CN115838675A - Lactobacillus rhamnosus and composition and application thereof - Google Patents
Lactobacillus rhamnosus and composition and application thereof Download PDFInfo
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- CN115838675A CN115838675A CN202211671694.2A CN202211671694A CN115838675A CN 115838675 A CN115838675 A CN 115838675A CN 202211671694 A CN202211671694 A CN 202211671694A CN 115838675 A CN115838675 A CN 115838675A
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- lactobacillus rhamnosus
- lactobacillus
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- sarcopenia
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
Lactobacillus rhamnosus S24 is preserved in China center for type culture Collection in Wuhan, china in 2021, 8 and 6 days, with the preservation number of CCTCC NO: M2021986. The composition comprises a mixture of one or more of a live strain of lactobacillus rhamnosus S24, an inactivated strain of lactobacillus rhamnosus S24, a metabolite of the strain and a postnatal lactobacillus rhamnosus S24. The invention also discloses the application of the lactobacillus rhamnosus S24 and the metazoans thereof in preparing functional food or medicines for preventing and treating sarcopenia, osteoporosis or musculoskeletal syndrome. The lactobacillus rhamnosus S24 has good safety, and the lactobacillus rhamnosus S24 has wide application, can be used for preparing functional food or medicine for preventing and treating sarcopenia, osteoporosis or musculoskeletal reduction syndrome, or functional food or medicine for inhibiting pathogenic bacteria, or can be used as a leaven for preparing fermented food and health-care food.
Description
Technical Field
The invention relates to the technical field of microorganisms and food and medicine, in particular to lactobacillus rhamnosus and a composition and application thereof.
Background
Sarcopenia (SP), a sarcopenia, is a disease of the muscular system characterized by progressive and systemic decline in muscle mass, muscle strength and muscle function, resulting in restricted activity and an increased risk of death due to easy falls. Osteoporosis (OP) is a systemic bone-driven disease characterized by decreased bone mass and increased bone fragility due to destruction of bone microarchitecture, and OP causes decreased quality of life, increased disability and mortality in patients. The muscular dystrophy and osteoporosis share common physiological mechanisms, such as mechanical factors, genetic factors, inflammation factors and endocrine factors. Meanwhile, sarcopenia and osteoporosis have strong relevance in the clinical manifestations of muscular atrophy, gait abnormality, pain, brittle fracture and the like. In 2009, binkley et al proposed the concept of Osteoporotic Syndrome (OS) based on the similar pathophysiological basis and close correlation between sarcopenia and osteoporosis, mainly referring to patients who met the diagnostic criteria for osteoporosis and had a concomitant decrease in muscle mass/function. Patients with OS present with decreased bone density (BMD), increased bone fragility, progressive and systemic decreases in muscle mass, muscle strength and muscle function, leading to increased patient weakness, falls, fractures, hospitalizations, disabilities and mortality risk.
According to different diagnostic criteria of the sarcopenia, the incidence rate of the sarcopenia in the population over 60 years old ranges from 5% to 37%. At present, no targeted medicine for preventing and treating the muscular-skeletal co-reduction syndrome exists, the effective prevention and treatment means at present comprises traditional Chinese medicine treatment mainly for strengthening spleen and tonifying 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 salted eucommia bark, and in addition, early intervention measures such as resistance and balance training, nutrition intake (vitamin D and calcium supplement) and the like can also effectively prevent and relieve the symptoms of the muscular-skeletal co-reduction syndrome. In recent years, numerous studies show that intestinal microbiota can be used as a medium for the influence of nutrition on muscle health and skeletal health, for example, CN 111748640B discloses the application of intestinal microbiota in sarcopenia, and by detecting the abundance of erysipelotrichiaceae _ UCG-003, whether a subject suffers from sarcopenia and/or the risk of sarcopenia can be diagnosed, so that noninvasive diagnosis of the subject is realized; CN 111718873B discloses a lactobacillus fermentum strain with osteoporosis relieving effect and application thereof, wherein the strain can be used for remarkably improving bone density and cortical volume of an ovariectomized rat. CN 103889241B discloses a composition for promoting healthy bone growth and/or for preventing and/or treating bone diseases comprising at least one long chain polyunsaturated fatty acid (LC-PUFA), at least one probiotic, and a mixture of 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 probiotic technology only carries out isolated research on sarcopenia and osteoporosis from a single visual angle, and no research report of specific bacterial strains and metazoans thereof on the sarcopenia is found, so that the development of new bacterial strains and compositions thereof for preparing a nutrient or medicament for conveniently and efficiently relieving and treating the sarcopenia has important significance and great market value.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention overcomes the defects of the prior art, provides lactobacillus rhamnosus which has high biological activity and wide carbon source utilization, can tolerate gastric juice, intestinal juice and bile salt and has the function of preventing or/and treating sarcopenia or osteoporosis or sarcopenia, also provides a composition consisting of the lactobacillus rhamnosus and provides a new application of the lactobacillus rhamnosus and the composition consisting of the lactobacillus rhamnosus in preventing or/and treating sarcopenia or osteoporosis or sarcopenia.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a Lactobacillus rhamnosus strain, named as Lactobacillus rhamnosus S24, is preserved in China Wuhan Chinese type culture Collection at 20/8 of 2021 with the preservation number of CCTCC NO: M2021986.
Description of biological preservation: lactobacillus rhamnosus (Lactobacillus rhamnosus) S24, deposited in the chinese type culture collection with the deposit address: eight-way 299 in Wuchang district, wuhan city, hubei province, the preservation organization is abbreviated as: CCTCC, preservation date: day 8 and 6 of 2021 (registration received at day 8 and 6 of 2021, survival was detected at day 8 and 20, and preservation), and the biological deposit number is CCTCCNO: m2021986, strain number: lactobacillus rhamnosus S24.
The lactobacillus rhamnosus S24 is separated from a plant source, and specifically, the lactobacillus rhamnosus S24 is obtained by screening and separating from Sichuan Pengzu mountain farmyard sour dough.
