CN116590357A - Application of lactobacillus reuteri in production of gamma-aminobutyric acid and sleep-aiding products - Google Patents

Application of lactobacillus reuteri in production of gamma-aminobutyric acid and sleep-aiding products Download PDF

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CN116590357A
CN116590357A CN202310677739.5A CN202310677739A CN116590357A CN 116590357 A CN116590357 A CN 116590357A CN 202310677739 A CN202310677739 A CN 202310677739A CN 116590357 A CN116590357 A CN 116590357A
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hcs02
lactobacillus reuteri
culture
sleep
use according
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林欣梅
宋佳
杨思程
余萍
矫艳平
汪海涛
陈雪
彭永振
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Jiangxi Renren Health Microecological Technology Co ltd
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Jiangxi Renren Health Microecological Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation. Glutamic Acid Decarboxylase (GAD) in lactobacillus reuteri HCS02-001 catalyzes sodium L-glutamate to produce gamma-aminobutyric acid, and the gamma-aminobutyric acid serving as a natural amino acid consisting of non-protein has good sleep improvement effect, and the yield of the gamma-aminobutyric acid can reach 5.637g/L through optimizing the culture condition of HCS 02-001. The GABA yield of the HCS02-001 is far higher than E9 and TR02, and the continuous sleep time of mice is also prolonged more effectively.

Description

Application of lactobacillus reuteri in production of gamma-aminobutyric acid and sleep-aiding products
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation.
Background
Insomnia, including difficulty initiating or maintaining sleep, is the most common sleep disorder in the population. Over 20% of adults suffer from chronic insomnia. Many factors that lead to chronic insomnia, such as shift work, irregular work time, time differences and stress, are related to modern lifestyles. Insufficient sleep can lead to memory loss, irritability, depression, inattention and fatigue. In addition to cognitive function, sleep disorders are also associated with metabolic syndrome, such as obesity, inflammation, diabetes and cardiovascular disease. While there are many drugs available for the treatment of insomnia, including benzodiazepine receptor agonists, antihistamines, melatonin receptor agonists, anxiolytics, antidepressants and antipsychotics, the potential problems of drug dependence and abuse are alarming. Furthermore, these drugs are often associated with side effects such as dizziness, headache, somnolence, amnesia and cognitive dysfunction, even leading to an increased risk of death. It is therefore important to find a potential hypnotic agent to replace or reduce the use of these hypnotics, in a safer option to improve sleep quality and efficiency without significant adverse effects.
Gamma-aminobutyric acid (Gamma-aminobutyric acid, GABA for short) is also called gamma-aminobutyric acid and piperidine acid, is a natural amino acid which is not protein, is usually obtained by catalyzing L-glutamic acid and sodium salt thereof by glutamate decarboxylase (GAD) in organisms, is widely used as an important neurotransmitter in organisms, and has a series of physiological and health care functions of treating epilepsy, strengthening liver and kidney, controlling hypertension, resisting anxiety, controlling asthma, promoting sleep and the like.
The preparation method of the gamma-aminobutyric acid mainly comprises two methods of chemical synthesis and biological synthesis, wherein a plant enrichment method and a microbial fermentation method are the two most commonly used biological synthesis methods. Although the chemical synthesis method has higher reaction speed, the reaction process is strong, the safety is poor, the yield is low, the cost is high, meanwhile, side reactions are more in the production process, and the production process is often carried out by toxic or highly corrosive dangerous solvents, so that the pollution to the environment is serious, generally, GABA prepared by the chemical synthesis method is not a natural food additive, and therefore, the GABA is difficult to apply to the food processing industry; the separation and extraction of GABA enriched in plants are relatively difficult, and the GABA content is relatively low, so that the GABA is not suitable for large-scale production of GABA; the product obtained by the microbial fermentation method has good safety, high yield and lower cost, but the microbial strain which is required to be obtained with high efficiency is relatively difficult, and the lactobacillus is a probiotic existing in human body, can ferment carbohydrates into lactic acid, can help digestion and is beneficial to the health of human intestinal tracts, so that the lactobacillus is often regarded as a health beneficial bacterium for people, is taken as a food-safe microorganism, is also rich in glutamate decarboxylase, has the capability of synthesizing GABA, has excellent health care efficacy, and has wide market application prospect.
