11 and application thereof in preparing an autism improving product.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Autism (autism), also known as autism or autism disorder (autistic disorder), etc., is a representative disease of pervasive developmental disorder (pervasive developmental disorder, PDD); at the same time, it is also a brain disorder disease.
There is a correlation between gut microbiota, performance and brain function. Enteric microorganisms can exchange sensory information with the host by producing a large number of metabolites in the intestinal lumen, including neurotransmitters, dopamine and norepinephrine, hormones (secretion of corticotropin releasing hormone in the hypothalamic-pituitary-adrenal axis), histamine, acetylcholine, catecholamines, and several vitamins and short chain fatty acids. Some of these molecules can enter the brain through the blood brain barrier and affect the neural circuit. Among these metabolites, intestinal microbiota can also affect the integrity of the intestinal barrier, controlling the passage of signal molecules from the intestinal lumen to the lamina propria (including immune cells and ends of ENS neurons) or portal venous circulation. The integrity of the intestinal barrier is compromised in certain neuropsychiatric disorders and the absence of intestinal microbiomes exacerbates anxiety-like behavior, such as autism. Thus, maintaining intestinal microbial balance plays a critical role in autistic patients.
Disclosure of Invention
According to the invention, in 8 th year 2005, lactobacillus is separated from fresh dendrobium candidum in Qingdao in Shandong, and identified as lactobacillus plantarum by a 16srRNA method, and researches show that the lactobacillus plantarum can regulate intestinal flora balance, promote digestion and absorption of human bodies, and also has the effects of conditioning and effectively relieving treatment anxiety, depression and improving sleep quality; meanwhile, the lactobacillus plantarum can generate endogenous sources in the intestinal canal reproduction and growth process, can continuously provide necessary amounts of beta-alanine, valine and docosahexaenoic acid, and is beneficial to preventing, improving and treating the occurrence of autism patients.
Based on the research results, the invention provides a lactobacillus plantarum strain and application thereof in preparing an autism improvement product, and specifically, the technical scheme of the invention is as follows:
on the one hand, the lactobacillus plantarum 9A-11 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2023625.
In another aspect, the method for culturing the lactobacillus plantarum 9A-11 comprises culturing the lactobacillus plantarum 9A-11.
In a third aspect, a microbial agent comprises lactobacillus plantarum 9A-11 or an inactivated thallus, a fermentation broth, an exosome and a metabolite thereof.
In a fourth aspect, the use of the lactobacillus plantarum 9A-11 or the microbial agent in preparing a product for preventing, improving and/or treating autism.
In a fifth aspect, the use of the lactobacillus plantarum 9A-11 in the fermentative preparation of beta-alanine, valine and/or docosahexaenoic acid.
Biological preservation: the lactobacillus plantarum 9A-11 is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023625, 25 th year of 2023, 04 th year of deposit address: chinese university of Wuhan and Wuhan.
The beneficial effects of the invention are as follows:
1. the lactobacillus plantarum 9A-11 provided by the invention is a probiotic, is also a main component part in normal flora of human intestinal tracts, has the characteristics of no toxicity, effectiveness, safety, green and the like, and can play an important role in human health.
2. Researches show that the lactobacillus plantarum 9A-11 provided by the invention is prepared into a finished product, and has a detail improving effect on autism of adults and children. Especially, the finished product of lactobacillus plantarum 9A-11 and the metabolite thereof can balance intestinal flora, promote digestion and absorption of human bodies, and simultaneously regulate and effectively relieve anxiety, depression and improve sleep quality. In addition, the lactobacillus plantarum 9A-11 provided by the invention can provide necessary amounts of beta-alanine, valine and docosahexaenoic acid, and is helpful for preventing, improving and treating the occurrence of autism patients.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
Beta-alanine content detection method: high Performance Liquid Chromatography (HPLC) and liquid mass spectrometry (LC-MS) techniques.
Valine assay: potentiometric titration.
The content detection method of docosahexaenoic acid comprises the following steps: gas chromatography.
Equipment used in production: strain incubator, ultra-clean bench, constant temperature incubator, various test tubes, triangular flask, seed tank, fermenter, vacuum freeze dryer, three-dimensional stirrer, low temperature pulverizer, and vacuum packaging machine.
In view of the symptoms of the patient with autism, which are facilitated by maintaining the balance of intestinal microorganisms, the invention provides a lactobacillus plantarum strain and application thereof in preparing a product for improving autism.
