CN115838675B - Lactobacillus rhamnosus and composition and application thereof - Google Patents
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Abstract
Lactobacillus rhamnosus (Lactobacillus rhamnosus) S24 is preserved in the China center for type culture collection of China, with the preservation number of CCTCC NO: M2021986, in 2021, 8 months and 6 days. The composition of the invention comprises a live strain of lactobacillus rhamnosus S24, an inactivated strain of lactobacillus rhamnosus S24, a strain metabolite and a mixture of one or more of lactobacillus rhamnosus S24 metants. The invention also discloses application of lactobacillus rhamnosus S24 and its metaplasia in preparing functional food or medicine for preventing and treating sarcopenia or osteoporosis or sarcopenia. The lactobacillus rhamnosus S24 has good safety, and the lactobacillus rhamnosus S24 has wide application, can be used for preparing functional foods or medicines for preventing and treating sarcopenia or osteoporosis or sarcoosseous co-subtraction syndrome, or functional foods or medicines for inhibiting pathogenic bacteria, or can be used as a starter for preparing fermented foods and health-care foods.
Description
Technical Field
The invention relates to the technical field of microorganisms and foods and medicines, in particular to lactobacillus rhamnosus and a composition and application thereof.
Background
Sarcopenia (SP) is a disease of the muscular system characterized by progressive and systemic decline in muscle mass, muscle strength and muscle function, leading to limited patient activity and an increased risk of mortality due to the tendency to fall. Osteoporosis (osteoporosis, OP) is a systemic osteotomy characterized by reduced bone mass and increased bone fragility due to destruction of bone microstructure, OP causes a decrease in patient quality of life, and increases in disability and mortality. Since sarcopenia and osteoporosis share common physiological mechanisms, such as mechano-mechanical factors, genetic genes, inflammatory factors, endocrine factors. Meanwhile, the sarcopenia and the osteoporosis have strong relevance in clinical manifestations of amyotrophy, gait abnormal, pain, falling to brittle fracture and the like. In 2009, binkley and the like, based on the pathophysiological basis and close correlation of sarcopenia and osteoporosis, proposed the concept of sarcopenia (osteosarcopenia, OS), mainly referring to patients who meet the diagnostic criteria of osteoporosis and simultaneously have reduced muscle mass/function. OS patients show a decrease in bone density (BMD), an increase in bone fragility, a progressive and systemic decrease in muscle mass, muscle strength and muscle function, leading to an increased risk of weakness, falls, fractures, hospitalization, disability and mortality in the patient.
According to different diagnostic criteria for sarcopenia, the incidence of sarcopenia in people over 60 years old ranges from 5% to 37%. At present, no targeted medicine for preventing and treating the sarcopenia syndrome exists, and the effective prevention and treatment means at present comprise traditional Chinese medicine treatment mainly for strengthening the spleen and tonifying the kidney, for example, CN202110982828.1 discloses a traditional Chinese medicine composition for preventing and treating sarcopenia osteoporosis, the composition comprises traditional Chinese medicines or extracts such as eucommia ulmoides, and the like, and in addition, early intervention measures such as resistance and balance training, nutrition intake (supplementing vitamin D and calcium) and the like can also effectively prevent and relieve the sarcopenia symptom. In recent years, numerous studies have shown that intestinal microbiota can be used as an intermediary for the influence of nutrition on muscle health and bone health, as CN 111748640B discloses the use of intestinal microbiota in sarcopenia, by detecting the abundance of Erysipelotrichaceae _ucg-003, it is possible to diagnose whether a subject is suffering from sarcopenia and/or the risk of suffering from sarcopenia, thus enabling a non-invasive diagnosis of a subject; CN 111718873B discloses a lactobacillus fermentum with osteoporosis relieving effect and application thereof, the strain obviously improves bone density and cortical volume of the ovariectomized rat. CN 103889241B discloses a composition for promoting healthy bone growth and/or for preventing and/or treating bone diseases comprising a mixture of at least one long chain polyunsaturated fatty acid (LC-PUFA), at least one probiotic, and oligosaccharides, said mixture containing at least one N-acetylated oligosaccharide, at least one sialylated oligosaccharide and at least one neutral oligosaccharide, said probiotic being lactobacillus or bifidobacterium. However, the existing probiotics technology only carries out isolation research on sarcopenia and osteoporosis at a single visual angle, and research reports on sarcopenia by specific strains and metaplasia thereof are not seen, so that new strains and compositions thereof are developed for preparing a convenient and efficient nutritional product or medicament for relieving and treating the sarcopenia, and the novel strains and compositions have important significance and great market value.
Disclosure of Invention
The invention aims to solve the technical problems that: the invention overcomes the defects of the prior art, provides lactobacillus rhamnosus which has high biological activity, wide carbon source utilization, can resist gastric juice, intestinal juice and bile salt and has the function of preventing or/and treating sarcopenia or osteoporosis or sarcoosseous co-reduction.
One of the technical schemes adopted for solving the technical problems is as follows:
Lactobacillus rhamnosus, named lactobacillus rhamnosus (Lactobacillus rhamnosus) S24, is preserved in China center for type culture Collection of Wuhan in China, with a preservation number of CCTCC NO: M2021986, at 8 months and 20 days of 2021.
Biological preservation description: lactobacillus rhamnosus (Lactobacillus rhamnosus) S24, deposited with the chinese collection of typical cultures, accession number: eight-path 299 of Wuchang district in Wuhan, hubei province, the preservation organization is abbreviated as: cctccc, date of preservation: 2021, 8, 20 (2021, 8, 6 received registration books, 8, 20 detected survival and preserved), biological preservation number cctccc NO: m2021986, strain number: lactobacillus rhamnosus S24.
Wherein the lactobacillus rhamnosus S24 is isolated from a plant source, in particular, the lactobacillus rhamnosus S24 is obtained by screening and separating from Sichuan Pengzushan farmyard acid dough.
The biological properties of lactobacillus rhamnosus S24 of the present invention are as follows:
1) Morphological features: the colony grows well on MRS agar culture medium, is in a nearly spherical rod shape, is arranged singly, in pairs or in a fence shape, is medium in size, is milky white, is raised upwards in gloss, is irregular in colony edge and easy to pick, is arranged in pairs or in a fence shape under a microscope, and is positive after gram staining.
