CN116676225B - Lactobacillus rhamnosus LR-28 strain with nerve soothing and sleep aiding effects, fermentation product, hypnotic fungus group mixture and application - Google Patents
Lactobacillus rhamnosus LR-28 strain with nerve soothing and sleep aiding effects, fermentation product, hypnotic fungus group mixture and application Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a lactobacillus rhamnosus LR-28 strain with the effects of soothing nerves and aiding sleep, a fermentation product, a hypnotic fungus group agent and application, wherein the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 7.464 in the 6 th month of 2022; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city. The lactobacillus rhamnosus LR-28 strain has high gastrointestinal tolerance, can enter intestinal tracts for smooth colonization, can be directly metabolized in the intestinal tract environment of human bodies to generate GABA, and can be applied to products for improving sleep quality, preventing sleep disorder of insomnia people and relieving dysphoria.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus rhamnosus LR-28 strain with a nerve soothing and sleep aiding effect, a fermentation product, a sleep-improving bacterial composition and application thereof.
Background
According to the statistics of the world health organization, the global sleep disorder rate reaches 27 percent. The sleep investigation result published by the China sleep research institute in 2016 shows that the incidence rate of insomnia of adults in China is as high as 38.2%, more than 3 hundred million Chinese people have sleep disorder, and the data is still rising year by year. Prolonged insomnia can pose a serious threat to physical health.
Several studies have shown that various neurotransmitters such as glutamate (Glu) and GABA are involved in central sleep regulation and play a very critical role. The microbial conversion method mainly uses sodium glutamate or glutamic acid as a substrate, utilizes the activity of microorganisms to convert the substrate into GABA, and needs to additionally provide sodium glutamate or glutamic acid substrate. In the prior art, strains for directly converting and producing GABA are cultured by microbial fermentation, but the fermentation process for producing GABA is carried out in an in-vitro fermentation tank, the culture medium used under the fermentation condition and the culture environment have great difference from the complex human intestinal environment, and the strains cannot be directly metabolized in the human intestinal environment to produce GABA. Therefore, the advantage of GABA production by the above strain needs to be exerted by means of in vitro fermentation, but is not necessarily applicable as a product of direct metabolism in human intestinal environment for GABA production.
Disclosure of Invention
Aiming at the technical problems, the invention provides the lactobacillus rhamnosus LR-28 strain with the effects of soothing nerves and aiding sleep, a fermentation product, a hypnotic fungus group agent and application, and the strain has good tolerance and strong antibacterial activity on gastric acid environment and bile salt, can produce gamma-aminobutyric acid in an intestinal in-vitro fermentation simulation system, and can play roles in preventing sleep disorder, improving sleep quality and relieving dysphoria after being directly taken or made into corresponding products.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
lactobacillus rhamnosus LR-28, which is classified under the name lactobacillus rhamnosus (Lactobacillus rhamnosus) and is preserved in the China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (CGMCC) at the date of 26, 6, 2022, and has a preservation number of CGMCC No.7.464; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
Compared with the prior art, the lactobacillus rhamnosus LR-28 with the preservation number of CGMCC No.7.464 provided by the invention has the following advantages:
(1) The lactobacillus rhamnosus LR-28 belongs to the genus Lactobacillus and has good tolerance to gastric acid environment and bile salt. Can survive in the harsh conditions of strong acidity and a large amount of digestive enzymes in the digestive tract of a human body, can be planted on the gastrointestinal tract wall, and can repair the damage of intestinal epithelial cells and repair the intestinal mucosa barrier.
(2) The living bacteria and metabolites of lactobacillus rhamnosus LR-28 provided by the invention can produce high-activity glutamate decarboxylase in intestinal tract environment, so as to catalyze in-vivo conversion of glutamate to generate GABA. GABA and other amino acids are continuously distributed to each tissue organ along with blood, wherein GABA enters nerve cells along with blood and is combined with GABA receptors among neurons to play a role in inhibiting nerve excitation, so that the effects of preventing sleep disorder, improving sleep quality and relieving dysphoria are exerted.
(3) The lactobacillus rhamnosus LR-28 provided by the invention also has strong antibacterial activity, and can regulate microecological balance, strengthen the intestinal resistance of a host, prevent and treat diarrhea and eliminate allergy.
The 16S rRNA sequence is shown below:
in a second aspect, the invention also provides application of the lactobacillus rhamnosus LR-28 strain in preparing a product for improving sleep quality.
The lactobacillus rhamnosus LR-28 strain provided by the invention still has higher activity of producing glutamate decarboxylase in a complex intestinal environment of a human body, and sodium glutamate or glutamic acid in the intestinal environment of the human body can be used as a substrate to produce GABA, so that the lactobacillus rhamnosus LR-28 strain can be added into a sleep-aiding product, and the produced GABA plays a role in inhibiting nerve excitation so as to achieve the aim of improving sleep quality. The lactobacillus rhamnosus LR-28 strain can also be used for metabolizing GABA through a culture medium, so that a method of culturing the strain through in vitro fermentation can be adopted to prepare a product for improving sleep quality by using a fermentation product or a fermentation extract of the strain.
In a third aspect, the invention also provides a fermentation product of the lactobacillus rhamnosus LR-28 strain, wherein the fermentation product is prepared by fermenting the lactobacillus rhamnosus LR-28 strain. The fermented product can be further processed to improve sleep quality.