The invention provides a lactobacillus plantarum 9A-11 which is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023625.
In another embodiment of the invention, a method for culturing the lactobacillus plantarum 9A-11 is provided, and the lactobacillus plantarum 9A-11 is obtained by strain culture.
In some embodiments, the culturing comprises an expansion culture comprising, per 1000 milliliters of water: 5-7 g of glucose, 11-13 g of mannooligosaccharide, 8-10 g of peptone, 9-11 g of beef extract powder, 1-3 g of white kidney bean extract, 0.5-1.5 g of sodium acetate, 1.5-2.0 g of oyster peptide, 0.5-1.5 ml of tween 80 and 1-3 g of manganese sulfate. The pH value of the culture medium for the expansion culture is natural. The culture conditions of the expansion culture are as follows: culturing for 20-30 h at 36-38 ℃.
In some embodiments, the culturing comprises a primary seed culture comprising, per 1000 milliliters of water: 9-11 g of glucose, 3-5 g of mannooligosaccharide, 5-7 g of peptone, 2-4 g of beef extract powder, 1-3 g of corn oligopeptide, 0.5-1.5 g of sodium acetate, 0.5-1.5 g of ammonium citrate and 0.5-1.5 ml of tween 80. The pH value of the culture medium for primary seed culture is natural. The culture conditions for the primary seed culture are as follows: culturing for 10-20 h at 37-39 ℃.
In some embodiments, the culturing comprises a seed tank culture comprising, in weight percent, in a medium: 1-3% of glucose, 0.5-1.5% of mannooligosaccharide, 0.3-0.7% of inulin, 0.1-0.5% of monopotassium phosphate, 0.5-1.5% of dipotassium phosphate, 1.0-1.4% of corn oligopeptide, 0.1-0.3% of sodium acetate, 0.2-0.6% of valine, 0.2-0.6% of tween 80, 1.2-1.6% of fructo-oligosaccharide, 0.05-0.15% of polyether defoamer and the balance of water. The pH value of the culture medium cultured in the seed tank is natural. The culture conditions of the seed tank culture are as follows: the pot pressure is 0.04-0.06 megapascals, and the culture is carried out for 20-30 hours at 37-39 ℃.
In some embodiments, the culturing comprises fermenting, the fermenting medium comprising, in weight percent: 1-3% of glucose, 1.0-2.0% of rice powder, 0.5-1.5% of xylooligosaccharide, 1-3% of oat beta-glucan, 0.2-0.6% of bonito elastin peptide, 0.1-0.3% of sodium acetate, 0.1-0.3% of tween 80, 2-4% of sucrose, 0.10-0.20% of creatine monohydrate, 0.05-0.15% of lactoferrin, 0.1-0.3% of magnesium sulfate, 1-3% of L-arabinose, 0.05-0.15% of polyether defoamer and the balance of water. The pH value of the fermentation culture medium is natural. The culture conditions of the fermentation are as follows: the pot pressure is 0.04-0.06 megapascals, and the culture is carried out for 20-30 hours at 37-39 ℃.
In some embodiments, the culturing method comprises sequentially expanding culturing, primary seed culturing, seed tank culturing, and fermenting.
In a third embodiment of the present invention, a microbial agent is provided, which comprises the lactobacillus plantarum 9A-11 or an inactivated thallus, a fermentation broth, an exosome and a metabolite thereof.
In some embodiments, the Lactobacillus plantarum 9A-11 described above and metabolites thereof are included. The mass ratio of the lactobacillus plantarum 9A-11 to the metabolic products is 10:5-7.
In one or more embodiments, the lactobacillus plantarum 9A-11 is a lyophilized powder. The preparation method of the freeze-dried bacterial powder comprises the following steps: adding maltodextrin into water, mixing, adding fermented bacterial mud of plant lactobacillus strain, citrus fiber, apple fiber, skimmed milk powder, sucrose, glycerol, and galactooligosaccharide, mixing, and lyophilizing. In order to increase the mixing efficiency of maltodextrin and water, the temperature in the mixing process is 50-55 ℃. The conditions for freeze-drying were: vacuum, the temperature is-50 to-40 ℃, and the drying time is 50-60 hours. Pre-freezing is performed before freeze-drying. The pre-freezing temperature is-35 to-30 ℃. The mass ratio of the zymophyte paste to the citrus fiber to the apple fiber to the skimmed milk powder to the sucrose to the maltodextrin to the galactooligosaccharide is 1000:40-60:30-50:150-250:50-150:50-150:350-450. The addition ratio of the zymophyte sludge to the glycerol is 1000:30-70, and g is ml.