2) Biological identification: the lactobacillus rhamnosus S24 is sent to a Sichuan province microorganism resource platform strain collection center for 16S rRNA identification, the 16S rRNA gene sequence of the lactobacillus rhamnosus S24 is shown as SEQ ID NO. 1, NCBI BLAST comparison is carried out on the 16S rRNA gene sequence, a Neighbor-Joing phylogenetic tree is constructed by taking B.subtilis NCDO 1769 (NR 118972) as an outer branch based on the 16S rRNA gene sequence comparison result, as shown in figure 2, the similarity with lactobacillus rhamnosus (Lactobacillus rhamnosus) in Genebank is 100%, and the strain is identified as lactobacillus rhamnosus (Lactobacillus rhamnosus) S24.
The culture medium for lactobacillus rhamnosus S24 fermentation is MRS or vitamin D 3 -MRS culture medium, wherein the vitamin D 3 -MRS culture medium comprises 10.0 g/L of peptone, 5.0 g/L of beef powder, 4.0 g/L of yeast powder, 20.0 g/L of glucose, 1.0 g/L of tween 80, 2.0 g/L of K 2HPO4·7H2 O, 5 g/L of anhydrous sodium acetate, 2.0 g/L of diammonium citrate, 0.038 g/L of MgSO 4·7H2O 0.2 g/L,MnSO4·H2 O and 1000-4000 IU of vitamin D 3 (agar powder 15 g/L is added as a solid culture medium), preferably, the vitamin D 3 in the vitamin D 3 -MRS culture medium can promote the increase of short-chain fatty acid, and particularly the butyric acid content playing an important role in intestinal myoaxis and intestinal bone axis is remarkably improved; vitamin D 3 can promote and improve the antibacterial ability of lactobacillus rhamnosus S24 to pathogenic microorganisms, and has antibacterial effects on helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and Candida albicans ATCC 10231.
The other technical scheme adopted by the invention for solving the technical problems is as follows:
A composition comprising the above-mentioned live strain of lactobacillus rhamnosus S24, inactivated strain and strain metabolite of lactobacillus rhamnosus S24, a mixture of one or more of a live strain of lactobacillus rhamnosus S24 and a live strain of lactobacillus plantarum, a live strain of lactobacillus fermentum, wherein lactobacillus plantarum is preferably lactobacillus plantarum LP550 and lactobacillus plantarum LP220, and lactobacillus fermentum is preferably lactobacillus fermentum GF1800. Preferably, the composition is lactobacillus rhamnosus S24 metant.
The lactobacillus rhamnosus S24 metagen disclosed by the invention refers to lactobacillus thallus and metabolic components after lactobacillus rhamnosus S24 fermentation broth is processed/inactivated, concentrated and dried, and the lactobacillus thallus and metabolic components are collectively called as the lactobacillus thallus components and metabolic products.
The composition includes, but is not limited to, a biological agent, a health product, or a medicament.
The medicament also contains a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carriers include, but are not limited to: one or more of a filler, binder, wetting agent, disintegrant, or lubricant.
The medicine is used for preventing and treating sarcopenia, osteoporosis or sarcopenia, preferably oral medicine.
The filler is one or more of fucoidin, trehalose, lactose, chitosan, starch or dextrin; the adhesive is one or more of liquid glucose, starch paste or syrup; the wetting agent is one or more of glycerol or ethanol; the disintegrating agent is one or more of crospovidone, sodium carboxymethyl starch or sodium cross-linked carboxymethyl starch; the lubricant is one or more of silicon dioxide magnesium stearate or sodium stearate fumarate.
The lactobacillus rhamnosus S24 and the S24 metaplasia thereof can obviously relieve weight loss of rats with sarcopenia (OS for short), and the lactobacillus rhamnosus S24 and the S24 metaplasia thereof can obviously improve the whole body bone density and lumbar vertebra bone density of the OS rats so as to prevent the formation of Osteoporosis (OP).
The lactobacillus rhamnosus S24 and the S24 metagen thereof can obviously improve the forelimb grabbing force, the whole body skeletal muscle quantity and the limb skeletal muscle quantity of an OS rat, and realize effective prevention and treatment of Sarcopenia (SP).
Lactobacillus rhamnosus S24 can also promote the expression of gastrocnemius PI3K protein of OS rats, and lactobacillus rhamnosus S24 prevents and treats sarcoosseous co-reduction OS by up-regulating the expression of PI3K signal pathway related proteins.
Lactobacillus rhamnosus S24 also promotes increasing the abundance of lactobacillus and bifidobacterium in the intestinal tract of OS rats, thereby achieving prevention and treatment of sarcoidosis OS.
Based on the above characteristics of the lactobacillus rhamnosus S24 of the present invention, one of the applications of the lactobacillus rhamnosus of the present invention and its metants is:
Use of lactobacillus rhamnosus S24 in the manufacture of a medicament for the prevention and treatment of sarcopenia or osteoporosis or sarcoossium co-subtractive syndrome; or the application of a composition formed by lactobacillus rhamnosus S24 and one or more of inactivated strains, strain metabolites or lactobacillus rhamnosus S24 metazoans in preparing a medicament for preventing and treating sarcopenia or osteoporosis or musculoskeletal co-subtraction syndrome.
In an exemplary embodiment, the amount of lactobacillus rhamnosus S24 is equal to or more than 6×10 8 CFU/per mouse/day, the amount of inactivated strain of lactobacillus rhamnosus S24 is 3-8×10 10 CFU/day, the amount of the post-metazoan of lactobacillus rhamnosus S24 is equal to or more than 6×10 8 per mouse/day, and the free amino acid is > 1mg/g.
Secondly, bacteriostasis experiments show that: lactobacillus rhamnosus S24 has a broad antibacterial activity against pathogenic bacteria. The specific expression is that the composition has good inhibition effect on helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC 26003, clostridium perfringens ATCC 13124 and candida albicans ATCC 10231, wherein the inhibition effect on streptococcus mutans CGMCC1.2499 is the best.