The biological properties of lactobacillus rhamnosus S24 of the present invention are as follows:
1) Morphological characteristics: the growth is good on MRS agar medium, the bacterial colony is in a nearly spherical rod shape, is arranged singly, in pairs or in a fence shape, is medium in size, is milk-white, has upward convex luster, is not neat in the edge of the bacterial colony, is easy to pick, is arranged in pairs or in a fence shape under a microscope, and is positive after gram staining.
2) Biological identification: lactobacillus rhamnosus S24 is sent to a microorganism resource platform strain preservation center in Sichuan province for 16S rRNA identification, the 16S rRNA gene sequence is shown as SEQ ID NO:1, NCBI BLAST comparison is carried out on the 16S rRNA sequence of Lactobacillus rhamnosus S24, and based on the comparison result of the 16S rRNA gene sequence, a Neighbor-Joining phylogenetic tree which is constructed by taking B.subtilis NCDO 1769 (NR 118972) as an outer branch is shown as figure 2, the similarity with Lactobacillus rhamnosus (Lactobacillus rhamnosus) in Genebank is 100%, and the strain is identified as Lactobacillus rhamnosus S (Lactobacillus rhamnosus) S24.
The culture medium for fermenting lactobacillus rhamnosus S24 is MRS or vitamin D 3 -MRS medium, said vitamin D 3 The MRS culture medium comprises 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 1.0g/L of Tween and K 2 HPO 4 ·7H 2 O2.0g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 0.038g/L of O, 1000-4000 IU of vitamin D 3 (agar powder 15g/L as solid medium), preferably, vitamin D 3 Vitamin D in MRS Medium 3 Can promote the increase of short-chain fatty acid, and particularly obviously improve the content of butyric acid playing an important role in the intestinal muscle axis and the intestinal bone axis; vitamin D 3 Can promote and improve the bacteriostatic ability of lactobacillus rhamnosus S24 on pathogenic microorganisms to be greatly improved, and has bacteriostatic effects on helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231.
The other technical scheme adopted by the invention for solving the technical problem is as follows:
a composition comprises the active strain of Lactobacillus rhamnosus S24, the inactivated strain and the metabolite of the strain of Lactobacillus rhamnosus S24, the postbiotic strain of Lactobacillus rhamnosus S24, the active strain of Lactobacillus plantarum and the active strain of Lactobacillus fermentum, wherein the Lactobacillus plantarum is preferably Lactobacillus plantarum LP550 and Lactobacillus plantarum LP220, and the Lactobacillus fermentum is preferably Lactobacillus fermentum GF1800. Preferably, the composition is lactobacillus rhamnosus S24 metazoa.
The lactobacillus rhamnosus S24 metagenesis of the invention refers to a general name of lactobacillus thallus and metabolic components after processing/inactivation, concentration and drying of lactobacillus rhamnosus S24 fermentation liquor, and comprises thallus components and metabolic products.
The composition includes but is not limited to a biological agent, a functional food, a health product or a medicament.
The functional food is any one of ferment, pickle, solid beverage, pill, tablet or microcapsule crystal ball.
The medicine also contains a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carriers include, but are not limited to: one or more of a filler, a binder, a wetting agent, a disintegrant, or a lubricant.
The medicament is a medicament for preventing and treating sarcopenia, osteoporosis or sarcopenia, and is preferably an oral medicament.
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 disintegrant is one or more of crospovidone, sodium carboxymethyl starch or sodium croscarmellose; the lubricant is one or more of magnesium stearate silicon dioxide or sodium fumarate stearate.
The lactobacillus rhamnosus S24 and the S24 metazoans thereof can obviously relieve the weight loss of rats with muscular-skeletal syndrome (OS for short), and the lactobacillus rhamnosus S24 and the S24 metazoans thereof can obviously improve the whole bone density and the lumbar vertebrae density of the rats with OS so as to prevent the formation of Osteoporosis (OP).
The lactobacillus rhamnosus S24 and the S24 metazoan thereof can obviously improve the forelimb holding power, the whole body skeletal muscle quantity and the four limb skeletal muscle quantity of an OS rat, and realize effective prevention and treatment of Sarcopenia (SP).
The lactobacillus rhamnosus S24 can also promote the expression of gastrocnemius PI3K protein of an OS rat, and the lactobacillus rhamnosus S24 can prevent and treat the muscular bone reduction OS by up-regulating the expression of related protein of a PI3K signal channel.
The lactobacillus rhamnosus S24 can also promote the increase of the abundance of lactobacillus and bifidobacterium in the intestinal tract of an OS rat, so as to prevent and treat the muscular-skeletal co-reduction OS.
Based on the above characteristics of lactobacillus rhamnosus S24 of the present invention, one of the applications of lactobacillus rhamnosus and its progeny of the present invention:
the application of lactobacillus rhamnosus S24 in preparing functional food or medicine for preventing and treating sarcopenia, osteoporosis or sarcopenia is provided; or the application of a composition formed by the lactobacillus rhamnosus S24 and one or more of inactivated strains, strain metabolites or lactobacillus rhamnosus S24 metagenes in preparing functional foods or medicines for preventing and treating sarcopenia, osteoporosis or sarcopenia.
In an exemplary embodiment, lactobacillus rhamnosus S24 is used in an amount of 6X 10 or more 8 The dosage of the inactivated strain of the CFU/per mouse/day and the Lactobacillus rhamnosus S24 is 3 to 8 multiplied by 10 10 The dosage of CFU/day and lactobacillus rhamnosus S24 metazoan is more than or equal to 6 multiplied by 10 8 Per mouse/day, free amino acids > 1mg/g.
Secondly, bacteriostatic experiments show that: lactobacillus rhamnosus S24 has a broad bacteriostatic activity against pathogenic bacteria. The inhibitor has better inhibition effects on helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231, wherein the inhibitor has the best inhibition effect on streptococcus mutans CGMCC 1.2499.