In a fourth aspect, the present invention also provides a method for producing gamma-aminobutyric acid by using the lactobacillus rhamnosus LR-28 strain, which specifically comprises the following operations: seed culturing the strain in an improved MRS liquid culture medium, and anaerobic fermentation culturing the cultured fermentation broth on an intestinal model culture medium (CMGM); the strain comprises the lactobacillus rhamnosus LR-28.
Further, the strain further comprises at least one of Lactobacillus plantarum (Lactobacillus plantarum) LP-28 and Bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) BAL-28. Wherein the lactobacillus plantarum LP-28 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.7.463 at the month of 2022 and the day of 6; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city. The bifidobacterium animalis subspecies of milk BAL-28 is preserved in the China general microbiological culture Collection center (CGMCC) at the date of 28, 6 and 28 in 2022, and the preservation number is CGMCC No.7.462; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
Further, the strain is lactobacillus rhamnosus LR-28 and lactobacillus plantarum LP-28. Experimental study shows that lactobacillus rhamnosus LR-28 and lactobacillus plantarum LP-28 can produce synergistic effect.
Optionally, the seed culture conditions are: the inoculation amount is 3 percent, and the culture is carried out for 24 hours at 37 ℃ and is sequentially and continuously activated for three generations.
Further, the anaerobic fermentation culture conditions are as follows: the fermentation temperature is 35-37 ℃, the pH is 5-7, and the culture is carried out for more than 48 hours.
Alternatively, the method may specifically employ the following procedure:
seed culture stage: inoculating lactobacillus rhamnosus LR-28 seed solution into an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing for 24 hours at 37 ℃, sequentially and continuously activating for three generations, taking 100ml of the cultured fermentation liquor, obtaining a fermentation liquor stock solution sample, and refrigerating at 4 ℃ for later use; when at least one of lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28 is also included in the strain, the lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28 are respectively activated by the same method, each fermentation broth is mixed according to a specific proportion to obtain a mixed fermentation broth, and the mixed fermentation broth is refrigerated at 4 ℃ for standby.
CMGM composition (g)/L): starch, 5.0; pectin, 2.0; guar gum, 1.0; mucin (porcine stomach type III), 4.0; xylan, 2.0; arabinogalactan, 2.0; inulin, 1.0; casein, 3.0; peptone water, 5.0; tryptone, 5.0; bile salts, 0.4; yeast extract, 4.5; feSO 4 ·7H 2 O,0.005; sodium chloride, 4.5; potassium chloride, 4.5; KH (KH) 2 PO 4 ，0.5；MgSO 4 ·7H 2 O，1.25；CaCl 2 ·6H 2 O，0.15；NaHCO 3 1.5; cysteine, 0.8; heme, 0.05; tween 80,1.0.
Placing an intestinal model culture medium into an intestinal in-vitro fermentation simulating system fermentation tank for carrying out fermentation culture on the fermentation broth: the human colon three-stage continuous culture intestinal model system consists of three fermentors, namely a near-end fermentor (V1, 280 mL), a transverse fermentor (V2, 300 mL) and a far-end colon fermentor (V3, 320 mL). Three fermentors in series were maintained at 37℃and pH values of 5.5 (V1), 6.2 (V2) and 6.8 (V3), respectively, and were maintained by continuous N 2 The introduction creates anaerobic conditions. V1, V2 and V3 are all introduced into CMGM by peristaltic pumps. Fresh healthy human faeces samples were collected and stored in an anaerobic cabinet (10% H) 2 、10％CO 2 、80％N 2 ) In (1:5) (w/w) faecal dilutions in anaerobic PBS (0.1 mol/L PBS pH 7.4) were prepared within 15 minutes after collection. Each fermenter of the intestinal model was inoculated with 100mL of fecal dilution. The total system transit time was set to 48 hours based on the average retention time of healthy individuals. After inoculation with fecal dilutions, the intestinal model was run for 24 hours to stabilize the bacterial population prior to injection into the culture medium. After 24 hours, the system was run for 8 full capacity cycles to achieve steady state, and then the above fermentation broth was added to V1 in an amount of 1% (w/V) of the volume of the contents in the tank, followed by eight volume cycles to reach steady state, and further culture for 24 hours.
In a fifth aspect, the present invention also provides a composition of a formulation of a Jia-sleep fungus, the hypnotic fungus group agent is prepared by fermenting lactobacillus rhamnosus LR-28 strain or the combination of lactobacillus rhamnosus LR-28 strain and other probiotics; the other probiotics include at least one of Lactobacillus plantarum LP-28 and Bifidobacterium animalis BAL-28.
Further, the preparation method of the Jia-sleep fungus group agent comprises the following steps: fermenting the lactobacillus rhamnosus LR-28 strain or the combination of the lactobacillus rhamnosus LR-28 strain and other probiotics, centrifuging after fermentation to obtain bacterial sludge, uniformly mixing the bacterial sludge with a freeze-drying protective agent, freeze-drying, and crushing to obtain the Jia-sleep bacterial group agent.