Based on the above characteristics of lactobacillus rhamnosus S24, another application of lactobacillus rhamnosus and its metants of the present invention:
Application of lactobacillus rhamnosus S24 and its metagen in preparing medicament for inhibiting pathogenic bacteria is provided.
The tolerance test of the lactobacillus rhamnosus S24 to gastric acid and bile salts shows that the strain survival rate of the lactobacillus rhamnosus S24 reaches 43.17% when the gastric juice pH value is 2.0; when the concentration of bile salt reaches 3.0g/L, the survival rate of the strain reaches 91.54 percent. Lactobacillus rhamnosus S24 has very good gastric acid and bile salt tolerance and is suitable for oral administration.
Lactobacillus rhamnosus S24 is able to utilize L-arabinose, fructose, galactose, glucose, mannose, raffinose, rhamnose, trehalose, ribose, sucrose, maltose, cellobiose, melibiose, D-tagatose, sorbose, sorbitol, mannitol, escin, N-acetyl-glucosamine, alpha-methyl-D-mannoside, with a broad spectrum of carbon source availability. The method has the advantages of strong acid production capacity, rapid acid production in the initial fermentation stage, large acid production amount, and stable acid production amount along with the increase of fermentation time. Based on the above characteristics, lactobacillus rhamnosus and a further application of its metants are disclosed:
the lactobacillus rhamnosus S24 and its metazoan are used as a starter in the preparation of fermented food, health food or dietary supplement.
The dosage of lactobacillus rhamnosus S24 is 1X 10 8~1×1010 CFU/mL or 1X 10 8~1×1010 CFU/g, and the dosage of metazoan is 1-50 g/kg or 1-50 g/L, and the free amino acid is more than 1mg/g.
The lactobacillus rhamnosus has the beneficial effects that:
The lactobacillus rhamnosus S24 grows well on MRS agar culture and vitamin D 3 -MRS culture medium, has high biological activity, wide carbon source utilization capacity and good acid production property, and can resist artificial gastric juice, intestinal juice and bile salt.
The lactobacillus rhamnosus S24 and the vitamin D 3 can play a role in synergistic promotion, including growth promotion, short-chain fatty acid content improvement and synergistic antibacterial effect.
The lactobacillus rhamnosus S24 and the metazoan group can obviously inhibit the weight reduction of an OS rat, obviously improve the skeletal muscle mass index, the limb skeletal mass index, the whole body bone density, the lumbar vertebra bone density and the forelimb gripping power, further delay or improve the sarcopenia or the osteoporosis or the sarcopenia syndrome, and prevent and treat the sarcopenia syndrome by increasing the PI3K protein expression, improving the abundance of intestinal lactobacillus and bifidobacteria and other various ways.
The lactobacillus rhamnosus S24 has good safety, and the lactobacillus rhamnosus S24 has wide application, and can be used for preparing medicines for preventing and treating sarcopenia or osteoporosis or sarcoosseous co-reduction syndrome, medicines for inhibiting pathogenic bacteria, or used as a starter for preparing fermented foods and health-care foods.
Drawings
FIG. 1 is a colony morphology of Lactobacillus rhamnosus S24 on MRS agar medium, wherein (A) plate colony morphology is shown, (B) microscopic strain morphology is shown;
FIG. 2 is a lactobacillus rhamnosus S24 phylogenetic tree;
FIG. 3 shows the gastric juice tolerance of Lactobacillus rhamnosus S24;
FIG. 4 shows the bile salt tolerance of Lactobacillus rhamnosus S24;
FIG. 5 is a graph showing the growth of Lactobacillus rhamnosus S24 in MRS, vitamin D 3 -MRS medium;
FIG. 6 is an acidogenic profile of lactobacillus rhamnosus S24 fermentation in MRS, vitamin D 3 -MRS medium;
FIG. 7 is a graph showing the comparison analysis of the total bone density and lumbar bone density of rats in different experimental groups; wherein, (A) the bone density of the whole body of the rat and (B) the lumbar bone density of the rat;
FIG. 8 is a graph of comparative analysis of forelimb grip of rats from different experimental groups;
FIG. 9 is a graph of comparative analysis of SMI (A) and RSMI (B) for rats from different experimental groups;
FIG. 10 is a graph showing analysis of gastrocnemius PI3K protein expression levels in rats of different experimental groups.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The formula of the culture medium in each embodiment of the invention comprises the following components:
MRS medium (Lactobacillus rhamnosus S24): 10.0 g/L peptone, 5.0 g/L beef powder, 4.0 g/L yeast powder, 20.0 g/L glucose, 1.0 g/L Tween 80, 2.0 g/L K 2HPO4·7H2 O, 5.5 g/L anhydrous sodium acetate, 2.0 g/L diammine citrate, 0.038 g/L MgSO 4·7H2O 0.2 g/L,MnSO4·H2 O (agar powder 15 g/L as solid medium).
Vitamin D 3 -MRS Medium (Lactobacillus rhamnosus S24) (abbreviated as VD 3 -MRS): 10.0 g/L peptone, 5.0 g/L beef powder, 4.0 g/L yeast powder, 20.0 g/L glucose, 1.0 g/L Tween 80, 2.0 g/L K 2HPO4·7H2 O, 5. 5 g/L anhydrous sodium acetate, 2.0 g/L diammine citrate, 0.038 g/L MgSO 4·7H2O 0.2 g/L,MnSO4·H2 O, and 4000 IU vitamin D 3 (agar-plus 15 g/L is solid medium).
Examples
Lactobacillus rhamnosus S24 (Lactobacillus rhamnosus S, 24) of the invention, accession number: CCTCC NO: M2021986.
Microorganism screening samples are collected from Sichuan Pengzu mountain farmhouse acid dough, the collected samples are sheared, then 1g is weighed, the samples are put into 9 mL sterile normal saline, and after full shaking and uniform mixing, 10-time gradient dilution is carried out, the samples are coated in MRS solid culture medium, and the samples are cultured for 48 hours at 37 ℃. Visually observing, picking single colonies with different forms and sizes in the culture medium, and repeatedly streaking, purifying and culturing; then the strain is preliminarily determined to be lactobacillus by gram staining and calcium dissolving method, and the purified strain is stored in a refrigerator at-80 ℃ for standby by 45% glycerol.