Lactobacillus reuteri is widely present in the intestinal tract of humans and animals and is available from a relatively wide variety of sources. The preparation has various probiotic effects, can regulate intestinal flora, effectively prevent diarrhea, inhibit the proliferation of pathogenic microorganisms, and reduce intestinal diseases. Lactobacillus reuteri has more probiotics, one of which is involved in cholesterol metabolism, enters the intestinal tract through the digestive system after being taken, has acid resistance and bile salt resistance, and plays a role in probiotics for organisms. Patent CN 115025131A discloses application of lactobacillus reuteri E9 in preparation of medicines for relieving anxiety and improving sleep, fermentation supernatant and bacterial suspension of lactobacillus reuteri E9 can obviously reduce movement distance, mania time and active time of zebra fish and obviously increase resting time in an in-vivo insomnia model, and has potential of being applied to relieving anxiety and improving sleep in vivo. Patent CN 114990011A discloses that lactobacillus reuteri (Lactobacillus reuteri) HCS02-001 has both cholesterol-lowering and gardnerella vaginalis-inhibiting functions. However, the application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid by fermentation is not disclosed in the prior art.
Disclosure of Invention
In order to solve the problems, the invention provides application of lactobacillus reuteri HCS02-001 (with the preservation number of CGMCC No. 19746) in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation. Glutamic Acid Decarboxylase (GAD) in lactobacillus reuteri HCS02-001 catalyzes sodium L-glutamate to produce gamma-aminobutyric acid, and the gamma-aminobutyric acid serving as a natural amino acid consisting of non-protein has good sleep improving effect, high safety and small side effect compared with other medicines. The gamma-aminobutyric acid obtained by the lactobacillus reuteri HCS02-001 fermentation method has the advantages of high yield, low cost and the like, and has great potential application prospect in improving sleep.
In the invention, the following components are added:
"fermentation broth" refers to a liquid that is cultured for a period of time by inoculating a strain into a culture medium.
The term "fermentation broth supernatant" refers to the supernatant of the fermentation broth after centrifugation. Contains rich metabolites and a part of thallus fragments in the growth and propagation process of bacteria, and acid substances secreted by the bacteria and bacteriocins have antagonism and killing effects on harmful bacteria; amino acids after decomposing food by bacteria, and synthetic vitamins are in the culture solution, and also include enzymes secreted by bacteria useful for human body; and part of thallus components have immunity promoting effect on human body.
The fungus suspension is a uniform suspension formed by pouring out the supernatant after centrifugation, adding water or buffer solution, and shaking or blowing and sucking to suspend the lower layer of fungus.
"fermentation broth sediment" refers to liquid sediment that is centrifuged and includes free protein, residual cells, broken cells, residues of culture medium, mainly protein, and intracellular medium.
The living bacteria are also called active flora, can colonize and reproduce in intestinal tracts, and are beneficial to increasing the number of beneficial bacteria.
The invention provides application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation.
In particular, the product can comprise one or more of fermentation liquor, fermentation liquor supernatant, fermentation liquor sediment, living bacteria and dead bacteria of lactobacillus reuteri HCS 02-001.
Specifically, the culture medium formula of the lactobacillus reuteri HCS02-001 is as follows: 10-20g/L of yeast peptone, 2-5g/L of beef powder, 4-6g/L of yeast extract, 1-2g/L of monopotassium phosphate, 15-20g/L of citric acid monohydrate, 4-5g/L of sodium acetate, 10-20g/L of anhydrous glucose, 0.5-0.6g/L of magnesium sulfate, 0.2-0.3g/L of manganese sulfate, 0.5-0.6g/L of tween 80, 1-2% (v/v) of tomato juice and 10-15g/L of L-sodium glutamate.