Alternatively, the culture medium used for fermentation in the preparation method can be prepared by the following operation process:
mixing 10-90g of glucose, 3-15g of soybean peptone, 15-19g of yeast extract powder, 0-14g of citric acid diamine, 2-26g of anhydrous sodium acetate, 0-10g of dipotassium hydrogen phosphate, 3-16g of isomaltooligosaccharide, 0-30g of starch and other food-grade materials, adding 1000g of pure water, uniformly mixing until no macroscopic particles exist, regulating pH to 5.0-6.8 after material melting, and sterilizing for 20min at 117.5-118.5 ℃ to obtain the culture medium for fermentation.
Mixing seed solution of lactobacillus rhamnosus LR-28 strain or combination of lactobacillus rhamnosus LR-28 strain and other probiotics according to a specific proportion, adding the mixture into the culture medium, culturing for 10-12 hours at 34-37 ℃, centrifuging for 10-30 minutes at 5000-9000r/min after fermentation to obtain bacterial mud, uniformly mixing the bacterial mud with a freeze-drying protective agent, freeze-drying, and crushing to obtain the Jia-sleep flora composite agent powder.
Further, the raw materials of the lyoprotectant comprise at least one of starch, glucose, maltodextrin, sucrose, lactose and skimmed milk powder; the preparation method of the freeze-drying protective agent comprises the following operations: dissolving the raw materials of the freeze-drying protective agent in water, regulating the pH to 5.0-6.8, sterilizing, and regulating the pH to 6.0-7.3 after sterilizing to obtain the freeze-drying protective agent.
Drawings
FIG. 1 is a microscopic image of Lactobacillus rhamnosus LR-28 of example 1;
FIG. 2 is a comparison of the tolerance of Lactobacillus rhamnosus LR-28 of example 1 to conventional commercial Lactobacillus rhamnosus to artificial gastric juice;
FIG. 3 is a comparison of bile salt tolerance of Lactobacillus rhamnosus LR-28 in example 1 with that of conventional commercial Lactobacillus rhamnosus;
FIG. 4 is a comparative analysis of the adhesion ability of Lactobacillus rhamnosus LR-28 of example 1 to conventional commercial Lactobacillus rhamnosus;
FIG. 5 is an analysis of the antibacterial activity of Lactobacillus rhamnosus LR-28 in example 1.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The sources of the strains used in the following examples of the invention are as follows:
lactobacillus plantarum LP-28, which is classified under the name Lactobacillus plantarum (Lactobacillus plantarum), is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 7.463 in the year 2022, month 6 and day 25; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
The bifidobacterium animalis BAL-28 is preserved in the China general microbiological culture Collection center (CGMCC) at the date of 28, 6 and 28 in 2022, and the preservation number is CGMCC No.7.462; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
Common commercial rhamnose (strain number ATCC 53103), escherichia coli (strain number ATCC 25922), staphylococcus aureus (strain number ATCC 43300).
Modified MRS liquid Medium (g/L): peptone 8.0-10.0; 9.0 to 10.0 portions of beef powder; 3.0 to 5.0 percent of yeast powder; glucose 15.0-20.0; magnesium sulfate, 0.08-0.1; sodium acetate 1.8-2.0; ammonium citrate, 4.0-5.0; 1.6 to 2.0 portions of dipotassium hydrogen phosphate; manganese sulfate, 0.03-0.05; tween 80,0.8-1.0; l-cysteine hydrochloride, 0.05-0.07 (pH 6.2.+ -. 0.2).
CMGM (g/L): starch, 5.0; pectin, 2.0; guar gum, 1.0; mucin (porcine stomach type III), 4.0; xylan, 2.0; arabinogalactan, 2.0; inulin, 1.0; casein, 3.0; peptone water, 5.0; tryptone, 5.0; bile salts, 0.4; yeast4.5 parts of extract; feSO 4 ·7H 2 O,0.005; sodium chloride, 4.5; potassium chloride, 4.5; KH (KH) 2 PO 4 ，0.5；MgSO 4 ·7H 2 O，1.25；CaCl 2 ·6H 2 O，0.15；NaHCO 3 1.5; cysteine, 0.8; heme, 0.05; tween 80,1.0.
Intestinal tract in vitro fermentation simulation system: the human colon three-stage continuous culture intestinal model system consists of three fermentors, namely a near-end fermentor (V1, 280 mL), a transverse fermentor (V2, 300 mL) and a far-end colon fermentor (V3, 320 mL). Three fermentors in series were maintained at 37℃and pH values were maintained at 5.5 (V1), 6.2 (V2) and 6.8 (V3) and were operated by continuous N 2 The introduction creates anaerobic conditions. V1, V2 and V3 are all introduced into CMGM by peristaltic pumps. Fresh healthy human faeces samples were collected and stored in an anaerobic cabinet (10% H) 2 、10％CO 2 、80％N 2 ) In (1:5) (w/w) faecal dilutions in anaerobic PBS (0.1 mol/L PBS pH 7.4) were prepared within 15 minutes after collection. Each fermenter of the intestinal model was inoculated with 100mL of fecal dilution. The total system transit time was set to 48 hours based on the average retention time of healthy individuals. After inoculation with fecal dilutions, the intestinal model was run for 24 hours to stabilize the bacterial population prior to injection into the culture medium. After 24 hours, the system was run for 8 full capacity cycles to achieve steady state, and then the above fermentation broth was added to V1 in an amount of 1% (w/V) of the volume of the contents in the tank, followed by eight volume cycles to reach steady state, and further culture for 24 hours.
Other experimental materials and instruments were commercially available unless otherwise specified.