A) Morphological observation
Purified lactobacillus rhamnosus S24 was streaked on MRS agar medium, and after culturing for 48 hours at 37 ℃ in an inverted state, the colony morphology of the strain was observed, and the result is shown in fig. 1: the strain grows well on MRS agar culture medium, and the bacterial colonies are in a nearly spherical rod shape, are arranged singly, in pairs or in a fence shape, are medium in size, are milky white, are raised upwards and glossy, are irregular in edges and easy to pick, are positive after gram staining, and are arranged in pairs or in a fence shape under a microscope.
B) Molecular biology identification of strains
The purified strain is sent to the Sichuan province microorganism resource platform bacterial collection center for 16S rRNA identification, the measured 16S rRNA sequence is subjected to NCBI BLAST comparison, the similarity with lactobacillus rhamnosus in Genebank is 100%, and the strain can be primarily identified as lactobacillus rhamnosus (Lactobacillus rhamnosus). The identification sequence of the 16S rRNA of the strain is shown as SEQ ID NO. 1, and is named as lactobacillus rhamnosus S24 (Lactobacillus rhamnosus S) and the lactobacillus rhamnosus S24 is based on the comparison result of the 16S rRNA gene sequence, and a Neighbor-Joining phylogenetic tree constructed by taking B.subtilis NCDO 1769 (NR 118972) as an outer branch is shown in figure 2.
Tolerance of lactobacillus rhamnosus S24 to gastric and intestinal fluids
Preparing simulated gastric juice: 2.0g/L NaCl, regulating pH to 2.0, 2.5, 3.0 and 4.0 with HCl, autoclaving, and pepsin 3.2g/L, wherein the pepsin is used at present in experiment; preparing simulated intestinal juice: 6.8g/L of potassium dihydrogen phosphate, regulating the pH value to 7.5 by NaOH, performing high-pressure sterilization, and adding 10.0g/L of trypsin which is currently used in experiments; inoculating lactobacillus rhamnosus S24 strain stored in a glycerol tube into MRS culture medium with 10% of inoculation amount, and activating at 37 ℃ for 24 hours; equal amount of lactobacillus rhamnosus S24 bacterial liquid is added into 50mL simulated gastric fluid of the system, initial viable bacteria are recorded, and the viable bacteria number is measured after the constant temperature culture at 37 ℃ for 3 hours. The detected live lactic acid bacteria were counted and the survival rate was calculated, strain survival rate=test group/control group×100%.
Gastric acid begins to secrete when food enters the stomach. The pH value of gastric acid concentration secreted by normal stomach of human body is about 0.5-1.5. The pH value is about 7.0-7.2 when the stomach is emptied, and rapidly drops to 2-3 when food enters the stomach. After meals, gastric juice is diluted and the pH rises to about 3.5. Referring to FIG. 3, at pH2.0, S24 survival was 43.17%; at pH2.50, S24 survival rate was 65.79%; at pH3.0, S24 survival was 87.12%; lactobacillus rhamnosus S24 strain taken after feeding is able to tolerate gastric juice and in the intestine lactobacillus rhamnosus S24 strain is able to tolerate intestinal juice.
Tolerance of lactobacillus rhamnosus S24 to bile salts
Lactobacillus rhamnosus S24 strain was inoculated into MRS liquid medium at an inoculum size of 5%, and subjected to activation culture at 37 ℃ for 24 hours, followed by two consecutive activations. Inoculating the activated S24 bacterial liquid into an MRS liquid culture medium according to an inoculum size of 5%, and standing and culturing for 15h at 37 ℃ in a constant temperature incubator. And (3) centrifuging the cultured bacterial liquid at 5000rpm for 10min to collect bacterial bodies, and vibrating the bacterial bodies uniformly with sterile physiological saline.
Adding the bacterial liquid with uniform vibration into MRS culture medium with bile salt concentration of 1.0 g/L, 2.0 g/L, 3.0 g/L and 0.0g/L (initial bacterial liquid) according to the addition amount of 10%, and taking the bile salt concentration of 0.0g/L as a control group. Then incubated in a constant temperature incubator at 37℃for 3h. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterile physiological saline, beating and uniformly mixing, and detecting the number of lactic acid bacteria; counting the detected live lactobacillus and calculating the survival rate, wherein the calculation formula is as follows:
Strain survival (%) = test/control x 100%.
Lactobacillus rhamnosus S24 bile salt tolerance data are shown in fig. 4: the viability of the strain was 99.87% and 94.09% when the bile salt concentration was 1.0g/L and 2.0g/L, respectively, but the viability of the strain was still 91.54% when the bile salt concentration was 3.0 g/L. The concentration of bile salts in the intestinal tract is not more than 3.0g/L, which indicates that the S24 strain can tolerate the bile salts in the intestinal tract.
Fermentation substrate utilization Capacity
Inoculating fresh lactobacillus to be tested by slant culture into culture medium, or dripping fresh lactobacillus into melted soft agar column (temperature 47+ -1deg.C), mixing, covering a layer of 2% agar with thickness of about 7mm, culturing at 37deg.C for 48 hr, and determining that strain produces acid by using carbon source.
As shown in Table 1, lactobacillus rhamnosus S24 strain can ferment and produce acid by using 23 carbon source substrates such as L-arabinose, fructose, galactose, glucose, mannose, melezitose, raffinose, rhamnose, trehalose, ribose, sucrose, maltose, cellobiose, melibiose, D-tagatose, sorbose, sorbitol, mannitol, escin, salix-alcohol, N-acetyl-glucosamine, alpha-methyl-D-mannoside and the like.
② Growth performance test of lactobacillus rhamnosus S24 in different media
Lactobacillus rhamnosus S24 strain is inoculated into MRS and vitamin D 3 -MRS culture medium liquid culture medium respectively according to the inoculation amount of 5%, and is activated and cultured for 24 hours at 37 ℃ and is activated twice continuously. Inoculating the activated lactobacillus rhamnosus S24 bacterial liquid into MRS and vitamin D 3 -MRS liquid culture medium according to 5% of inoculum size, mixing uniformly, and sub-packaging into sterile test tubes (18 mm×180 mm) according to 8 ml/branch; placing the subpackaged lactobacillus rhamnosus S24 bacterial liquid in a constant temperature incubator at 37 ℃ for static culture, taking 3 test tubes to measure absorbance value OD 600 of the bacterial liquid, and calculating an average value; at regular intervals, 3 test tubes are taken to measure the absorbance value OD 600 of the S24 bacterial liquid, and the average value is calculated; and drawing a growth curve by taking time as an abscissa and an absorbance value OD 600 as an ordinate.