The tomato juice is prepared by weighing tomatoes, crushing, putting the tomatoes into a juicer for juicing, filtering, discarding filter residues, weighing, supplementing distilled water to the original mass according to the weight, subpackaging into 10mL centrifuge tubes, and putting 5mL of each tube into a refrigerator at the temperature of minus 20 ℃ for later use.
Preferably, the culture medium formula of the lactobacillus reuteri HCS02-001 is as follows: 20g/L of yeast peptone, 3g/L of beef powder, 6g/L of yeast extract, 2g/L of monopotassium phosphate, 15g/L of citric acid monohydrate, 5g/L of sodium acetate, 10g/L of anhydrous glucose, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.6g/L of tween 80, 1% (v/v) of tomato juice and 15g/L of L-sodium glutamate.
Specifically, the pH of the culture medium is 6-7; preferably pH6.5.
Specifically, the sterilization condition of the culture medium is 115 ℃ for 30min.
Specifically, the culture method of the lactobacillus reuteri HCS02-001 comprises the following steps:
(1) Resuscitates the frozen strain, resuscitates for 15-30s at 35-37 ℃;
(2) Culturing at 35-37deg.C for 16-20 hr;
(3) Secondary culturing, namely transferring bacterial suspension obtained by primary culturing into the culture medium according to the inoculum size of 4-8%, and carrying out shaking culturing for 16-20h at the temperature of 35-37 ℃ with the liquid loading amount of 40-60%;
(4) And (3) carrying out tertiary culture, namely transferring the bacterial suspension obtained by the secondary culture into the culture medium according to the inoculation amount of 4-8%, carrying out shaking culture for 16-20h at the temperature of 35-37 ℃ and the liquid loading amount of 40-60%.
Preferably, the seed recovery time in the step (1) is 30S.
Preferably, the bacterial suspension in steps (3) - (4) is inoculated at 5%.
Preferably, the culture temperature in steps (2) - (4) is 37 ℃.
Preferably, the liquid loading in the steps (3) - (4) is 50%.
Preferably, the incubation time described in steps (2) - (4) is 20 hours.
The invention has the technical effects that: the invention provides application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation. Glutamic Acid Decarboxylase (GAD) in lactobacillus reuteri HCS02-001 catalyzes sodium L-glutamate to produce gamma-aminobutyric acid, and the gamma-aminobutyric acid serving as a natural amino acid consisting of non-protein has good sleep improvement effect, and the yield of the gamma-aminobutyric acid can reach 5.637g/L through optimizing the culture condition of HCS 02-001. The GABA yield of the HCS02-001 is far higher than E9 and TR02, and the continuous sleep time of mice is also prolonged more effectively.
Drawings
FIG. 1 is a graph of gamma-aminobutyric acid standard curve.
Figure 2 is the effect on the sleep duration of mice.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
1.1 Medium formulation
MRS medium: 10g/L of yeast peptone, 3g/L of beef powder, 4g/L of yeast extract, 2g/L of monopotassium phosphate, 20g/L of citric acid monohydrate, 4g/L of sodium acetate, 20g/L of anhydrous glucose, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.6g/L of Tween 80, 1% (v/v) of tomato juice, and sterilizing at 115 ℃ for 30min.
Preparing tomato juice: weighing tomato, grinding, squeezing in a juicer, filtering, discarding residue, weighing, supplementing with distilled water to original mass, packaging into 10mL centrifuge tubes with 5mL concentration, and placing into a refrigerator at-20deg.C.
MRS-S liquid medium: 10g/L of sodium L-glutamate is added on the basis of MRS liquid culture medium as a precursor substance for GABA synthesis.