Example 1
The embodiment provides a lactobacillus rhamnosus LR-28 strain with the effects of soothing nerves and aiding sleep, which is obtained by screening the following steps:
1. separation and screening of lactobacillus rhamnosus LR-28
1.1 separation
10g of traditional yoghurt (from Tashikuer Ganjik county, tashikuer) in the family of herdsman is taken and added into 100ml of physiological saline to be fully and uniformly mixedRespectively diluting with PBS to 10 -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 Each concentration was 100. Mu.L to the modified MRS solid medium supplemented with calcium carbonate and was spread evenly, and each concentration was repeated. Placing the strain in a constant temperature incubator at 37 ℃ for culturing for 48 hours, and observing the conditions of colony and transparent ring generation, thereby being convenient for further separation and purification.
1.2 purification culture
Selecting a proper gradient from the above culture concentration gradients, selecting 15-25 single colonies with clear boundaries and transparent circles, subculturing the selected colonies on a newly prepared MRS solid culture medium by a streaking method for 24-48h, and repeating the steps three times again to obtain the monoclonal strain. After the strain is cultured for 24 hours by an MRS liquid culture medium, bacterial liquid and 50% glycerol are uniformly mixed according to the ratio of 1:1 (the final concentration of the glycerol is 25%), and the strain is preserved at the temperature of minus 75 ℃ and simultaneously inoculated to the inclined surface of an MRS solid culture medium test tube for temporary preservation.
1.3 preliminary screening of Strain
The glycerol-preserved bacterial liquid was inoculated into 1.5mL of MRS liquid medium at an inoculum size of 2% and placed in a constant temperature incubator at 37℃for 10 hours as seed bacterial liquid for the next experiment.
Inoculating the seed bacterial liquid into 2mL EP tube filled with MRS liquid culture medium according to the inoculation amount of 2%, and culturing for 48h at 37 ℃; picking single colony on MRS liquid culture medium, and culturing at 37deg.C for 16 hr; transferring to GYP liquid culture medium, standing at 37deg.C for 48 hr; taking 1 mu L of supernatant fluid of the fermentation liquor, spotting the supernatant fluid on filter paper (each 10 samples of +1 standard/piece of filter paper), centrifuging the supernatant fluid for 10min at 2000rpm, drying the filter paper after 4h of chromatography in a developing agent, spraying 8g/L ninhydrin on the filter paper, developing the color in a baking oven at 70 ℃ for 5min, and primarily screening 18 strains with gamma-aminobutyric acid (GABA) production.
1.4 strain double Screen
Inoculating the 18 strain seed bacterial liquid obtained by primary screening into 2mL EP tube filled with MRS liquid culture medium according to the inoculum size of 2%, and culturing for 48h at 37 ℃; picking single colony on MRS liquid culture medium, and culturing at 37deg.C for 16 hr; then transferring to GYP liquid culture medium, standing and culturing for 48h at 37 ℃. Centrifuging to obtain supernatant, and quantitatively determining GABA production capacity of 18 strains by adopting high performance liquid chromatography. The experimental result shows that the strain is in the shape of a fungus under a microscope as shown in the figure 1, 18 strains obtained by separation all have the capacity of producing GABA, wherein the capacity of producing GABA of the strain LR-28 is strongest, and the GABA yield is as high as 3.838g/L.
1.5 identification of Lactobacillus rhamnosus LR-28 Strain
1.5.1 Colony and fungus body shape characteristics of LR-28 strain
Bacterial colony morphology observation: the surface is smooth and glossy.
Characteristics of the cell morphology: gram positive, polymorphobacilli.
1.5.2 Sequencing analysis of 16S rRNA of LR-28 Strain
TABLE 1 similarity of 16S rRNA sequences of LR-28 strains and reference strains
In summary, the identified LR-28 was identified as a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) by combining the 16S rRNA sequence analysis and the physiological and biochemical identification results.
Lactobacillus rhamnosus LR-28 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 7.464 in the 6 th month of 2022; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
2. Probiotic property test of lactobacillus rhamnosus LR-28 strain
2.1 gastric acid and bile salt resistance test
Acid resistance test: the activated lactobacillus rhamnosus LR-28 strain screened in example 1 and the common lactobacillus rhamnosus are respectively inoculated into an MRS liquid culture medium with the pH value of 3.0 according to the inoculation amount of 2 percent, cultured in a 37 ℃ incubator, and the viable count of 0 and 4 hours is measured (plate counting method). The results are shown in FIG. 2.
Bile salt resistance test: the activated lactobacillus rhamnosus LR-28 strain screened in example 1 and the commonly used lactobacillus rhamnosus strain (strain number: ATCC 53103) are respectively inoculated into an MRS liquid culture medium containing 0.03% bovine bile salt, placed into a 37 ℃ incubator for culture, and the viable count of 0h and 4h is respectively measured (plate count method). The results are shown in FIG. 3.
Compared with common commercial strains, the lactobacillus rhamnosus LR-28 has very high acid resistance and bile salt resistance, can resist artificial gastric juice and artificial intestinal juice, and ensures that enough viable bacteria are planted in intestinal tracts after digestion of stomach and intestines.