The growth curve of the lactobacillus rhamnosus S24 strain in the MRS and vitamin D 3 -MRS culture medium is shown in the attached figure 5, and as can be seen from the figure 5, the lactobacillus rhamnosus grows in the vitamin D3-MRS liquid culture medium obviously better than that in the MRS liquid culture medium, OD 600 is increased by 35% in 18h, and the vitamin D3 obviously promotes the growth of the lactobacillus rhamnosus S24.
③ Antibacterial ability test of lactobacillus rhamnosus S24 on pathogenic bacteria
Bacteriostasis experiment for pathogenic bacteria: pouring 10 mL of water agar culture medium in a sterile flat plate, placing oxford cup after cooling and solidifying, adding indicator bacteria suspension (helicobacter pylori ATCC 26695, streptococcus mutans CGMCC 1.2499, staphylococcus aureus CMCC 26003, clostridium perfringens ATCC 13124 and candida albicans ATCC 10231) into agar culture medium which is cooled to 50 ℃ and corresponds to indicator bacteria, enabling the concentration of the indicator bacteria to be 10 6 CFU/mL, uniformly mixing, pouring the indicator bacteria on bottom water agar, taking out oxford cup after solidifying, forming holes by forceps, adding 200 mu L of sample to be tested (sample to be tested comprises lactobacillus rhamnosus S24 fermentation liquor of MRS culture medium and lactobacillus rhamnosus S24 fermentation liquor of vitamin D 3 -MRS) into each hole, diffusing for 30min, and culturing 15-24 h at 37 ℃. And (3) observing whether a bacteriostasis ring appears around the culture hole, measuring the diameter of the culture hole by using a vernier caliper, recording the diameter of the bacteriostasis ring, and finally evaluating the bacteriostasis activity according to the existence and the size of the bacteriostasis ring.
TABLE 2 antibacterial ability analysis of Lactobacillus rhamnosus S24 fermentation broths obtained by different media culture
As shown in table 2, compared with lactobacillus rhamnosus S24 obtained by fermentation of an MRS medium, lactobacillus rhamnosus S24 obtained by fermentation of the MRS medium after adding vitamin D 3 has the antibacterial ability to pathogenic microorganisms greatly improved, and the antibacterial ability of lactobacillus rhamnosus S24 in vitamin D 3 -MRS medium is obviously improved due to the fact that the antibacterial contents of helicobacter pylori ATCC 26695, streptococcus mutans CGMCC 1.2499, staphylococcus aureus CMCC 26003, clostridium perfringens ATCC 13124 and candida albicans ATCC 10231 have the antibacterial ring of more than 20mm and the number of bacteria in the vitamin D 3 -MRS medium is increased in combination with lactobacillus rhamnosus, but total acid is reduced.
④ Lactobacillus rhamnosus S24 acid production capability test
Lactobacillus rhamnosus S24 strain is inoculated into MRS and vitamin D 3 -MRS culture medium liquid culture medium respectively according to the inoculation amount of 5%, and is activated and cultured for 24 hours at 37 ℃ and is activated twice continuously. The activated S24 bacterial liquid is inoculated into MRS and vitamin D 3 -MRS culture medium liquid culture medium according to the inoculation amount of 5%, and the mixture is split into sterile test tubes (18 mm multiplied by 180) according to 8 ml/branch after being uniformly mixed. Placing the subpackaged lactobacillus rhamnosus S24 bacterial liquid in a constant temperature incubator at 37 ℃ for static culture, taking 3 test tubes to measure total acid of the bacterial liquid, and calculating an average value; at regular intervals, 3 test tubes are taken to measure the total acid of the bacterial liquid, the time is taken as an abscissa, the acid yield is taken as an ordinate, and an acid production curve is drawn, and is shown in figure 6.
Lactobacillus rhamnosus S24 is inhibited from producing acid in vitamin D 3 -MRS liquid medium, and since vitamin D 3 is stable in neutral environment but is easily absorbed in acidic environment, less acid production is beneficial to the stability of vitamin D 3, further analysis by HPLC shows that lactobacillus rhamnosus S24 has significantly improved short chain fatty acid content, in particular butyric acid content (table 3), compared to the total acid fermented in vitamin D 3 -MRS liquid medium, which is 186.35%, the above results indicate that: vitamin D 3 can obviously inhibit the generation of total acid of lactobacillus rhamnosus S24, but can improve the increase of short-chain fatty acid, in particular to obviously improve the content of butyric acid playing an important role in intestinal myoaxis and intestinal bone axis.
TABLE 3 short chain fatty acid detection results
Note that: short chain fatty acid quantitative unit is μg/ml
Examples
A composition comprising lactobacillus rhamnosus S24 metazoan, wherein the metazoan contains lactobacillus rhamnosus bacteria 500 hundred million/g and free amino acid more than 1mg/g, and the preparation method of the metazoan comprises the following steps: lactobacillus rhamnosus S24 is fermented in vitamin D 3 -MRS liquid medium to obtain fermentation broth, and the fermentation broth is concentrated and sprayed to dry solid powder containing thalli and metabolites.
Examples
The application of lactobacillus rhamnosus S24 and its metaplasia in preparing medicine for preventing and treating sarcopenia or osteoporosis or musculoskeletal co-subtraction syndrome.
Sample to be tested: lactobacillus rhamnosus S24 powder (viable count 500 hundred million CFU/g) in example 1 was obtained by fermentation culture using MRS medium; lactobacillus rhamnosus S24 metazoan (number of cells 500 hundred million/g, free amino acid >1 mg/g) in example 2 was prepared by: lactobacillus rhamnosus S24 is fermented in vitamin D 3 -MRS liquid medium to obtain fermentation broth, and the fermentation broth is concentrated and sprayed to dry solid powder containing thalli and metabolites.