Based on the above characteristics of lactobacillus rhamnosus S24, another application of lactobacillus rhamnosus and its progeny of the present invention:
application of Lactobacillus rhamnosus S24 and its metazoan in preparing functional food or medicine for inhibiting pathogenic bacteria is provided.
A tolerance test of the lactobacillus rhamnosus S24 disclosed by the invention to gastric acid and bile salt shows that the survival rate of the strain reaches 43.17% when the pH of gastric acid of the lactobacillus rhamnosus S24 is 2.0; when the concentration of bile salt reaches 3.0g/L, the survival rate of the strain reaches 91.54 percent. The 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-turanose, D-tagatose, sorbose, sorbitol, mannitol, esculetin, N-acetyl-glucosamine, alpha-methyl-D-mannoside, with a broad spectrum of carbon source utilization. The acid production capacity is strong, the acid production is rapid in the initial stage of fermentation, the acid production amount is large, and the acid production amount tends to be stable along with the increase of the fermentation time. Based on the characteristics, the lactobacillus rhamnosus and the subsequent application of the lactobacillus rhamnosus are as follows:
application of lactobacillus rhamnosus S24 and its metazoan as leaven in preparing fermented food, health food or dietary supplement is provided.
The amount of Lactobacillus rhamnosus S24 is 1 × 10 8 ～1×10 10 CFU/mL or 1X 10 8 ～1×10 10 CFU/g, the dosage of the postbiotic 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 is cultured in MRS agar and vitamin D 3 The strain grows well on an 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 vitamin D 3 Can achieve synergistic promotion effects, including growth promotion, short-chain fatty acid content increase, and synergistic antibacterial effect.
The lactobacillus rhamnosus S24 and the anagen group can obviously inhibit the weight loss of an OS rat, obviously improve the skeletal muscle quality index, the limb skeletal quality index, the whole body bone density, the lumbar vertebra density and the forelimb holding power, further delay or improve sarcopenia or osteoporosis or sarcopenia, and prevent and treat the sarcopenia by increasing the expression of PI3K protein, improving the abundance of intestinal lactobacillus and bifidobacterium and the like.
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, osteoporosis or sarcopenia, or inhibiting pathogenic bacteria, or can be used as a leavening agent 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) is a plate colony morphology diagram, and (B) is a strain morphology diagram under a microscope;
FIG. 2 is a phylogenetic tree of Lactobacillus rhamnosus S24;
FIG. 3 shows the tolerance of Lactobacillus rhamnosus S24 to gastric juices;
FIG. 4 shows the tolerance of Lactobacillus rhamnosus S24 to bile salts;
FIG. 5 shows the activity of Lactobacillus rhamnosus S24 in MRS and vitamin D 3 -growth curve in MRS medium;
FIG. 6 shows the presence of Lactobacillus rhamnosus S24 in MRS and vitamin D 3 -acid production profile of fermentation in MRS medium;
FIG. 7 is a graph of comparative analysis of bone density and lumbar vertebrae density of rats in different experimental groups; wherein, (A) the whole body bone density of the rat, (B) the lumbar vertebrae density of the rat;
FIG. 8 is a graph showing comparative analysis of the front limb grasping force of rats in different experimental groups;
FIG. 9 is a graph of SMI (A) and RSMI (B) comparative analyses of rats in different experimental groups;
FIG. 10 is a graph showing the analysis of the expression level of the gastrocnemius PI3K protein in rats of different experimental groups.
Detailed Description
The invention is further explained with reference to the drawings and the embodiments.
The culture medium formulation involved in the examples of the present invention:
MRS medium (lactobacillus rhamnosus S24): 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose,tween 80.0 g/L, K 2 HPO 4 ·7H 2 O2.0g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038g/L (agar powder 15g/L as solid medium).
Vitamin D 3 MRS Medium (Lactobacillus rhamnosus S24) (abbreviation: VD) 3 -MRS): 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 1.0g/L of Tween-80 and K 2 HPO 4 ·7H 2 O2.0g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038g/L,4000IU vitamin D 3 (15 g/L of agar powder is added as a solid culture medium).
Example 1
The Lactobacillus rhamnosus S24 (Lactobacillus rhamnosus S24) has the preservation number: CCTCC NO: M2021986.
1) Separation and screening of lactobacillus rhamnosus S24
Collecting a microorganism screening sample from the agricultural sourdough dough of the Sichuan Pengzushan, shearing the collected sample, weighing 1g of the sample, putting the sample into 9mL of sterile physiological saline, fully shaking and uniformly mixing, diluting by 10 times, coating the diluted sample in an MRS solid culture medium, and culturing for 48 hours at 37 ℃. Observing with naked eyes, picking single colonies with different morphologies and sizes in the culture medium, and repeatedly streaking, purifying and culturing; then primarily determining the strain as lactobacillus by gram staining and calcium dissolving method, and storing the purified strain in a refrigerator at-80 deg.C with 45% glycerol for use.
a) Morphological observation
The purified lactobacillus rhamnosus S24 is streaked on an MRS agar culture medium, and after the lactobacillus rhamnosus S24 is subjected to inverted culture at 37 ℃ for 48 hours, the colony morphology of the strain is observed, and the result is shown in the attached figure 1: the bacterial strain grows well on an MRS agar culture medium, 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, have upward convex gloss, are irregular in edges, are easy to pick, are positive after gram staining, and are arranged in pairs or in a fence shape under a microscope.
b) Molecular biological identification of strains
The purified strain is sent to a strain collection center of a microorganism resource platform of Sichuan province for 16S rRNA identification, and the measured 16S rRNA sequence is compared with NCBI BLAST, so that the similarity of the strain and the Lactobacillus rhamnosus in Genebank is 100 percent, and the strain can be preliminarily identified as the Lactobacillus rhamnosus (Lactobacillus rhamnosus). The 16S rRNA identification sequence of the strain is shown in SEQ ID NO 1 and is named as Lactobacillus rhamnosus S24 (Lactobacillus rhamnosus S24), and the Neighbor-Joining phylogenetic tree of the Lactobacillus rhamnosus S24 which is constructed by taking B.subtilis NCDO 1769 (NR 118972) as an outgrowth based on the 16S rRNA gene sequence comparison result is shown in figure 2.