2.2 adhesion test
Inoculating caco-2 cells with the concentration of 1×10 cells/mL at one passage to a 12-well plate, culturing to obtain single-layer cells under the environmental condition of 5% CO at 37 ℃, discarding the culture solution, lightly washing three times with sterile PBS buffer solution, adding 1mL of DMEM stock solution, and respectively adding lactobacillus rhamnosus LR-28 strain and common lactobacillus rhamnosus strain suspension (suspended with DMEM, the concentration is about 10 0 cfu/mL) was prepared, three wells were made per strain, after 2h incubation, the supernatant was discarded, washed three times with sterile PBS, digested 5-8min with 300 μl of 0.25% pancreatin per well, then stopped with 300 μl of incomplete DMEM medium (serum containing no diabody) and the number of adherent strains was counted by plate dilution coating.
Adhesion (%) = number of adhesion strains/initial inoculation number x 100%.
The results are shown in fig. 4, and compared with common commercial lactobacillus rhamnosus, lactobacillus rhamnosus LR-28 can adhere to intestinal mucosa more effectively, form biological barriers, protect intestinal tracts and provide important preconditions for colonisation in human intestinal tracts.
2.3 bacteriostasis test
The activated lactobacillus rhamnosus LR-28 was inoculated into 2mL EP tubes with MRS liquid medium at an inoculum size of 2% and placed in a 37 ℃ incubator for 18h. And (5) taking 500g of lactobacillus rhamnosus LR-28 culture solution, and centrifuging for 10min to obtain a supernatant for later use.
With E.coli (ATCC 25922) and Staphylococcus aureus(ATCC 43300) is an indicator bacterium. Taking 100 μl of activated indicator bacteria, and adjusting the concentration of indicator bacteria to 1.0X10 5 CFU/mL, coating plates on corresponding culture mediums, placing the coated culture dishes on an ultra-clean workbench for half opening for 10min, adding 200 mu L of lactobacillus rhamnosus LR-28 supernatant into oxford cups with well-placed solid culture mediums, culturing at 37 ℃ for 16h steadily, observing and measuring the diameter of a bacteriostasis ring of each bacterial liquid, and performing three groups of experiments in parallel.
The results show that lactobacillus rhamnosus LR-28 can inhibit escherichia coli and staphylococcus aureus, has antibacterial property, and can regulate intestinal flora imbalance and maintain intestinal flora balance by inhibiting pathogenic bacteria.
2.4 antioxidant Capacity analysis
2.4.1 Strain culture method and sample preparation
Inoculating preserved lactobacillus rhamnosus LR-28 strain and common commercial lactobacillus rhamnosus to an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing at 37 ℃ for 24 hours, sequentially and continuously activating for three generations to obtain fermentation liquor, taking 100ml of each fermentation liquor after culturing, and refrigerating at 4 ℃ for later use to obtain fermentation liquor stock solution samples.
2.4.2 functional test of Lactobacillus rhamnosus LR-28 for scavenging DPPH free radical
DPPH was dissolved in 95% ethanol to prepare a solution of 0.2 mmol/L. The sample group is that 0.5mL DPPH solution is respectively mixed with 0.5mL lactobacillus rhamnosus LR-28 fermentation stock solution or common commercial lactobacillus rhamnosus fermentation stock solution, and after shaking and mixing, standing for 30min at room temperature, centrifuging for 2min at 10000rpm, collecting supernatant, and measuring absorbance (OD value) at 517nm, wherein each group is 2 paralleled. An equal volume of physiological saline is used for replacing fermentation stock solution as a control group, and a mixture of the physiological saline and absolute ethyl alcohol with equal volume is used as a blank for zeroing. The DPPH radical scavenging rate was calculated according to the following formula:
DPPH radical scavenging = (a Control -A Sample of )/A Control ×100％。
TABLE 2 DPPH radical scavenger of Lactobacillus rhamnosus LR-28
| Strain | DPPH radical scavenging rate |
| Lactobacillus rhamnosus LR-28 | 21％ |
| Commonly used commercial lactobacillus rhamnosus | 11％ |
2.4.3 functional test of Lactobacillus rhamnosus LR-28 for scavenging hydroxyl free radicals
2.0mmol/L ferrous sulfate solution 2.0mL,1.0mmol/L H are added in sequence into a 10mL cuvette 2 O 2 2.0mL of the solution is shaken uniformly, 2.0mL of lactobacillus rhamnosus LR-28 fermentation stock solution or common commercial lactobacillus rhamnosus fermentation stock solution is respectively added, uniformly mixed, and then 2.0mL of 6.0mmol/L salicylic acid solution is added. The absorbance was measured at 510nm after 15min in a water bath at 37℃and 2 replicates were made for each group. Clearance was calculated by the following formula with equal volume:
clearance (%) = [ a 0 -(A 1 -A 2 )]/A 0 ×100％。
Wherein: a is that 0 Absorbance measured for the unfermented stock solution; a is that 1 Absorbance of the fermentation stock solution; a is that 2 To add no H 2 O 2 Absorbance of the solution.
TABLE 3 hydroxyl radical clearance of Lactobacillus rhamnosus LR-28
| Strain | Hydroxyl radical scavenging rate |
| Lactobacillus rhamnosus LR-28 | 28％ |
| Commonly used commercial lactobacillus rhamnosus | 16％ |
The result shows that the lactobacillus rhamnosus LR-28 has higher antioxidant activity than the common commercial lactobacillus rhamnosus, can effectively remove the number of free radicals in a body, and relieve oxidative stress reaction of the body, so that body cells are prevented from being damaged by the free radicals.