Test grouping: 30 SPF grade SD rats purchased from duda company are randomly divided into 5 groups by body mass, namely a normal group (CON group), a sham group (SHM group), a musculoskeletal co-reduction model group (OS group), OS lactobacillus rhamnosus S24 (os+s24 group) and OS lactobacillus rhamnosus S24 metazoan (os+s24 metazoan group), wherein: the rats of the OS group, the OS+S24 group and the OS+S24 metaplasia group are molded by adopting a castration method, the SHM group is molded by adopting a sham operation, and 8 ten thousand units/unit of penicillin sodium are injected into all the rats of the other groups except the CON group after operation, and 3 d continuous. The rats in the OS group, OS+S24 group and OS+S24 metagroup were intraperitoneally annotated with dexamethasone sodium phosphate injection (DXM) 1 mg (/ kg.d) for 2 weeks after 7d, 7d post-operative recovery. After the molding is finished, the drug intervention is started. CON group, SHM group and OS group rats were perfused with 10 mL/kg physiological saline daily, OS+S24 group was perfused with lactobacillus rhamnosus S24 powder (6×10 8 CFU/per mouse/day) for 12 weeks continuously, OS+S24 metagenome was perfused with lactobacillus rhamnosus S24 metagenome (6×10 8/per mouse/day) for 12 weeks continuously, and data were analyzed after the experimental results were completed, compared with CON group, a P <0.05; b P <0.05 compared to SHM group; c P <0.05 compared to OS group; d P <0.05 compared to os+s24 group.
① Rat general condition observation and weight change analysis
Feeding activity, mental state, abnormal behavior, etc. of each group of rats were observed and recorded daily during the gavage. After the end of the intervention, the Body Weight (BW) of each group of rats was measured weekly by an electronic scale.
During the experiment, no mice in each group died. The OS group of rats had a dry skin and hair, a messy hair and a reduced gloss compared with the other groups. The os+s24 group and os+s24 metagroup rats had a luster, soft hair and normal spirit and activity compared with the OS group. The change in rat body weight for the different test groups is shown in table 4. No significant difference (P > 0.05) between each dose group and control group, SHM group; the weight rise amplitude of the OS group is obviously smaller than that of the CON group and the SHM group, which is consistent with weight loss which is one of clinical manifestations of patients suffering from muscular and bone co-reduction; compared with the OS group, the weight change of the OS+S24 group and the OS+S24 metaplasia group has obvious difference, which indicates that the S24 metaplasia group and the S24 metaplasia group can effectively relieve the weight drop of an OS rat, namely, the long-term oral administration of the lactobacillus rhamnosus S24 live strain or the metaplasia group thereof can effectively prevent the muscular bone co-reduction disease of animals, and the growth safety is good.
Table 4 weight change in rats in each experimental group
Annotation: a P <0.05 compared to CON group; b P <0.05 compared to SHM group; c P <0.05 compared to OS group; d P <0.05 compared to os+s24 group.
The test method comprises the following steps: after the intervention, before the material is obtained, the rats of each group are anesthetized, and the total Bone density (Bone MINERAL DENSITY, BMD) and lumbar BMD of the rats of each group are measured by a dual-energy X-ray Bone densitometer.
Referring to fig. 7, the test results indicate that: compared with CON group and SHM group, the OS group rats have significantly reduced BMD (P < 0.05) and lumbar BMD, which indicates successful modeling; compared with the OS group, the total body BMD and the lumbar vertebra BMD of the rats with the OS+S24 and OS+S24 metazoans are obviously increased (P < 0.05), which shows that lactobacillus rhamnosus S24 and metazoans thereof can obviously improve the bone density of the rats with the sarcopenia (OS group), thereby effectively preventing and treating osteoporosis OP.
The test method comprises the following steps: after the intervention was completed, rat forelimb grip was measured using a grip tester for a fixed period of time per week (Forelimb GRIP STRENGTH). Rats were allowed to acclimate on the grab instrument for 5 minutes prior to testing, then the rat tail level was grabbed and pulled slowly back until the rat forelimbs were unable to grab the rail, and the maximum grab force reading on the instrument was recorded. Three measurements were made and recorded, with the average of the three grips being used as an indicator reflecting the muscle strength of the forelimb. The dual energy X-ray densitometer measures the total skeletal muscle mass (Lean mass, LM) and the skeletal muscle mass of the extremities (appendicular Lean mass, ALM) of each group of rats. And calculates the rat skeletal muscle mass index (smi=lm/BW) and the limb skeletal muscle mass index (RSMI =alm/BW).
Referring to fig. 8 and 9, the OS group rats had significantly reduced forelimb grip (P < 0.05), and SMI and RSMI were significantly reduced (P < 0.05) compared to the CON and SHM groups; compared with the OS group and the CON group, the rat forelimb holding power, the SMI and RSMI of the OS+S24 group and the OS+S24 metagroup are obviously increased (P < 0.05), compared with the rat forelimb holding power, the SMI and RSMI of the OS group, the rat forelimb holding power, the SMI and RSMI of the OS group are respectively improved by 29.40 percent, 43.48 percent and 45.74 percent, and even compared with the rat forelimb holding power, the SMI and RSMI of the CON group, the rat forelimb holding power, the SMI and the RSMI of the CON group are respectively improved by 15.85 percent, 13.6 percent and 45 percent. The following is indicated: the OS+S24 group and the OS+S24 metaplasia group can remarkably improve the forelimb grabbing force and skeletal muscle quantity of a rat with muscular-skeletal co-reduction (OS), so that the forelimb grabbing force and skeletal muscle quantity of the rat with muscular-skeletal co-reduction can be recovered, the further enhancement of the forelimb grabbing force and skeletal muscle quantity can be promoted, and the prevention and treatment of the muscular-skeletal co-reduction syndrome or osteoporosis can be effectively prevented.