2) Test for human gastrointestinal tolerance of lactobacillus rhamnosus S24
(1) Tolerance of lactobacillus rhamnosus S24 to gastric juice and intestinal juice
Preparing simulated gastric juice: naCl 2.0g/L, adjusting pH to 2.0, 2.5, 3.0 and 4.0 with HCl, respectively, and autoclaving with pepsin 3.2g/L, wherein pepsin is added at present during the experiment; preparing simulated intestinal juice: 6.8g/L potassium dihydrogen phosphate, adjusting the pH value to 7.5 by NaOH, and autoclaving, wherein the concentration of trypsin is 10.0g/L, and the trypsin is added at the moment of experiment; inoculating lactobacillus rhamnosus S24 strain stored in glycerin tube into MRS culture medium at 37 deg.C for activating for 24 hr at an inoculation amount of 10%; adding an equivalent amount of lactobacillus rhamnosus S24 bacterial liquid into a simulated gastric juice of a system of 50mL, recording initial viable bacteria, and determining the number of viable bacteria after culturing at the constant temperature of 37 ℃ for 3h. Counting the detected live lactobacillus, and calculating the survival rate, wherein the survival rate of the strain = test group/control group × 100%.
When food enters the stomach, gastric acid begins to be secreted. The pH value of the gastric acid which is normally secreted by the stomach of a human body is about 0.5-1.5. The pH of the stomach is between about 7.0 and 7.2 when empty, and rapidly drops to 2-3 when food enters the stomach. After meal, the gastric juice is diluted and the pH rises to about 3.5. Referring to FIG. 3, the survival rate of S24 was 43.17% at pH 2.0; the survival rate of S24 is 65.79 percent when the pH value is 2.50; at ph3.0, the survival rate of S24 was 87.12%; the lactobacillus rhamnosus S24 strain taken after eating is able to tolerate gastric juices and, in the intestine, lactobacillus rhamnosus S24 strain is able to tolerate intestinal juices.
(2) Tolerance of lactobacillus rhamnosus S24 to bile salts
Inoculating lactobacillus rhamnosus S24 strain into MRS liquid culture medium according to the inoculation amount of 5%, activating and culturing at 37 ℃ for 24 hours, and continuously activating twice. Inoculating the activated S24 bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 5%, and performing static culture for 15h at 37 ℃ in a constant-temperature incubator. And centrifuging the cultured bacterial liquid at 5000rpm for 10min to collect thalli, and shaking the thalli uniformly by using sterile physiological saline.
The uniformly shaken bacterial liquid is added into MRS culture media with cholate concentrations of 1.0g/L, 2.0g/L, 3.0g/L and 0.0g/L (initial bacterial liquid) according to the addition of 10 percent, and the cholate concentration of 0.0g/L is taken as a control group. Then incubated in a 37 ℃ incubator for 3h. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterile normal saline, beating and uniformly mixing, and detecting the number of lactic acid bacteria; counting the detected live lactobacillus, and calculating the survival rate according to the following formula:
strain survival (%) = test group/control group × 100%.
The Lactobacillus rhamnosus S24 bile salt tolerance data are shown in the attached figure 4: when the concentration of the bile salt is 1.0g/L and 2.0g/L, the survival rate of the strain is 99.87 percent and 94.09 percent respectively, but when the concentration of the bile salt reaches 3.0g/L, the survival rate of the strain still reaches 91.54 percent. The concentration of the 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.
3) Physiological and biochemical characteristics of lactobacillus rhamnosus S24
(1) Fermentation substrate utilization capacity
Puncturing and inoculating a fresh slant culture of the lactobacillus to be tested into a culture medium, or dripping a fresh culture bacterial solution into a melted soft agar column (the temperature is 47 +/-1 ℃), mixing uniformly, covering a layer of 2% agar with the thickness of about 7mm on the upper part, placing the mixture at the temperature of 37 ℃ for culturing for 48 hours, and then producing acid by utilizing a carbon source through the purple strain Huang Panding in the culture medium.
As shown in Table 1, the Lactobacillus rhamnosus S24 strain can utilize 23 carbon source substrates such as L-arabinose, fructose, galactose, glucose, mannose, pinsanose, raffinose, rhamnose, trehalose, ribose, sucrose, maltose, cellobiose, melibiose, D-turanose, D-tagatose, sorbose, sorbitol, mannitol, esculetin, saligenin, N-acetyl-glucosamine, alpha-methyl-D-mannoside, etc. to ferment and produce acid.
TABLE 1 physiological and biochemical characteristics of Lactobacillus rhamnosus S24 Strain-production of acid Using carbon sources
Note: +: carrying out positive reaction; negative reaction; w-weakly positive reaction
(2) Growth performance test of lactobacillus rhamnosus S24 in different culture media
Respectively inoculating lactobacillus rhamnosus S24 strain strains to MRS and vitamin D according to the inoculation amount of 5 percent 3 Activating and culturing for 24 hours at 37 ℃ in an MRS culture medium liquid culture medium, and continuously activating twice. Correspondingly inoculating the activated lactobacillus rhamnosus S24 bacterial liquid to MRS and vitamin D according to the inoculation amount of 5 percent 3 MRS liquid culture medium, mixed well and dispensed into sterile test tubes (18 mm × 180 mm) at an amount of 8 ml/tube; standing and culturing the subpackaged lactobacillus rhamnosus S24 bacterial liquid in a constant-temperature incubator at 37 ℃, and measuring the absorbance value OD of the bacterial liquid by taking 3 test tubes 600 Calculating an average value; at regular intervals, 3 test tubes are taken to determine the absorbance value OD of S24 bacterial liquid 600 Calculating an average value; absorbance value OD with time as abscissa 600 The growth curve is plotted as ordinate.