1.2 preparation of test Strain fermentation broth
(1) Primary culture: taking out a freeze-preserving tube of the lactobacillus reuteri HCS02-001 strain preserved at the temperature of minus 80 ℃, thawing at room temperature, uniformly mixing, streaking 1-cycle bacteria liquid on an MRS solid flat plate, and standing and culturing at the temperature of 37 ℃ for 48 hours;
(2) Secondary culture: selecting single colony to 5mL MRS liquid culture medium, and culturing at 37 ℃ for 20h;
(4) And (3) three-stage culture: inoculating 5mL of bacterial liquid into 100mL of MRS-S liquid culture medium, and culturing at 37 ℃ for 24h;
centrifuging the cultured bacterial liquid at 5000r/min for 20min, collecting 1mL of supernatant, diluting with ultrapure water for 20 times, and filtering with a 0.22 μm filter membrane for later use.
1.4 drawing of Standard Curve
Taking 0.4mL of prepared standard solution, adding 0.1mL of 0.1mol/L sodium carbonate, 0.5mL of 0.2mol/L borate buffer solution with pH of 9.0, 1mL of 6% phenol and 1mL of 10% sodium hypochlorite solution, uniformly mixing, standing for 4-8min, carrying out ice bath for 20min after boiling water bath for 10min, adding 2mL of 60% ethanol solution after the solution has blue-green color, standing for 30min after uniformly mixing, and measuring absorbance at 640 nm. The standard curve is plotted with GABA concentration on the abscissa and OD640 on the ordinate as shown in FIG. 1.
1.5 determination of GABA content in sample
0.4mL of the test strain fermentation broth is added with 0.1mL of 0.1mol/L sodium carbonate, 0.5mL of 0.2mol/L borate buffer solution with pH of 9.0, 1mL of 6% phenol and 1mL of 10% sodium hypochlorite solution, the mixture is uniformly mixed, the mixture is placed for 4 to 8min, the mixture is subjected to ice bath for 20min after 10min of boiling water bath, 2mL of 60% ethanol solution is added after the mixture is subjected to blue-green color, the mixture is uniformly mixed, the mixture is placed for 30min, and the absorbance value is measured at 640 nm.
Absorbance values of the sample system measured at 640nm were taken into a standard curve to calculate the GABA content in the sample, GABA content (g/L) = 3.267g/L.
Example 2 optimization of fermentation Medium
(1) Resuscitates the frozen strain: taking a strain freezing tube stored in a low-temperature refrigerator, immediately placing the strain freezing tube into a water bath kettle at 37 ℃ for strain resuscitation for 30s until the solid in the freezing tube is completely melted;
(2) First-stage culture
Transferring 1mL of recovered strain into 10mL of basic culture medium, culturing at 37deg.C under shaking at 100rpm for 20h, and preserving at 4deg.C in refrigerator.
(3) Second-stage culture
The bacterial suspension obtained by the primary culture was transferred to 100 mM RS-S liquid medium at an inoculum size of 5%, the liquid loading amount was 50%, and the culture was carried out at 37℃and 100rpm for 20 hours.
(4) Three-stage culture
The bacterial suspension obtained by the secondary fermentation is transferred into 300mLMRS-S liquid culture medium with the inoculum size of 5 percent, the liquid loading amount is 50 percent, and the shaking culture is carried out at the temperature of 37 ℃ and the rpm of 100 for 20 hours.
Specific formulations of MRS-S liquid medium and GABA content are shown in the following table:
the experimental results show that the GABA content of the lactobacillus reuteri HCS02-001 can reach 5.637g/L.