The test method comprises the following steps: and detecting the expression level of the PI3K protein by adopting a western blotting method. The specific operation is as follows: frozen gastrocnemius muscle of each group of test rats is taken, ground and crushed under liquid nitrogen, and is cracked by a lysate to extract total protein, and protein concentration is measured by a BCA method, and protein samples are denatured. Preparing gel, electrophoresis, transferring film and sealing. PI3K (1:1000) and GAPDH (1:1000) were added sequentially and incubated overnight at 4 ℃. TBST was washed 3 times, 15 min at a time. HRP-labeled goat anti-rabbit IgG antibody was incubated for 1h at room temperature, washed 3 times with tbst, 15 min each. Development was performed using a full-scale gel imaging system and protein bands were quantitatively analyzed using Image J software.
As shown in fig. 10, the OS group rats showed significantly reduced gastrocnemius PI3K protein expression compared to the CON group and the SHM group; os+s24 and os+s24 metazoan rats gastrocnemius PI3K protein expression was significantly increased compared to the OS group (P < 0.05). It was shown that lactobacillus rhamnosus S24 and lactobacillus rhamnosus S24 metants may prevent and treat sarcopenia (OS) by up-regulating PI3K signaling pathway related protein expression.
Referring to the method of patent CN113234622A for detecting lactobacillus and bifidobacterium in feces, the test results are shown in Table 5, and compared with CON group and SHM group, the lactobacillus and bifidobacterium in the intestinal tract of the rat in the OS group are obviously reduced; compared with the OS group, the OS+S24 and OS+S24 metazoan groups have significantly increased lactobacillus and bifidobacterium in the intestinal tract of the rat (P < 0.05), which indicates that lactobacillus rhamnosus S24 and lactobacillus rhamnosus S24 metazoan can prevent and treat the musculoskeletal co-subtraction syndrome (OS) by increasing the abundance of lactobacillus and bifidobacterium in the intestinal tract.
TABLE 5 Lactobacillus rhamnosus S24 regulates the intestinal flora effect (lg cfu/g) in rats
In summary, the total BMD, lumbar BMD, SMI and RSMI were significantly reduced in the OS group compared to the CON and SHM groups, indicating that castration in combination with DXM resulted in the development of sarco-osseous co-reduction syndrome (OS). Compared with the OS group, the os+s24 group, os+metas24 group rats had significantly increased body weight, whole body BMD, lumbar BMD, forelimb grip, SMI and RSMI, indicating that: lactobacillus rhamnosus S24 and its metaplasia can significantly improve OS rat weight loss, BMD, muscle strength and muscle mass, thereby effectively preventing and treating musculoskeletal co-subtraction syndrome. In addition, the lactobacillus rhamnosus S24 and the metazoan thereof obviously improve the expression level of PI3K protein in gastrocnemius of the OS rat, which indicates that the effect of the lactobacillus rhamnosus S24 and the metazoan thereof on preventing and controlling the OS may be related to up-regulating the expression level of the protein related to a PI3K signal path, and meanwhile, the lactobacillus rhamnosus S24 and the metazoan S24 can increase the abundance of lactobacillus and bifidobacterium in intestinal tracts to regulate intestinal flora to prevent and control the sarcoossium co-reduction syndrome.
Examples
A composition of this embodiment comprising a live strain of lactobacillus rhamnosus S24 and an inactivated strain of lactobacillus rhamnosus S24, wherein the live strain of lactobacillus rhamnosus S24 is as described in example 2; the inactivated strain of lactobacillus rhamnosus S24 is obtained by culturing for 48 hours in vitamin D 3 -MRS liquid culture medium at 37 ℃ to obtain lactobacillus rhamnosus S24 bacterial liquid, and sterilizing the bacterial liquid at 125 ℃ for 5 minutes; centrifuging 8000g of the inactivated bacterial liquid for 5min to obtain an inactivated strain of lactobacillus rhamnosus S24; the weight ratio of the live strain of lactobacillus rhamnosus S24 to the inactivated strain of lactobacillus rhamnosus S24 is 1:3.
The application of the composition in preparing the medicament for treating sarcopenia, osteoporosis or sarcopenia syndrome in the embodiment is that the dosage of lactobacillus rhamnosus S24 in the composition is 1.0 x 10 11 CFU/day and the dosage of lactobacillus rhamnosus S24 in the composition is 6.0 x 10 10 CFU/day.
Examples
Application of lactobacillus rhamnosus S24 in preparing a medicament for preventing and treating OS is disclosed, wherein the dosage of lactobacillus rhamnosus S24 is 1.0X10 11 CFU/day.
Wherein lactobacillus rhamnosus S24 was obtained by culture with MRS medium as described in example 1.
Examples
A composition of the embodiment comprises a lactobacillus rhamnosus S24 live strain, a lactobacillus rhamnosus S24 inactivated strain and lactobacillus rhamnosus S24 metazoan in a mass ratio of 1:2:1.
As shown by the antibacterial capability test of 3- ③ lactobacillus rhamnosus S24 of the embodiment 1 on pathogenic bacteria, the live lactobacillus rhamnosus S24 strain has good inhibition effect on common pathogenic bacteria such as helicobacter pylori ATCC 26695, streptococcus mutans CGMCC 1.2499, staphylococcus aureus CMCC 26003, clostridium perfringens ATCC 13124, candida albicans ATCC 10231 and the like.
The composition of the embodiment has antibacterial effect and is applied to preparation of medicines for inhibiting pathogenic bacteria, wherein pathogenic helicobacter includes helicobacter pylori ATCC 26695, streptococcus mutans CGMCC 1.2499, staphylococcus aureus CMCC 26003, clostridium perfringens ATCC 13124 and candida albicans ATCC 10231. When the strain is used, the dosage of the live strain of the lactobacillus rhamnosus S24 is 1 multiplied by 10 10 CFU/day, the dosage of the inactivated strain of the lactobacillus rhamnosus S24 is 2 multiplied by 10 10 CFU/day, and the dosage of the metazoan is 100mg/day.
Examples
A composition of this embodiment comprises a live strain of lactobacillus rhamnosus S24 and a metabolite of lactobacillus rhamnosus S24 strain.
According to the embodiment 1, the lactobacillus rhamnosus S24 live strain cultured on the MRS culture medium has better acid production and bacteriostasis capacity, and the composition of the embodiment is applied to preparing fermented foods and health-care foods as a starter.