The lactobacillus rhamnosus S24 strain is MRS and vitamin D 3 The growth curve in MRS medium is shown in figure 5, and it can be seen from figure 5 that Lactobacillus rhamnosus grows significantly better than MRS liquid medium in vitamin D3-MRS liquid medium, OD 600 The increase of 35% in 18h shows that the vitamin D3 remarkably promotes the growth of the lactobacillus rhamnosus S24.
(3) Test of bacteriostatic ability of lactobacillus rhamnosus S24 on pathogenic bacteria
Bacteriostatic experiments on pathogenic bacteria: pouring 10mL of water agar culture medium in an aseptic plate, placing an Oxford cup after cooling and solidifying, respectively adding indicator bacterium suspensions (helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC 10231) into the agar culture medium correspondingly grown by the indicator bacterium cooled to 50 ℃ to ensure that the concentration of the indicator bacterium is 10 6 Mixing CFU/mL, pouring onto water agar, coagulating, taking out Oxford cup with forceps to form holes, adding 200 μ L of sample to be tested (sample to be tested comprises MRS culture medium lactobacillus rhamnosus S24 fermentation liquid, and vitamin D) into each hole 3 Lactobacillus rhamnosus S24 fermentation broth of MRS), spread for 30min, and cultured at 37 ℃ for 15-24h. Observing whether a bacteriostatic circle appears around the culture hole, measuring the diameter of the bacteriostatic circle by using a vernier caliper, recording the diameter of the bacteriostatic circle, and finally evaluating bacteriostatic activity according to the existence and the size of the bacteriostatic circle.
TABLE 2 analysis of the bacteriostatic ability of the fermentation broth of Lactobacillus rhamnosus S24 cultured in different media
The results are shown in Table 2, in which vitamin D was added to MRS medium relative to Lactobacillus rhamnosus S24 obtained by fermentation of MRS medium 3 The lactobacillus rhamnosus S24 obtained by post-fermentation has greatly improved bacteriostatic ability on pathogenic microorganisms, has bacteriostatic circle reaching more than 20mm for helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231, and combines with lactobacillus rhamnosus in vitamin D 3 Increase in the number of bacteria in MRS medium, but decrease in the total acid, indicating that Lactobacillus rhamnosus S24 is in vitamin D 3 -MRThe content of antibacterial substances such as antibacterial peptide in the S culture medium is improved, and the bacteriostatic performance of the lactobacillus rhamnosus S24 is obviously improved.
(4) Acid production capacity test of lactobacillus rhamnosus S24
Respectively inoculating lactobacillus rhamnosus S24 strain strains to MRS and vitamin D according to the inoculation amount of 5 percent 3 Activating and culturing for 24 hours at 37 ℃ in an MRS culture medium liquid culture medium, and continuously activating twice. Correspondingly inoculating the activated S24 bacterial liquid to MRS and vitamin D according to the inoculation amount of 5 percent 3 MRS medium liquid medium, mixed well and distributed into sterile test tubes (18 mm × 180) according to 8 ml/tube. Placing the subpackaged lactobacillus rhamnosus S24 bacterial liquid in a constant-temperature incubator at 37 ℃ for standing culture, taking 3 test tubes to measure the total acid of the bacterial liquid, and calculating the average value; at regular intervals, 3 test tubes are taken to measure the total acid of the bacterial liquid, and an acid production curve is drawn by taking the time as the abscissa and the acid production amount as the ordinate, and is shown in fig. 6.
Lactobacillus rhamnosus S24 in vitamin D 3 Acid production in MRS liquid medium is inhibited due to vitamin D 3 Is stable in a neutral environment, but is easily absorbed in an acidic environment, so that less acid production is beneficial to vitamin D 3 Further analysis by HPLC showed that Lactobacillus rhamnosus S24 is in vitamin D 3 Short-chain fatty acids in total acids fermented by the MRS liquid culture medium are remarkably improved, particularly, the content of butyric acid (Table 3) is improved by 186.35 percent compared with the content of butyric acid fermented in the MRS liquid culture medium, and the results show that: vitamin D 3 Can obviously inhibit the generation of total acid of the lactobacillus rhamnosus S24, but can improve the increase of short-chain fatty acid, and especially obviously improve the content of butyric acid playing an important role in the intestinal muscle axis and the intestinal bone axis.
TABLE 3 short-chain fatty acid test results
Note: the unit of short chain fatty acid is mug/ml
Example 2
A composition comprises a lactobacillus rhamnosus S24 metagen, wherein the metagen contains 500 hundred million lactobacillus rhamnosus thalli/g, and free amino acid is more than 1mg/g, and the preparation method of the metagen comprises the following steps: adding Lactobacillus rhamnosus S24 in vitamin D 3 And (3) fermenting in an MRS liquid culture medium to obtain a fermentation liquid, concentrating, and spraying dry solid powder containing thalli and metabolites.
Example 3
Application of lactobacillus rhamnosus S24 and its metazoans in preparing functional food or medicine for preventing and treating sarcopenia, osteoporosis and sarcopenia is provided.
And (3) testing a sample: the lactobacillus rhamnosus S24 bacterial powder (the number of viable bacteria is 500 hundred million CFU/g) in the embodiment 1 is obtained by adopting an MRS culture medium for fermentation culture; the lactobacillus rhamnosus S24 metazoan in example 2 (the number of thalli is 500 hundred million/g, free amino acid is more than 1 mg/g) is prepared by the following steps: adding Lactobacillus rhamnosus S24 in vitamin D 3 And (3) fermenting in an MRS liquid culture medium to obtain a fermentation liquid, concentrating, and spraying dry solid powder containing thalli and metabolites.