In one or more embodiments, the metabolic product is prepared by: concentrating the liquid after the fermentation broth is centrifuged, concentrating to paste, adding a carrier, uniformly mixing, and drying to obtain the fermentation broth. The carrier is preferably a pharmaceutically acceptable water-soluble corn starch. The mass ratio of the concentrated paste to the carrier is 1000:350-450.
In some embodiments, an adjunct is included. Such excipients include, but are not limited to, microcrystalline cellulose, pharmaceutically acceptable water soluble corn starch, casein phosphopeptide, magnesium stearate. The auxiliary materials are preferably microcrystalline cellulose, medicinal water-soluble corn starch, casein phosphopeptide and magnesium stearate. The mass ratio of the crystalline cellulose to the pharmaceutical water-soluble corn starch to the casein phosphopeptide to the magnesium stearate is 10:30-50:5-7:5-15. The adding ratio of the lactobacillus plantarum strain (especially the freeze-dried bacterial powder) to the auxiliary materials is 10:40-75.
The fourth embodiment of the invention provides application of the lactobacillus plantarum 9A-11 or the microbial agent in preparing products for preventing, improving and/or treating autism.
In particular, the products of the invention include pharmaceuticals.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
The strain adopted in the following examples is Lactobacillus plantarum 9A-11, which is preserved in China center for type culture Collection with a preservation number of CCTCC NO: m2023625, 25 th year of 2023, 04 th year of deposit address: chinese university of Wuhan and Wuhan. Preserving the strain in a refrigerator at-80 ℃.
Beta-alanine content detection method: high Performance Liquid Chromatography (HPLC) and liquid mass spectrometry (LC-MS) techniques.
Valine assay: potentiometric titration.
The content detection method of docosahexaenoic acid comprises the following steps: gas chromatography.
Equipment used in production: strain incubator, ultra-clean bench, constant temperature incubator, various test tubes, triangular flask, seed tank, fermenter, vacuum freeze dryer, three-dimensional stirrer, low temperature pulverizer, and vacuum packaging machine.
Examples
1. Culturing strains:
(1) And (3) performing expansion culture:
preparing a culture medium: glucose 6 g, mannooligosaccharide 12 g, peptone 9 g, beef extract 10 g, white kidney bean extract 2 g, sodium acetate 1 g, oyster peptide 1.5 g, tween 80 1ml, manganese sulfate 2 g, tap water 1000 ml and natural pH value.
Adding the raw materials such as glucose and the like in the formula into 1000 ml of water before and after the raw materials are not separated, stirring for 30 minutes to dissolve the raw materials, then subpackaging the raw materials into 18 x 180 test tubes, filling 10 ml of each test tube, sealing a rubber plug kraft paper, placing the test tubes into a medical sterilizer for sterilization for 25 minutes at 0.1-0.12 megapascals, and finishing the sterilization of the culture medium.
Opening a freezing tube preserved at-80 ℃ under aseptic condition (in an ultra-clean workbench), melting at room temperature, taking 1 count of sterilized test tube culture medium, adding 1 milliliter of melted preserved fungus liquid at-80 ℃ into a test tube, sealing a tube orifice with rubber plugs and kraft paper, shaking by hands uniformly, and placing into a constant-temperature incubator for standing culture at 37 ℃ for 24 hours to obtain the expanded fungus liquid.
(2) Primary seed culture:
preparing a culture medium: 10 g of glucose, 4 g of mannooligosaccharide, 6 g of peptone, 3 g of beef extract powder, 2 g of corn oligopeptide, 1 g of sodium acetate, 1 g of ammonium citrate, 1ml of tween 80, 1000 ml of tap water and natural pH value.
The raw materials are added into tap water before and after the raw materials are not separated, a stirrer is started, and the raw materials are stirred for 20 minutes to be completely dissolved, so that a first-stage seed culture solution is obtained.