Example 2
The embodiment provides a fermentation product of lactobacillus rhamnosus LR-28 strain, which is prepared by the following steps: inoculating seed solution of lactobacillus rhamnosus LR-28 strain into an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing at 37 ℃ for 24 hours, and sequentially and continuously activating for three generations to obtain fermentation liquor.
Taking the cultured fermentation liquor, adding the fermentation liquor into a fermentation tank (V1) which is connected with an intestinal in-vitro fermentation simulation system into which CMGM and fecal diluent are introduced and is completed for 8 times of full-capacity turnover according to the adding amount of 1% (w/V), performing fermentation culture, performing volume turnover for eight times, reaching a steady state, centrifuging, and taking supernatant.
Considering the operating volume (900 mL) and retention time (48 h) of the colon model system, the fermentation broth was added to V1 at an addition level of 1% (w/V) of the volume of the contents of the tank every day, and eight additional volume turnovers were performed to reach steady state, centrifuged, and the supernatant was taken.
Example 3
The embodiment provides a fermentation product of lactobacillus plantarum LP-28 and lactobacillus rhamnosus LR-28 strain combination, and the preparation method comprises the following steps: seed solutions of lactobacillus plantarum LP-28 and lactobacillus rhamnosus LR-28 strains are respectively inoculated into an improved MRS liquid culture medium according to the inoculum size of 3 percent, and are uniformly mixed by shaking, cultured for 24 hours at 37 ℃, sequentially activated for three generations continuously to obtain fermentation liquor, and the obtained fermentation liquor is mixed according to the volume ratio of 2:5 to obtain mixed fermentation liquor.
Taking mixed fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal tract in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the compound fermentation liquor.
Example 4
The embodiment provides a fermentation product of lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28 strain combination, and the preparation method comprises the following steps: inoculating seed solutions of lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28 strain into an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing at 37 ℃ for 24 hours, sequentially and continuously activating for three generations to obtain fermentation liquor, and mixing the obtained fermentation liquor according to a volume ratio of 5:1 to obtain mixed fermentation liquor.
Taking mixed fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal tract in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the compound fermentation liquor.
Example 5
The embodiment provides a fermentation product of lactobacillus plantarum LP-28, lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28, which is prepared by the following steps: seed solutions of lactobacillus plantarum LP-28, lactobacillus rhamnosus LR-28 and bifidobacterium animalis BAL-28 are respectively inoculated into an improved MRS liquid culture medium according to the inoculation amount of 3 percent, and are uniformly mixed by shaking, cultured for 24 hours at 37 ℃, sequentially activated for three generations continuously to obtain fermentation liquor, and the obtained fermentation liquor is mixed according to the volume ratio of 2:5:1 to obtain mixed fermentation liquor.
Taking mixed fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal tract in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the compound fermentation liquor.
Comparative example 1
Inoculating seed solution of lactobacillus plantarum LP-28 strain into an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing for 24 hours at 37 ℃, and sequentially and continuously activating for three generations to obtain fermentation liquor.
Taking the cultured fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the fermentation liquor.
Comparative example 2
Inoculating the seed solution of the bifidobacterium animalis BAL-28 strain into an improved MRS liquid culture medium according to the inoculation amount of 3%, shaking and mixing uniformly, culturing for 24 hours at 37 ℃, and sequentially and continuously activating for three generations to obtain fermentation liquor.
Taking the cultured fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the fermentation liquor.
Comparative example 3
Inoculating seed solutions of lactobacillus plantarum LP-28 and bifidobacterium animalis BAL-28 strain into an improved MRS liquid culture medium according to an inoculum size of 3%, shaking and mixing uniformly, culturing at 37 ℃ for 24 hours, sequentially and continuously activating for three generations to obtain fermentation liquor, and mixing the obtained fermentation liquor according to a volume ratio of 5:1 to obtain mixed fermentation liquor.
Taking mixed fermentation liquor, and carrying out fermentation culture in a fermentation tank (V1) of an intestinal tract in-vitro fermentation simulation system according to the method of the embodiment 1 to obtain the compound fermentation liquor.
Test example 1
The total GABA content of the fermentation broths obtained in examples 2 to 5 and comparative examples 1 to 3 was quantitatively determined by high performance liquid chromatography.
The experimental results are as follows:
table 4: examples 2 to 5 and comparative examples 1 to 3 were carried out in terms of total GABA content in the fermentation broths
The results show that the lactobacillus plantarum LP-28, the lactobacillus rhamnosus LR-28 and the bifidobacterium animalis BAL-28 can all generate GABA under the in-vitro simulated fermentation condition of intestinal tracts, but the lactobacillus rhamnosus LR-28 has the strongest GABA production capacity, and the lactobacillus rhamnosus LR-28 can obviously improve the GABA content under the condition of mixed fermentation with lactobacillus plantarum LP-28 and/or lactobacillus rhamnosus LR-28 strain, and has obvious synergistic effect.