EXAMPLE 3 mouse sleep improvement function experiment
1. Preparation of lactobacillus reuteri HCS02-001 bacterial powder
(1) Resuscitates the frozen strain: taking a lactobacillus plantarum fungus mud freezing tube stored in a low-temperature refrigerator, immediately placing the tube into a water bath kettle at 37 ℃ for strain recovery until liquid in the freezing tube is completely melted;
(2) Activating and expanding culture of strains: directly inoculating the recovered 1-2mL of bacterial sludge mixed solution into a triangular flask A filled with a basic culture medium, sealing the triangular flask, and carrying out stationary culture at a constant temperature in a 37 ℃ incubator for 16+/-0.5 h; inoculating the bacterial suspension after the primary culture into a triangular flask B filled with a basic culture medium according to the inoculation amount of 2-6%, sealing the triangular flask, and carrying out stationary culture at a constant temperature in a 37 ℃ incubator for 8-10 hours;
(3) And (3) strain fermentation: inoculating the bacterial suspension into a fermentation tank filled with MRS-S liquid culture medium according to the inoculum size of 2-6%, starting a stirring paddle of the fermentation tank, rotating at 100rpm, introducing air volume of 0, and culturing at 37 ℃ for 7-12 hours at constant temperature; inoculating the bacterial suspension into a fermentation tank filled with MRS-S liquid culture medium according to the inoculation amount of 2-8%, starting a stirring paddle of the fermentation tank, rotating at 100rpm, introducing air volume of 0, and culturing at 37 ℃ for 6-12 hr until monitoring bacterial liquid OD 600 The value stops growing or grows negatively, and fermentation is stopped immediately;
(4) And (3) centrifuging fermentation liquor: centrifuging after fermentation, wherein the centrifugal speed is 10000-15000rpm;
(5) And (3) freeze drying: after centrifugation, collecting bacterial sludge in the rotary drum, placing the bacterial sludge in a freeze dryer, and continuously freeze-drying the bacterial sludge for 43-58 hours at the vacuum degree of 0-1.0Pa and the temperature of-25 ℃; collecting lyophilized powder, mixing with maltodextrin, and collecting powder with viable count greater than 1.0X10 9 cfu/g。
The lactobacillus reuteri HCS02-001 freeze-dried powder is used for the following animal experiments.
2. Animal experiment group
50 SPF-class 6-week-old male mice were randomly divided into 5 groups, each of which was 10, based on body weight, into a blank group, a GABA solution group, a low-dose group of Lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001, a medium-dose group of Lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001, and a high-dose group of Lactobacillus reuteri (Limosilactobacillus reuteri) HCS 02-001. The blank control group is filled with sterile physiological saline, the GABA solution group is filled with 8.83 mg/kg.bw, the low-dose group is filled with 8.83 mg/kg.bw, the medium-dose group is filled with 16.67 mg/kg.bw, the high-dose group is filled with 33.33 mg/kg.bw, the filling amount of the mice is 20 mL/kg.bw for 1 time a day, and the continuous 4 weeks are filled with stomach. The ambient temperature is maintained at about 25 ℃ and the humidity is controlled at about 50%.
2. Sleep improvement experiment
The experiment is evaluated by referring to the sleep improvement function specification part in the technical specification for health food inspection and evaluation (2003 edition).
(1) Sleep time experiment for prolonging sodium pentobarbital hypnotic mice
After the mice are subjected to gastric lavage, the mice are injected for inducing sleep for 30min, the injection quantity is 10 mL/kg.bw, the time when the inversion and the disappearance of the positive reflection appear again is the sleep time of the mice, and whether the sleep time of each group of mice is prolonged is recorded. The results are shown in Table 1, and compared with the blank control group, the sleep time of mice taking medium-dose and high-dose Lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001 is significantly higher than that of the blank control group, and the sleep time of the high-dose group is significantly higher than that of the GABA group. Demonstrating that lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001 plays an extended role in sleep in mice.
TABLE 1 influence on sleep duration in mice
(2) Sub-threshold dose hypnotic experiment with sodium pentobarbital
Animals were dosed in the same manner as (1), and mice which disappeared with the flip-flop for more than 1min were considered to enter sleep, and the number of falling asleep and the rate of falling asleep of the mice within 30min were recorded. The experimental results are shown in table 2, and the high dose group mice fall asleep at 40%. The rate of sleep of mice in the medium dose group reached 20% after gastric lavage, indicating that lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001 was able to promote the mice to fall asleep.