Adding the stirred and melted primary seed culture solution into a triangular flask according to the amount, adding a layer of kraft paper to bundle the bottle mouth, placing the bottle mouth into a medical sterilizer for sterilization, opening a vent valve on the sterilizer and the like before sterilization, closing when a small amount of steam is discharged, opening the vent valve on the sterilizer for venting for 6-8 minutes when the pressure on the sterilizer reaches 0.05 megapascals, then closing the vent valve, continuing to heat, starting timing when the steam pressure in the sterilizer reaches 0.1-0.12 megapascals, sterilizing for 25 minutes, leaving a heat source after the completion, naturally cooling, opening the sterilizer when a pressure meter on the sterilizer returns to zero, taking out the sterilized culture medium, placing the sterilized culture medium into an ultra-clean workbench for natural cooling, transferring the cultured and expanded strain solution into the sterilized culture solution of the triangular flask when the temperature is reduced to 36 ℃, wherein the inoculation amount is 1.5% respectively, namely 100 milliliters of the primary seed culture solution is accessed into the expanded strain solution, starting to time when the pressure in the sterilizer reaches 0.1.5 milliliters, sealing the primary gauze and the kraft paper after the sterilization is completed, and then placing the primary seed culture solution into a small-shaking incubator by hands, and uniformly mixing the primary seed culture solution with the strain culture solution under the condition of 38, and shaking the primary culture solution under the condition of shaking condition.
(3) Seed pot culture:
sterilization of fermentation equipment, tubing, and sterile filtration systems:
firstly, opening valves of each inlet and outlet pipeline and a sterile air pipeline, introducing steam of 0.12-0.14 megapascals, enabling the steam to be communicated with the pipeline valves and a small amount of steam to be discharged, ventilating for 40 minutes, enabling the steam pressure in the pipeline to be kept at 0.12-0.14 megapascals for 40 minutes, and then closing each inlet and outlet pipeline valve for standby.
Closing each valve, opening the drain valve at the bottom of the tank and the drain valve in the interlayer of the tank, opening the direct steam valve to introduce 0.12-0.14 megapascals of steam into the tank, starting timing when the temperature in the tank reaches 121 ℃, sterilizing for 40 minutes, then closing the drain valve at the bottom of the tank and the drain valve in the interlayer, and naturally reducing the temperature in the tank to 38 ℃ for standby.
Preparing a culture medium: glucose 2%, mannooligosaccharide 1%, inulin 0.5%, potassium dihydrogen phosphate 0.3%, dipotassium hydrogen phosphate 1%, corn oligopeptide 1.2%, sodium acetate 0.2%, valine 0.4%, tween 800.4%, fructo-oligosaccharide 1.4%, polyether defoamer 0.1%, tap water 85.5% and natural pH value.
Putting tap water used in the formula into a seed tank sterilized by an empty tank, starting a stirrer, respectively adding raw materials required by the formula (before and after the adding sequence is not divided), then introducing steam for heating in continuous stirring, firstly heating to 95 ℃ by utilizing tank interlayer steam, then heating by direct steam, keeping for 30 minutes when the temperature in the tank reaches 121 ℃, achieving the sterilizing effect, then closing the steam, beginning to cool down by tap water in the tank interlayer, and standing by when the temperature in the tank reaches 38 ℃, thus obtaining the seed culture medium.
Inoculating the cultured primary strain culture solution into a seed tank under aseptic condition, wherein the inoculum size is 5% of that of the culture medium in the tank, namely, 100 kg of the culture medium in the seed tank is respectively inoculated with 5 kg of the primary strain solution. Then, the inoculating cap on the pot is covered to start stirring culture, and the culture conditions are as follows: the temperature is 38 ℃, the pH value is natural, the stirring rotation speed is 100 revolutions per minute, the tank pressure is 0.05 megapascal, the culture time is 24 hours, if the tank pressure is reduced, sterile air can be introduced to keep the tank pressure, and the tank pressure is too high and can be regulated by a deflation valve at the top of the tank, so that the seed tank strain is obtained.
(4) Fermentation:
and (3) sterilizing the fermentation equipment, the pipeline and the sterile filtration system.
Preparing a culture medium: 2% of glucose, 1.5% of rice powder, 1% of xylooligosaccharide, 2% of oat beta-glucan, 0.4% of bonito elastin peptide, 0.2% of sodium acetate, 0.2% of Tween 80, 3% of sucrose, 0.15% of creatine monohydrate, 0.1% of lactoferrin, 0.2% of magnesium sulfate, 2% of L-arabinose, 0.1% of polyether defoamer and 87.15% of tap water, wherein the total weight of the composition is 100 kg.