Research experiment for effect of lactobacillus rhamnosus S24 on muscular bone reduction
And (3) test grouping: 30 SPF-grade SD rats purchased from toddalo corporation were randomly divided into 5 groups by physical mass, namely, normal group (CON group), sham group (SHM group), 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 OS group, the OS + S24 group and the OS + S24 metazoan group rats are molded by a castration method, the SHM group is molded by a pseudo operation, and except the CON group, the rest groups of rats are injected with 8 ten thousand units of penicillin sodium per day in an intramuscular mode every day after the operation and are continuously 3 days. After surgical recovery of 7d,7d 1mg (/ kg. D) dexamethasone sodium phosphate injection (DXM) was annotated to the abdominal cavity of OS, OS + S24 and OS + S24 metazoan rats for 2 weeks. And after the molding is finished, starting the medicine intervention. Rats of CON group, SHM group and OS group were gavaged with 10mL/kg physiological saline daily, and rats of OS + S24 group were gavaged with Lactobacillus rhamnosus S24 powder (6 × 10) for 12 weeks 8 CFU/per mouse/day), OS + S24 subsequent tuple continuous 12 weeks intragastric Lactobacillus rhamnosus S24 anagen (6X 10) 8 Per mouse/day), the data were analyzed after completion of the experimental results, compared to the CON group, a P<0.05; in contrast to the SHM group, b P<0.05; in contrast to the OS group, c P<0.05; in contrast to the OS + S24 group, d P<0.05。
(1) general condition observation and body weight change analysis of rats
During the gavage period, feeding activity, mental state, abnormal behavior, etc. of the rats in each group were observed and recorded daily. After the intervention was completed, the Body Weight (BW) of each group was measured periodically on an electronic scale every week.
During the experiment, no mice died. The OS group rats had dry, messy hair and reduced gloss compared to the other groups. The OS + S24 group and the OS + S24 metaplastic rats had softer hair and normal spirit and activity than the OS group skin Mao Guangze. The body weight changes of the rats in the different test groups are shown in Table 4. No significant difference exists between each dose group and a control group and an SHM group (P is more than 0.05); 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 the weight loss which is one of clinical manifestations of a patient with muscular-skeletal loss; compared with the OS group, the weight changes of the OS + S24 group and the OS + S24 metazoan group have significant differences, which shows that the S24 metazoan group and the S24 metazoan group can effectively relieve the weight loss of the OS rat, namely, the long-term oral administration of the lactobacillus rhamnosus S24 viable strain or the metazoan thereof can effectively prevent the animal musculoskeletal co-reduction disease, and the growth safety is good.
TABLE 4 weight change of rats in each experimental group
Note that: in contrast to the CON group, a P<0.05; in contrast to the SHM group, b P<0.05; in contrast to the OS group, c P<0.05; in contrast to the OS + S24 group, d P<0.05。
(2) bone Density Change analysis in rats of Each group
The test method comprises the following steps: after the intervention and before the material is drawn, the rats in each group are anesthetized, and the Bone density (BMD) of the whole body and the BMD of the lumbar vertebra of the rats in each group are measured by a dual-energy X-ray Bone densitometer.
Referring to fig. 7, the test results show that: compared with the CON group and the SHM group, the BMD of the whole body and the BMD of the lumbar vertebra of the rats in the OS group are obviously reduced (P < 0.05), which indicates that the modeling is successful; compared with the OS group, the total body BMD and lumbar vertebra BMD of the rats with the metazoan group of OS + S24 and OS + S24 are obviously increased (P < 0.05), which indicates that the lactobacillus rhamnosus S24 and the metazoan thereof can obviously improve the bone density of the rats with the muscular-bone co-reduction disease (the OS group), and further effectively prevent and treat the osteoporosis OP.
(3) Comparative analysis of forelimb holding power, SMI and RSMI of rats in each group
The test method comprises the following steps: after the intervention was completed, the front-limb grip (Forelimb grip strength) of the rat was measured using a grip meter at fixed times per week. The rats were allowed to acclimate on the grab-force instrument for 5 minutes before testing, then the tail of the rat was grasped and pulled back slowly until the forelimb of the rat failed to grasp the cross-bar and the maximum grab-force reading on the instrument was recorded. Three times are measured and recorded, and the average of three grasping powers is used as an index reflecting the strength of forelimb muscles. The dual-energy X-ray bone densitometer measures the skeletal muscle mass (LM) of the whole body and the skeletal muscle mass (ALM) of the extremities of each group of rats. And rat skeletal muscle mass index (SMI = LM/BW) and limb skeletal muscle mass index (RSMI = ALM/BW) were calculated.
Referring to fig. 8 and 9, the anterior limb holding power of the OS group rats was significantly decreased (P < 0.05), and SMI and RSMI were also significantly decreased (P < 0.05) compared to the CON and SHM groups; compared with the OS group and the CON group, the front limb holding power, SMI and RSMI of the rats of the OS + S24 group and the OS + S24 hind birth group are obviously increased (P < 0.05), the front limb holding power, SMI and RSMI of the rats of the OS group are respectively increased by 29.40%, 43.48% and 45.74%, and even the front limb holding power, SMI and RSMI of the rats of the CON group are respectively increased by 15.85%, 13.6% and 45%. Shows that: the OS + S24 group and the OS + S24 metazoan group can obviously improve the forelimb holding power and the skeletal muscle mass of a rat with the muscular-skeletal-hypofunction (OS), not only can recover the forelimb holding power and the skeletal muscle mass of the rat with the muscular-skeletal-hypofunction, but also can promote the further enhancement of the forelimb holding power and the skeletal muscle mass, and effectively prevent and treat sarcopenia or osteoporosis or muscular-skeletal hypofunction.
(4) Comparative analysis of expression of proteins related to gastrocnemius PI3K signal channel of rats in each group
The test method comprises the following steps: and detecting the expression level of the PI3K protein by adopting a protein immunoblotting method. The specific operation is as follows: frozen gastrocnemius muscles of each group of experimental rats are taken, ground and crushed under liquid nitrogen, cracked by using a lysate, total protein is extracted, and the protein concentration and the protein sample denaturation are measured by using a BCA method. Preparing gel, electrophoresis, transferring a membrane and sealing. PI3K (1) and GAPDH (1) were added sequentially and incubated overnight at 4 ℃. TBST was washed 3 times for 15min each. HRP-labeled goat anti-rabbit IgG antibody was incubated at room temperature for 1h, and washed 3 times in TBST for 15min each. The protein bands were developed using a totipotent gel imaging system and quantitatively analyzed using Image J software.