The fermented food is pickle, and the application method of the composition as a starter in preparing pickle is as follows:
Fresh vegetables are selected, washed and added into 4-5 times of drinking water, then edible glucose with the total volume of 1% and edible sodium chloride with the total volume of 0.5-2.0% are added, then lactobacillus rhamnosus S24 prepared in the embodiment 1 of the invention is inoculated, the concentration of the lactobacillus rhamnosus S24 reaches more than 10 7 CFU/mL, and fermentation is carried out for 5-24 hours at room temperature, so that fermented pickle containing the composition containing lactobacillus rhamnosus S24 and lactobacillus rhamnosus S24 strain metabolites is obtained. The fermented pickle has crisp and unique flavor, contains lactobacillus rhamnosus S24 thallus and metabolites, and has good safety and probiotic function.
Examples
The composition of the embodiment comprises, by weight, 14 parts of lactobacillus rhamnosus S24 powder (4.0X10 10 CFU/g), 10 parts of lactobacillus plantarum LP550 (4.0X10 10 CFU/g), 4 parts of lactobacillus plantarum LP220 (4.0X10 10 CFU/g), 1 part of lactobacillus rhamnosus S24 metazoan (cell number 4.0X10 10/g) and auxiliary materials, wherein the auxiliary materials comprise 10 parts by weight of skimmed milk powder, 10 parts by weight of resistant starch, 10 parts by weight of maltodextrin, 10 parts by weight of sodium hyaluronate, 10 parts by weight of cherry extract, 5 parts by weight of vitamin C, 3 parts by weight of folic acid and 1 part by weight of vitamin D 3.
Firstly, weighing 10 parts by weight of skimmed milk powder, 10 parts by weight of resistant starch, 10 parts by weight of maltodextrin, 10 parts by weight of sodium hyaluronate, 10 parts by weight of cherry extract, 5 parts by weight of vitamin C, 3 parts by weight of folic acid and 3 parts by weight of vitamin D, uniformly mixing, granulating into wet granules by adopting a 20-mesh screen with the concentration of 30% alcohol wet method, drying at 55 ℃ for 3.5 hours, finishing the granules by adopting a 20-mesh screen, adding 14 parts by weight of lactobacillus rhamnosus S24 bacterial powder (4.0X10 10 CFU/g), 10 parts by weight of lactobacillus plantarum LP550 (4.0X10 10 CFU/g), 4 parts by weight of lactobacillus plantarum LP220 (4.0X10 10 CFU/g), 1 part by weight of lactobacillus rhamnosus S24 metaplague (cell number of 4.0X10 10/g) and 2 parts by weight of magnesium stearate, uniformly mixing, and tabletting by a rotary tabletting machine, thus obtaining tablets with lactobacillus rhamnosus S24 dietary supplements.
The composition of the embodiment is used for preparing the medicine for preventing and treating the musculoskeletal co-reduction syndrome.
Examples
A composition of the present example comprises, by weight, 15 parts of Lactobacillus rhamnosus S24 (4.0X10 10 CFU/g), 1 part of Lactobacillus rhamnosus S24 metagen (4.0X10 10/g), 9 parts of Lactobacillus plantarum LP220 (4.0X10 10 CFU/g), 10 parts of Lactobacillus fermentum GF1800 (4.0X10 10 CFU/g), 10 parts of maltodextrin, 10 parts of sorbitol, 10 parts of corn peptide, 10 parts of sodium hyaluronate, 4 parts of anserine, 4 parts of soybean peptide, 2 parts of xylooligosaccharide, 2 parts of broccoli seed aqueous extract, 2 parts of selenium-enriched yeast, 2 parts of folic acid, 2 parts of malic acid, 2 parts of glutathione, 2 parts of vitamin E, 2 parts of vitamin C, and 3 parts of vitamin D351.
The raw materials of the composition are firstly sieved by a 40-mesh screen, then uniformly mixed according to a proportion, and packaged by a screw back-packaging machine to prepare 2-10 g/bag of solid beverage for preventing and treating the muscular-bone co-reduction syndrome.
Examples
Application of lactobacillus rhamnosus S24 in preparing a medicament for preventing and treating sarcopenia is provided, wherein the dosage of lactobacillus rhamnosus S24 is 1.0X10 11 CFU/day.
Wherein lactobacillus rhamnosus S24 was obtained by culturing on MRS medium as described in example 1.
Examples
The application of lactobacillus rhamnosus S24 metagen in preparing a medicament for preventing and treating sarcopenia or osteoporosis or sarcoosseous co-subtraction syndrome is that the dosage of lactobacillus rhamnosus S24 metagen is 1.0 x 10 11/day when in use.
Wherein lactobacillus rhamnosus S24 metazoan was prepared as described in example 2.
Examples
The application of lactobacillus rhamnosus S24 and its metants in the medicine for preventing and treating OS, wherein the dosage of lactobacillus rhamnosus S24 is 5 x 10 10 CFU/day, and the dosage of lactobacillus rhamnosus S24 metants is 5 x 10 10/day. The lactobacillus rhamnosus was obtained by culturing in vitamin D 3 -MRS medium as described in example 1, and the lactobacillus rhamnosus metazoan was obtained by culturing as described in example 2.
Claims (6)
1. Lactobacillus rhamnosus (Lactobacillus rhamnosus) S24 was deposited with the chinese classical cultures collection of chinese wuhan at 2021, 8 and 6, with the deposit number cctccc NO: m2021986.
2. A composition comprising one of the live strain of lactobacillus rhamnosus S24, the lactobacillus rhamnosus S24 metazoan of claim 1.
3. Use of lactobacillus rhamnosus S24 according to claim 1 and/or the composition according to claim 2 for the manufacture of a medicament for the treatment of sarcopenia or osteoporosis or sarcoosseous co-subtraction syndrome.
4. The use of claim 3, wherein the medicament treats sarcopenia or osteoporosis or sarcopenia by one or more of weight loss, increasing total bone density and lumbar bone density in rats, increasing forelimb grip in rats, total skeletal muscle mass and skeletal muscle mass in limbs.
5. The use of lactobacillus rhamnosus S24 as claimed in claim 1 and its metants for the manufacture of a medicament for inhibiting pathogenic bacteria.
6. Use of lactobacillus rhamnosus S24 according to claim 1 and/or the composition according to claim 2 as a starter in the preparation of fermented food products, health food products.
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