Putting tap water used in a formula into a fermentation tank sterilized by an empty tank, starting a stirrer, putting raw materials used in the formula into the fermentation tank (the adding sequence is not sequential), then introducing steam for heating, firstly heating to 95 ℃ by using interlayer steam, then heating by using direct steam, starting timing when the temperature in the tank reaches 121 ℃, maintaining for 30 minutes to achieve a sterilization effect, then cooling down by using the tap water of the interlayer, starting inoculating strains when the temperature in the tank is reduced to 37 ℃, firstly increasing the tank pressure of a seed tank to 0.1 megapascal, keeping the tank pressure of the fermentation tank to 0.05 megapascal, then opening an inoculating pipeline valve, conveying the seeds cultured in the seed tank into the fermentation tank in a pressure difference mode, and then closing the inoculating pipeline valve.
The inoculation amount is 5 kg of strain cultured in a seed tank after inoculating each 100 kg of culture medium in the fermentation tank, and the culture conditions are as follows: the fermentation temperature is 38 ℃, the stirring rotation speed is 120 r/min, the fermentation time is 20 hours, the tank pressure is kept at 0.05 megaPa, the pH value is natural, if the tank pressure is reduced, sterile air can be introduced to keep the tank pressure, and the tank pressure is excessively high and can be regulated by an exhaust valve at the top of the tank, so that fermentation liquid is obtained. The contents of beta-alanine, valine and docosahexaenoic acid in the fermentation broth during the fermentation are shown in Table 1.
TABLE 1 content of beta-alanine, valine and docosahexaenoic acid in fermentation broth during fermentation
(5) Centrifuging fermentation liquor, and collecting wet bacterial sludge:
after fermentation, the fermentation broth is pumped into a storage tank, and the centrifugal rotation speed is very important to ensure the integrity of thalli in the fermentation broth.
Starting a tubular centrifuge, adjusting the rotating speed to 9000 r/min, centrifuging at a feeding speed of 15 kg/min, collecting wet bacterial sludge after centrifuging, freeze-drying the wet bacterial sludge collected by centrifuging, concentrating the centrifuged fermentation liquor at a low temperature in vacuum, adding a carrier, and compounding a finished product after vacuum drying.
2. Compounding a finished product:
(1) Preparing freeze-dried bacterial powder:
the preparation of the suspension is to take 1000 g of wet bacterial mud, and add 2500 ml of sterile distilled water, 50g of citrus fiber, 40g of apple fiber, 200 g of skimmed milk powder, 100 g of sucrose, 50ml of 98% glycerol, 100 g of maltodextrin and 400 g of galacto-oligosaccharide. The preparation method comprises heating 2000 ml of water in the formula to 55deg.C, adding maltodextrin under stirring, dissolving, adding the rest water, and adding other raw materials (before and after the addition). Stirring was carried out for 30 minutes (stirring at 35 ℃ C.). The bacterial suspension is prepared, and then the bacterial suspension is put into a thermostatic chamber or box with the temperature of 30 ℃ for 30 minutes to be pre-frozen.
Pre-freezing before freeze drying, packaging the suspension with constant temperature for 30 min in freeze-drying tray, pre-freezing at-30deg.C for 3 hr in pre-freezing chamber of freeze dryer, and vacuum drying after freezing.
And (3) placing the pre-frozen material tray into a vacuum drying chamber to start vacuumizing and drying under the conditions that the vacuum degree is 2-5 Pa, the temperature is-45 ℃, the temperature of the drying chamber is 28 ℃, and the drying time is 55 hours. After freeze-drying, collecting freeze-dried materials, crushing the freeze-dried materials into bacterial powder with fineness of 100 meshes by using a sterile crusher, and sealing and packaging the bacterial powder for later use, thus obtaining the freeze-dried bacterial powder.
(2) Preparation of metabolite dry powder:
concentrating the centrifuged fermentation liquor at low temperature, specifically: concentrating to paste with vacuum degree of 0.06 megapascal and temperature of 55deg.C until solid content is 80% and water content is 20%, adding medicinal water-soluble corn starch as carrier, mixing, and vacuum drying to obtain concentrated extract.