TABLE 2 influence on the sleep rate of mice
(3) Barbiturana sleep latency experiment
The animals are dosed in the same way as (1), and after the barbital sodium intraperitoneal injection is injected, the incubation period of the mice is the time from the injection of the barbital sodium to the disappearance of the eversion, and the influence of the test substances on the incubation period of the barbital sodium is observed. The results are shown in Table 3, and the significantly reduced sleep latency in the low, medium and high dose groups of Lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001 compared to the placebo group, indicate that the test subjects can shorten the sleep latency of the mice and allow the mice to fall asleep more rapidly.
TABLE 3 sodium barbiturate mice sleep latency experiments
In conclusion, the results of three experiments of the sleep time experiment, the pentobarbital sodium subthreshold dose hypnotic experiment and the barbital sleep latency experiment of the pentobarbital sodium hypnotic mice are all positive, and the lactobacillus reuteri (Limosilactobacillus reuteri) HCS02-001 has the effect of improving sleep.
Comparative examples 1 to 2
Referring to the experimental methods of example 2 and example 3, the following comparative experiments were set up:
the results show that the GABA yield of HCS02-001 is far higher than E9 and TR02, and the continuous sleep time of mice is also prolonged more effectively.

Claims (10)

1. The application of lactobacillus reuteri HCS02-001 in preparing gamma-aminobutyric acid and sleep-aiding products by fermentation.
2. The use according to claim 1, wherein the product comprises one or more of lactobacillus reuteri HCS02-001 broth, broth supernatant, broth pellet, viable bacteria, dead bacteria.
3. The use according to claim 2, wherein the medium formulation of lactobacillus reuteri HCS02-001 is: 10-20g/L of yeast peptone, 2-5g/L of beef powder, 4-6g/L of yeast extract, 1-2g/L of monopotassium phosphate, 15-20g/L of citric acid monohydrate, 4-5g/L of sodium acetate, 10-20g/L of anhydrous glucose, 0.5-0.6g/L of magnesium sulfate, 0.2-0.3g/L of manganese sulfate, 0.5-0.6g/L of tween 80, 1-2% (v/v) of tomato juice and 10-15g/L of L-sodium glutamate.
4. The use according to claim 3, wherein the medium formulation of lactobacillus reuteri HCS02-001 is: 20g/L of yeast peptone, 3g/L of beef powder, 6g/L of yeast extract, 2g/L of monopotassium phosphate, 15g/L of citric acid monohydrate, 5g/L of sodium acetate, 10g/L of anhydrous glucose, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.6g/L of tween 80, 1% (v/v) of tomato juice and 15g/L of L-sodium glutamate.
5. The use according to claim 2, wherein the method for culturing lactobacillus reuteri HCS02-001 comprises:
(1) Resuscitates the frozen strain, resuscitates for 15-30s at 35-37 ℃;
(2) Culturing the first stage, transferring the resuscitated strain into the culture medium according to claim 3 or claim 4, culturing at 35-37 ℃ for 16-20h by shaking;
(3) Second-stage culture, namely transferring the bacterial suspension obtained by the first-stage culture into the culture medium according to claim 3 or claim 4 in an inoculum size of 4-8%, filling the culture medium with 40-60% of liquid, culturing at 35-37 ℃ and shaking-culturing for 16-20h;
(4) The bacterial suspension obtained by the third-stage culture is transferred into the culture medium according to claim 3 or claim 4 in an inoculum size of 4-8%, the liquid loading amount is 40-60%, the culture temperature is 35-37 ℃, and the shaking culture is carried out for 16-20h.
6. The use according to claim 5, wherein the resuscitation time of step (1) is 30s.
7. The use according to claim 5, wherein the bacterial suspension according to steps (3) to (4) is inoculated in an amount of 5%.
8. The use according to claim 5, wherein the incubation temperature in steps (2) - (4) is 37 ℃.
9. The use according to claim 5, wherein the liquid loading in steps (3) to (4) is 50%.
10. The method according to claim 5, wherein the incubation time in steps (2) to (4) is 20h.
CN202310677739.5A 2023-06-08 2023-06-08 Application of lactobacillus reuteri in production of gamma-aminobutyric acid and sleep-aiding products Pending CN116590357A (en)

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