The test results are shown in fig. 10, and compared with the CON group and the SHM group, the rat gastrocnemius PI3K protein expression of the OS group is significantly reduced; OS + S24 and OS + S24 metaplastic rats had significantly increased gastrocnemius PI3K protein expression compared to the OS group (P < 0.05). It is shown that lactobacillus rhamnosus S24 and lactobacillus rhamnosus S24 metagenes can prevent and treat musculoskeletal reduction syndrome (OS) by up-regulating PI3K signaling pathway-related protein expression.
(5) Detection and analysis of lactobacillus and bifidobacterium abundance in feces of rats in each group
According to the method of patent CN113234622A, lactobacillus and bifidobacterium in excrement are detected, the test results are shown in Table 5, and compared with CON group and SHM group, lactobacillus and bifidobacterium in intestinal tracts of rats in OS group are remarkably reduced; compared with the OS group, lactobacillus and bifidobacterium in intestinal tracts of rats are remarkably increased in OS + S24 and OS + S24 metagenes (P < 0.05), and the lactobacillus rhamnosus S24 and lactobacillus rhamnosus S24 metagenes can prevent and treat the muscular bone loss syndrome (OS) by increasing the abundance of the lactobacillus and the bifidobacterium in the intestinal tracts.
TABLE 5 Lactobacillus rhamnosus S24 Regulation of the intestinal flora in rats (lg cfu/g)
In conclusion, in the OS group rats, the total BMD, lumbar BMD, SMI and RSMI were significantly reduced compared to the CON group and the SHM group, indicating that castration in combination with DXM may cause the rats to develop the osteopenia syndrome (OS for short). Rats in the OS + S24 group, OS + metazoan S24 group had significantly increased body weight, total body BMD, lumbar BMD, forelimb grip, SMI and RSMI compared to the OS group, indicating: the lactobacillus rhamnosus S24 and the metazoans thereof can obviously improve the weight loss, BMD, muscle strength and muscle mass of OS rats, thereby effectively preventing and treating the muscular-bone reduction syndrome. In addition, the expression level of PI3K protein in gastrocnemius of an OS rat is obviously improved by the lactobacillus rhamnosus S24 and the metagenes thereof, which indicates that the effect of the lactobacillus rhamnosus S24 and the metagenes thereof on preventing and treating OS is probably related to the up-regulation of the expression level of the protein related to a PI3K signal pathway, and simultaneously, the lactobacillus rhamnosus S24 and the lactobacillus rhamnosus S24 metagenes can increase the abundance of lactobacillus and bifidobacterium in intestinal tracts to regulate intestinal flora so as to prevent and treat musculoskeletal reduction syndrome.
Example 4
A composition of this example 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 vitamin D 3 Culturing in MRS liquid culture medium at 37 deg.C for 48 hr to obtain Lactobacillus rhamnosus S24 bacterial liquid, and sterilizing at 125 deg.C for 5 min; 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 composition of the embodiment is applied to the preparation of functional food or medicine for treating sarcopenia, osteoporosis or sarcopenia, and when in use, the dosage of the lactobacillus rhamnosus S24 in the composition is 1.0 x 10 11 The amount of the CFU/day, inactivated Lactobacillus rhamnosus S24 is 6.0 x 10 10 CFU/day。
Example 5
Function of lactobacillus rhamnosus S24 in preparation of medicines for preventing and treating OSApplication in sexual food or medicine, the dosage of Lactobacillus rhamnosus S24 is 1.0 × 10 11 CFU/day。
Wherein, the lactobacillus rhamnosus S24 is obtained by adopting MRS culture medium to culture as described in example 1.
Example 6
The composition comprises a viable lactobacillus rhamnosus S24 strain, an inactivated lactobacillus rhamnosus S24 strain and a metazoan lactobacillus rhamnosus S24 strain in a mass ratio of 1.
As shown in the bacteriostatic ability tests of the lactobacillus rhamnosus S24 in the embodiments 3 to 3 of example 1 on pathogenic bacteria, the lactobacillus rhamnosus S24 viable strains have good inhibitory action on common pathogenic bacteria such as helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231.
The composition of the embodiment has bacteriostatic action and is applied to the preparation of functional food or drugs for inhibiting pathogenic bacteria, wherein the pathogenic helicobacter comprises helicobacter pylori ATCC 26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231. When in use, the dosage of the active lactobacillus rhamnosus S24 strain is 1 × 10 10 The inactivated strain of CFU/day, lactobacillus rhamnosus S24 is 2X 10 10 CFU/day, the amount of metazoan is 100mg/day.
Example 7
A composition of this example, comprising a viable strain of lactobacillus rhamnosus S24 and a metabolite of the strain of lactobacillus rhamnosus S24.
According to the embodiment 1, the viable lactobacillus rhamnosus S24 strain cultured on the MRS culture medium has better acid production and bacteriostasis capabilities, and the composition of the embodiment is used as a leavening agent to be applied to the preparation of fermented food and health-care food.
The fermented food is pickled vegetables, and the application method of the composition serving as the leavening agent in preparing the pickled vegetables specifically comprises the following steps:
cleaning fresh vegetables, adding into 4-5 times of drinking water, and adding edible glucose 1% of the total volumeEdible sodium chloride with the total volume of 0.5-2.0 percent is inoculated into the lactobacillus rhamnosus S24 prepared in the embodiment 1 of the invention to ensure that the concentration reaches 10 7 Fermenting at room temperature for 5-24 hr at CFU/mL above to obtain fermented sauerkraut containing composition containing Lactobacillus rhamnosus S24 and Lactobacillus rhamnosus S24 strain metabolite. The fermented sauerkraut has crisp and tasty taste, unique flavor, contains Lactobacillus rhamnosus S24 thallus and metabolite, and has good safety and probiotic function.