Taking 1000 g of concentrated paste, adding 400 g of medicinal water-soluble corn starch, uniformly mixing, and drying in a vacuum drying oven under the conditions of: vacuum degree 0.06 megapascal, temperature 55-60 deg.C, drying time 50 hours, moisture content 3% is qualified, then pulverizing it to 100 mesh powder, sealing and packaging for standby, obtaining metabolite dry powder.
(3) Compounding of finished products:
taking 10 g of 100-mesh freeze-dried bacterial powder, 6 g of metabolite dry powder, 10 g of microcrystalline cellulose, 40g of medicinal water-soluble corn starch, 6 g of casein phosphopeptide and 10 g of magnesium stearate.
The materials are put into a solid stirring mixer to be mixed uniformly, then the materials are packaged into capsules under the aseptic condition, and the finished product (carrier fungus powder) is obtained after bottling.
The obtained carrier bacterial powder contains 70 hundred million CFU and 100 mg of metabolite of live lactobacillus plantarum.
Application of prepared carrier fungus powder in improvement of autism
In the application test, the recruited subjects are autism patients who are treated in a release army ninth and sixth hospital, the age is 3-45 years, the selected patients all accord with the diagnosis standard of the autism, serious organ dysfunction is not combined, and mental intelligence and cognitive function are normal. By 5 months of 2023, a total of 200 were enrolled in the experiment, 50 individuals for adult males, adult females, boys, girls, each of which was classified into control and experimental groups according to a random number, each of which signed a written informed consent, and if the participants were unable to sign, by an agent (typically a family member).
The nursing method comprises the following steps:
control group: using conventional treatment methods and care
Experimental group: based on the conventional treatment method and nursing, the lactobacillus plantarum carrier fungus powder prepared above is taken by patients, 0.2 gram is taken once before sleeping every day, and each gram contains 70 hundred million CFU of live lactobacillus plantarum and 100 milligrams of metabolite.
TABLE 2 patient and Carrier powder administration conditions
Judgment standard:
unstable condition improvement: the physical pain caused by the emotion pain is relieved, or the emotion abnormality is relieved;
the unstable condition is obviously improved: communication with the outside is increased, or interaction and communication between language and mind are increased;
the improvement of the instability is extremely obvious: the repeated behavior of the engraving plate is properly changed;
the instability situation is completely improved: the happy emotion rise is shared with other people, the happy emotion rise can be communicated with eyes of other people, and the facial expression is various.
The results illustrate:
adult male 50 began statistics at 5 days of care, experimental group: the unstable condition of 8 patients with autism of different ages is improved after 5 days of administration, and the physical pain caused by emotional pain is relieved; after taking for 10 days, the unstable condition of 15 patients suffering from autism is obviously improved, and communication with the outside is increased. The unstable condition of 18 patients suffering from autism is obviously improved after 15 days of taking, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 21 patients suffering from autism is completely improved after taking the medicine for 30 days, and the feeling of happiness shared with other people is increased, so that the medicine can be communicated with eyes of other people, and the facial expression is various; a total of 4 people have failed to improve autism by taking Lactobacillus plantarum. Control group: the unstable condition of 2 patients with autism of different ages is improved after the conventional nursing for 5 days, and the physical pain caused by emotional pain is relieved; after 10 days of routine nursing, the unstable condition of 4 patients suffering from autism is obviously improved, and communication with the outside is increased. The improvement of the instability of 7 patients suffering from autism in 15 days of routine nursing is extremely obvious, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 8 patients suffering from autism in 30 days of routine nursing is completely improved, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 17 failed to improve autism after routine treatment and care.
Adult female 50 people began statistics at 5 days of care, experimental group: the unstable condition of 7 patients with autism of different ages is improved after 5 days of administration, and the physical pain caused by emotional pain is relieved; after taking for 10 days, the unstable condition of the patient with 12 persons suffering from autism is obviously improved, and communication with the outside is increased; the unstable condition of 16 patients suffering from autism is obviously improved after 15 days of taking, and the repeated behavior of the engraving plate is properly changed; the unstable condition of 19 patients suffering from autism is completely improved after 30 days of taking the medicine; a total of 6 people can not improve autism by taking lactobacillus plantarum. Control group: the unstable condition of 3 patients with autism of different ages is improved after the conventional nursing for 5 days, and the physical pain caused by emotional pain is relieved; after 10 days of routine nursing, the unstable condition of 5 patients suffering from autism is obviously improved, and communication with the outside is increased. The improvement of the instability of 8 patients suffering from autism in 15 days of routine nursing is extremely obvious, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 9 patients suffering from autism in 30 days of routine nursing is completely improved, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 16 failed to improve autism after conventional treatment and care.