Example 6
The present embodiment provides a method of a composition of a Jia-ban fungus group, is prepared by fermenting lactobacillus rhamnosus LR-28 strain obtained in the example 1. The preparation method comprises the following steps:
lactobacillus rhamnosus LR-28 strain seed solution is added into a culture medium according to the inoculation amount of 3 percent for fermentation culture, and the culture conditions are as follows: culturing at 37deg.C for 12 hr, centrifuging at 6000r/min for 30min to obtain bacterial mud, mixing bacterial mud with freeze-drying protecting agent, freeze drying, and pulverizing to obtain Jia-Gang fungus group mixture (1.0X10) 11 CFU/g)。
The preparation method of the culture medium comprises the following steps: mixing 40g of glucose, 4g of soybean peptone, 3g of yeast extract powder, 2g of diamine hydrogen citrate, 6g of anhydrous sodium acetate, 3g of dipotassium hydrogen phosphate, 1g of isomaltooligosaccharide, 3g of starch and other food-grade materials, adding 1000g of pure water, uniformly mixing until no macroscopic particles exist, regulating the pH value to 5.0-6.8 after material melting, and sterilizing for 20min at 117.5-118.5 ℃ to obtain the culture medium.
The preparation method of the freeze-drying protective agent comprises the following steps: mixing 100g of starch, 20g of glucose, 60g of maltodextrin, 30g of sucrose, 10g of lactose, 50g of skimmed milk powder and other food raw materials uniformly, adding 1000g of pure water, dissolving until no macroscopic particles exist, adjusting the pH to 5.0-6.8 after material melting, sterilizing for 20min at 117.5-118.5 ℃, and adjusting the pH to 6.0-7.3 after sterilization to obtain the freeze-drying protective agent.
Example 7
The present embodiment provides a method of a composition of a Jia-ban fungus group, is prepared by fermenting lactobacillus rhamnosus LR-28 strain and lactobacillus plantarum LP-28 strain obtained in the example 1. The preparation method comprises the following steps:
mixing seed solutions of lactobacillus rhamnosus LR-28 strain and lactobacillus plantarum LP-28 strain according to the volume ratio of 2:5, and adding 3% of inoculum size into a culture medium for fermentation culture, wherein the culture conditions are as follows: 37 ℃ for 12 hours; centrifuging at 6000r/min for 30min to obtain bacterial mud, mixing bacterial mud with freeze-drying protecting agent, lyophilizing, and pulverizing to obtain Jia-Gang fungus composition (1.1X10) 11 CFU/g)。
The preparation method of the culture medium comprises the following steps: mixing 40g of glucose, 4g of soybean peptone, 3g of yeast extract powder, 2g of diamine hydrogen citrate, 6g of anhydrous sodium acetate, 3g of dipotassium hydrogen phosphate, 1g of isomaltooligosaccharide, 3g of starch and other food-grade materials, adding 1000g of pure water, uniformly mixing until no macroscopic particles exist, regulating the pH value to 5.0-6.8 after material melting, and sterilizing for 20min at 117.5-118.5 ℃ to obtain the culture medium.
The preparation method of the freeze-drying protective agent comprises the following steps: mixing 100g of starch, 20g of glucose, 60g of maltodextrin, 30g of sucrose, 10g of lactose, 50g of skimmed milk powder and other food raw materials uniformly, adding 1000g of pure water, dissolving until no macroscopic particles exist, adjusting the pH to 5.0-6.8 after material melting, sterilizing for 20min at 117.5-118.5 ℃, and adjusting the pH to 6.0-7.3 after sterilization to obtain the freeze-drying protective agent.
Example 8
The present embodiment provides a method of a composition of a Jia-ban fungus group, is prepared by fermenting lactobacillus rhamnosus LR-28 strain, lactobacillus plantarum LP-28 strain and bifidobacterium animalis BAL-28 strain obtained in the example 1. The preparation method comprises the following steps:
mixing seed solutions of lactobacillus rhamnosus LR-28 strain, lactobacillus plantarum LP-28 strain and bifidobacterium animalis BAL-28 strain according to the volume ratio of 3:5:1, and adding 3% of inoculum size into a culture medium for fermentation culture, wherein the culture conditions are as follows: 37 ℃ for 12 hours; centrifuging at 6000r/min for 30min to obtain bacterial mud, mixing bacterial mud with freeze-drying protecting agent, lyophilizing, and pulverizing to obtain Jia-Gang fungus composition (1.0X10) 11 CFU/g)。
The preparation method of the culture medium comprises the following steps: mixing 40g of glucose, 4g of soybean peptone, 3g of yeast extract powder, 2g of diamine hydrogen citrate, 6g of anhydrous sodium acetate, 3g of dipotassium hydrogen phosphate, 1g of isomaltooligosaccharide, 3g of starch and other food-grade materials, adding 1000g of pure water, uniformly mixing until no macroscopic particles exist, regulating the pH value to 5.0-6.8 after material melting, and sterilizing for 20min at 117.5-118.5 ℃ to obtain the culture medium.
The preparation method of the freeze-drying protective agent comprises the following steps: mixing 100g of starch, 20g of glucose, 60g of maltodextrin, 30g of sucrose, 10g of lactose, 50g of skimmed milk powder and other food raw materials uniformly, adding 1000g of pure water, dissolving until no macroscopic particles exist, adjusting the pH to 5.0-6.8 after material melting, sterilizing for 20min at 117.5-118.5 ℃, and adjusting the pH to 6.0-7.3 after sterilization to obtain the freeze-drying protective agent.