The child male 50 began counting at 5 days of care, the experimental group: the unstable condition of 5-person autism patients with different ages is improved after taking for 5 days, and the mood abnormality is relieved; after taking for 10 days, the unstable condition of 9 patients suffering from autism is obviously improved, and the interaction and communication between language and eye spirit are increased. The unstable condition of 14 patients suffering from autism is obviously improved after 15 days of taking, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 18 patients suffering from autism is completely improved after 30 days of taking, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 7 people have failed to improve autism on administration of lactobacillus plantarum. Control group: the unstable condition of 1 patient suffering from autism with different ages is improved after the conventional nursing for 5 days, and the physical pain caused by emotional pain is relieved; after 10 days of routine nursing, the unstable condition of the patient with 3 persons suffering from autism is obviously improved, and communication with the outside is increased. The improvement of the instability of 7 patients suffering from autism in 15 days of routine nursing is extremely obvious, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 9 patients suffering from autism in 30 days of routine nursing is completely improved, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 16 failed to improve autism after conventional treatment and care.
Children 50 began statistics at 5 days of care, experimental group: the unstable condition of 6 patients with autism of different ages is improved after 5 days of taking, and the mood abnormality is relieved; after taking for 10 days, the unstable condition of 10 patients suffering from autism is obviously improved, and the interaction and communication between language and eye spirit are increased. The unstable condition of 13 patients suffering from autism is obviously improved after 15 days of taking, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 19 patients suffering from autism is completely improved after 30 days of taking, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 6 people can not improve autism by taking lactobacillus plantarum. Control group: the unstable condition of 2 patients with autism of different ages is improved after the conventional nursing for 5 days, and the physical pain caused by emotional pain is relieved; after 10 days of routine nursing, the unstable condition of 4 patients suffering from autism is obviously improved, and communication with the outside is increased. The improvement of the instability of 7 patients suffering from autism in 15 days of routine nursing is extremely obvious, and the repeated behavior of the engraving plate is properly changed. The unstable condition of 8 patients suffering from autism in 30 days of routine nursing is completely improved, and the patients share happy emotion with other people, can communicate with eyes of other people, and have various facial expressions; a total of 17 failed to improve autism after routine treatment and care.
The strain sequences identified by the 16srRNA method were as follows:
AGTCGTACGAACTCTGTGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAG
TTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCGTTAAA, SEQ ID NO.1.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. Lactobacillus plantarum strainLactobacillus plantarum) 9A-11, characterized in that the material is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023625.
2. A method for culturing lactobacillus plantarum 9A-11 according to claim 1, wherein the lactobacillus plantarum 9A-11 is obtained by strain culture, and the culturing method sequentially comprises expansion culture, primary seed culture, seed tank culture and fermentation.
3. The method for culturing lactobacillus plantarum 9A-11 according to claim 2, wherein the culturing includes an enlarged culturing; the medium for the expansion culture contains per 1000 ml of water: 5-7 g of glucose, 11-13 g of mannooligosaccharide, 8-10 g of peptone, 9-11 g of beef extract, 1-3 g of white kidney bean extract, 0.5-1.5 g of sodium acetate, 1.5-2.0 g of oyster peptide, 0.5-1.5 ml of tween 80 and 1-3 g of manganese sulfate; the culture conditions of the expansion culture are as follows: culturing for 20-30 h at 36-38 ℃;
or, the culturing includes primary seed culturing; the primary seed culture medium comprises the following components in every 1000 ml of water: 9-11 g of glucose, 3-5 g of mannooligosaccharide, 5-7 g of peptone, 2-4 g of beef extract powder, 1-3 g of corn oligopeptide, 0.5-1.5 g of sodium acetate, 0.5-1.5 g of ammonium citrate and 0.5-1.5 ml of tween 80; the culture conditions for the primary seed culture are as follows: culturing for 10-20 h at 37-39 ℃;
or, the culture comprises a seed tank culture, wherein the culture medium for the seed tank culture comprises the following components in percentage by weight: 1-3% of glucose, 0.5-1.5% of mannooligosaccharide, 0.3-0.7% of inulin, 0.1-0.5% of monopotassium phosphate, 0.5-1.5% of dipotassium phosphate, 1.0-1.4% of corn oligopeptide, 0.1-0.3% of sodium acetate, 0.2-0.6% of valine, 0.2-0.6% of tween 80, 1.2-1.6% of fructo-oligosaccharide, 0.05-0.15% of polyether defoamer and the balance of water; the culture conditions of the seed tank culture are as follows: the tank pressure is 0.04-0.06 megapascals, and the culture is carried out for 20-30 hours at 37-39 ℃;
or, the culturing comprises fermentation, wherein the fermentation medium comprises the following components in percentage by weight: 1-3% of glucose, 1.0-2.0% of rice powder, 0.5-1.5% of xylooligosaccharide, 1-3% of oat beta-glucan, 0.2-0.6% of bonito elastin peptide, 0.1-0.3% of sodium acetate, 0.1-0.3% of tween 80, 2-4% of sucrose, 0.10-0.20% of creatine monohydrate, 0.05-0.15% of lactoferrin, 0.1-0.3% of magnesium sulfate, 1-3% of L-arabinose, 0.05-0.15% of polyether defoamer and the balance of water; the culture conditions of the fermentation are as follows: the pot pressure is 0.04-0.06 megapascals, and the culture is carried out for 20-30 hours at 37-39 ℃.
4. A microbial agent comprising the fermentation broth of lactobacillus plantarum 9A-11 of claim 1 or lactobacillus plantarum 9A-11 of claim 1 and a metabolite thereof;
the preparation method of the metabolite comprises the following steps: concentrating the liquid after the fermentation broth is centrifuged, concentrating into paste, adding a carrier, uniformly mixing, and drying to obtain the fermentation broth; the carrier is medicinal water-soluble corn starch; the mass ratio of the concentrated paste to the carrier is 1000:350-450.
5. The microbial agent according to claim 4, comprising lactobacillus plantarum 9A-11 and its metabolites; the mass ratio of the lactobacillus plantarum strain to the metabolic products is 10:5-7.
6. The microbial agent according to claim 5, wherein the lactobacillus plantarum 9A-11 is a freeze-dried bacterial powder; the preparation method of the freeze-dried bacterial powder comprises the following steps: adding maltodextrin into water, mixing, adding fermented bacterial mud of lactobacillus plantarum, citrus fiber, apple fiber, skimmed milk powder, sucrose, glycerol and galactooligosaccharide, mixing, and freeze drying; the temperature of maltodextrin in the process of adding water and mixing is 50-55 ℃; the conditions for freeze-drying were: vacuum, the temperature is-50 to-40 ℃, and the drying time is 50-60 hours; pre-freezing before freeze drying; the pre-freezing temperature is minus 35 to minus 30 ℃; the mass ratio of the zymophyte paste to the citrus fiber to the apple fiber to the skimmed milk powder to the sucrose to the maltodextrin to the galactooligosaccharide is 1000:40-60:30-50:150-250:50-150:50-150:350-450; the addition ratio of the zymophyte sludge to the glycerol is 1000:30-70, and g is ml.
7. The microbial agent of claim 4, comprising an adjuvant; the auxiliary materials comprise one or more of microcrystalline cellulose, medicinal water-soluble corn starch, casein phosphopeptide and magnesium stearate; or the auxiliary materials are microcrystalline cellulose, medicinal water-soluble corn starch, casein phosphopeptide and magnesium stearate; the mass ratio of the crystalline cellulose to the pharmaceutical water-soluble corn starch to the casein phosphopeptide to the magnesium stearate is 10:30-50:5-7:5-15; the adding ratio of the lactobacillus plantarum 9A-11 to the auxiliary materials is 10:40-75.
8. Use of the microbial agent according to any one of claims 6 to 7 in the preparation of a medicament for improving and/or treating autism.
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| CN114748515A (en) * | 2022-03-25 | 2022-07-15 | 光明乳业股份有限公司 | Application of lactobacillus plantarum ST-III in preparation of products for treating autism |
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| CN114748515A (en) * | 2022-03-25 | 2022-07-15 | 光明乳业股份有限公司 | Application of lactobacillus plantarum ST-III in preparation of products for treating autism |
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| Title |
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| Effects of Lactobacillus plantarum PS128 on Children with Autism Spectrum Disorder in Taiwan: A Randomized, Double-Blind, Placebo-Controlled Trial;Yen-Wenn Liu等;Nutrients;第4卷;doi:10.3390/nu11040820 * |
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