Example 9
The embodiment provides application of lactobacillus rhamnosus LR-28 strain serving as a raw material in preparation of a product for improving sleep quality.
1. Test procedure
Groups of 195 insomnia patients (screening criteria: PSQI (Pisburgh index) > 7 were enrolled, grouped by age, group A (20-35 years): 60 people; group B (35-59 years): 70 people; group C (over 60 years): 65 persons. The composition of the present invention was administered to insomnia patients in an amount of 5-6 g/day containing 150-180mg of gamma-aminobutyric acid for 2 weeks. And carrying out sleep investigation on the three groups, and carrying out sleep quality index investigation of Pisburg in America.
2. Test results and analysis
2.1 Sleep findings for 2 weeks:
the sleep quality of 60 persons in the group A is obviously improved for 58 persons, no obvious effect is achieved for 2 persons, and the effective rate is 96.7%; the sleep quality of 66 persons in the group B is obviously improved, 4 persons have no obvious effect, and the effective rate is 94.3%; the 65 persons in group C all reflect obvious improvement effect, and the effective rate is 100%.
2.2 A, B, C results of three group of symptomatic improvement studies
Table 5A group results of symptomatic improvement study
Table 6B group results of symptomatic improvement study
Table 7C group results of symptomatic improvement study
2.3 measurement of improvement Using (PSQI) American Pisburgh sleep quality index investigation
Tables 8A and B, C three groups of sleep quality index investigation and measurement improvement amounts using (PSQI) U.S. Pisburgh
| Group numbering | Before taking | For 1-7 days | For 8-14 days |
| Group A | 11 | 7 | 5 |
| Group B | 13 | 9 | 7 |
| Group C | 15 | 9 | 8 |
From the above tables 5 to 8, it can be seen that the agent for promoting sleep provided by the invention has good effects of preventing sleep disorder, improving sleep quality and relieving dysphoria.
In conclusion, the lactobacillus rhamnosus LR-28 strain provided by the invention has the activity of high-yield GABA in human intestinal environment, has strong antibacterial activity and gastrointestinal fluid tolerance, and can be used for preparing health-care products, foods or food additives related to sleep improvement.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A lactobacillus rhamnosus LR-28 strain with effects of tranquillizing and aiding sleep is characterized in that the strain is classified as lactobacillus rhamnosus @ or lactobacillus rhamnosus @ and has the functions of tranquillizing and aiding sleepLactobacillus rhamnosus) The microbial strain is preserved in China general microbiological culture collection center (CGMCC) at 26 days of 6.2022, and the preservation number is CGMCC No.7.464; the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
2. Use of lactobacillus rhamnosus LR-28 strain of claim 1 in the preparation of a food product.
3. The fermentation product of lactobacillus rhamnosus LR-28 strain of claim 1, wherein the fermentation product is produced by fermentation of the lactobacillus rhamnosus LR-28 strain.
4. Method for producing gamma-aminobutyric acid using lactobacillus rhamnosus LR-28 strain according to claim 1, characterized in that it comprises in particular the following operations: seed culture is carried out on the strain in an improved MRS liquid culture medium, and anaerobic fermentation culture is carried out on the cultured fermentation liquor on an intestinal model culture medium; the strain comprises the lactobacillus rhamnosus LR-28.
5. The method of claim 4, wherein the strain further comprises Lactobacillus plantarumLactobacillus plantarum) LP-28 and Bifidobacterium animalis subspecies LactobacilliBifidobacterium animalis subsp.lactis) At least one of BAL-28;
wherein, the LP-28 and the BAL-28 are both preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.7.463 and the preservation number of the BAL-28 is CGMCC No.7.462.
6. The method of claim 4, wherein the strain is lactobacillus rhamnosus LR-28 or lactobacillus plantarum LP-28.
7. The method for producing gamma-aminobutyric acid by lactobacillus rhamnosus LR-28 strain according to claim 4, wherein the anaerobic fermentation culture conditions are as follows: the fermentation temperature is 35-37 ℃, the pH is 5-7, and the culture is carried out for more than 48 hours.
8. A bacterial composition comprising lactobacillus rhamnosus LR-28 strain according to claim 1 or a combination thereof with other probiotics; the other probiotics comprise at least one of lactobacillus plantarum LP-28 and bifidobacterium animalis subspecies BAL-28;
wherein, the LP-28 and the BAL-28 are both preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.7.463 and the preservation number of the BAL-28 is CGMCC No.7.462.
9. The microbial community composition according to claim 8, wherein the preparation method of the microbial community composition comprises the following steps: fermenting the lactobacillus rhamnosus LR-28 strain or the combination of the lactobacillus rhamnosus LR-28 strain and other probiotics, centrifuging after fermentation to obtain bacterial sludge, uniformly mixing the bacterial sludge with a freeze-drying protective agent, freeze-drying, and crushing to obtain the bacterial group mixture.
10. The microbial composition of claim 9, wherein the lyoprotectant comprises at least one of starch, glucose, maltodextrin, sucrose, lactose, whey protein powder and skimmed milk powder as a raw material; the preparation method of the freeze-drying protective agent comprises the following operations: dissolving the raw materials of the freeze-drying protective agent in water, regulating the pH to 5.0-6.8, sterilizing, and regulating the pH to 6.0-7.3 after sterilizing to obtain the freeze-drying protective agent.
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