Example 8
The composition of this example comprises Lactobacillus rhamnosus S24 powder (4.0 × 10 by weight) 10 CFU/g) 14 parts, lactobacillus plantarum LP550 (4.0X 10) 10 CFU/g) 10 parts, lactobacillus plantarum LP220 (4.0X 10) 10 CFU/g) 4 parts, lactobacillus rhamnosus S24 metazoa (cell number 4.0X 10) 10 Per gram) 1 part 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 vitamin D 3 1 part by weight.
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 vitamin D 3 1 part by weight, mixing uniformly, granulating with 30% alcohol wet method 20 mesh sieve to obtain wet granules, drying at 55 deg.C for 3.5 hr, grading with 20 mesh sieve, and adding Lactobacillus rhamnosus S24 powder (4.0 × 10) 10 CFU/g) 14 parts, lactobacillus plantarum LP550 (4.0X 10) 10 CFU/g) 10 parts, lactobacillus plantarum LP220 (4.0X 10) 10 CFU/g) 4 parts, lactobacillus rhamnosus S24 metazoa (cell number 4.0X 10) 10 Per g) 1 part, and magnesium stearate 2 parts, mixing uniformly, and tabletting by a rotary tablet press to obtain tablets with lactobacillus rhamnosus S24 dietary supplement.
The composition of the embodiment is used for preparing a medicine for preventing and treating the muscular-bone reduction syndrome.
Example 9
A composition of this example, in parts by weight, comprisesLactobacillus rhamnosus S24 (4.0X 10) 10 CFU/g) 15 weight portions, lactobacillus rhamnosus S24 metazoan (4.0X 10) 10 Per g) 1 part by weight of Lactobacillus plantarum LP220 (4.0X 10) 10 CFU/g) 9 parts by weight of Lactobacillus fermentum GF1800 (4.0X 10) 10 CFU/g) 10 parts by weight, maltodextrin 10 parts by weight, sorbitol 10 parts by weight, corn peptide 10 parts by weight, sodium hyaluronate 10 parts by weight, anserine 4 parts by weight, soybean peptide 4 parts by weight, xylo-oligosaccharide 2 parts by weight, broccoli seed water extract 2 parts by weight, selenium-enriched yeast 2 parts by weight, folic acid 2 parts by weight, malic acid 2 parts by weight, glutathione 2 parts by weight, vitamin E2 parts by weight, vitamin C2 parts by weight, vitamin D 3 1 part by weight.
The raw materials of the composition are uniformly mixed according to the proportion after passing through a 40-mesh screen, and are bagged by a screw back sealing packaging machine to prepare 2-10g of solid beverage for preventing and treating musculoskeletal syndrome per bag.
Example 10
Application of lactobacillus rhamnosus S24 in preparing functional food or medicine for preventing and treating muscular-bone reduction syndrome, wherein the dosage of lactobacillus rhamnosus S24 is 1.0 × 10 11 CFU/day。
Wherein, the lactobacillus rhamnosus S24 is obtained by culturing on MRS culture medium as described in example 1.
Example 11
Application of lactobacillus rhamnosus S24 metazoan in preparing functional food or medicine for preventing and treating sarcopenia, osteoporosis or sarcopenia syndrome, wherein when in use, the dosage of lactobacillus rhamnosus S24 metazoan is 1.0 x 10 11 Person/day.
Wherein the lactobacillus rhamnosus S24 metazoan is prepared as described in example 2.
Example 12
Application of lactobacillus rhamnosus S24 and metazoan thereof in functional food or medicine for preventing and treating OS, wherein the dosage of lactobacillus rhamnosus S24 is 5 × 10 10 The consumption of post-natal lactobacillus rhamnosus S24 is 5 × 10 10 Person/day. The Lactobacillus rhamnosus strain was treated with vitamin D as described in example 1 3 MRS medium such as Lactobacillus rhamnosus metazoaObtained by culturing as described in example 2.
Claims (10)
1. The Lactobacillus rhamnosus is named as Lactobacillus rhamnosus S24 and is preserved in China Wuhan China center for type culture Collection in 8 months and 6 days of 2021, wherein the preservation number is CCTCC NO: m2021986.
2. The lactobacillus rhamnosus strain of claim 1, wherein the 16S rRNA gene sequence of lactobacillus rhamnosus S24 is shown in SEQ ID No. 1.
3. The lactobacillus rhamnosus strain of claim 1 wherein the lactobacillus rhamnosus S24 fermentation medium is MRS or vitamin D 3 -MRS medium, said vitamin D 3 The MRS culture medium comprises 10.0g/L of peptone, 5.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 1.5g/L of Tween and K 2 HPO 4 ·7H 2 O2.0g/L, anhydrous sodium acetate 5g/L, triammonium citrate 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 0.038g/L of O and 1000-4000 IU of vitamin D 3 。
4. A composition comprising a mixture of one or more of the viable strains of lactobacillus rhamnosus S24, the inactivated strains and metabolites of lactobacillus rhamnosus S24, the postnatal elements of lactobacillus rhamnosus S24, the viable strains of lactobacillus plantarum and lactobacillus fermentum according to any one of claims 1 to 3.
5. Use of lactobacillus rhamnosus S24 according to claim 1 or 2 and/or a composition according to claim 4 for the preparation of a functional food or medicament for the prevention or treatment of sarcopenia or osteoporosis or sarcopenia.
6. The use of claim 5, wherein the Lactobacillus rhamnosus S24 or the composition comprising it prevents or treats sarcopenia or osteoporosis or sarcopenia by one or more of alleviating weight loss, increasing the systemic bone density and lumbar vertebrae density in OS rats, increasing the forelimb grip, the systemic skeletal muscle mass and the skeletal muscle mass of the limbs in OS rats.
7. The use of claim 5, wherein the Lactobacillus rhamnosus S24 or the composition containing it prevents or treats sarcopenia or osteoporosis or sarcopenia by up-regulating the expression level of PI3K protein and/or increasing the abundance of Lactobacillus plantarum and Bifidobacterium enteron.
8. The use of claim 5, wherein the amount of Lactobacillus rhamnosus S24 is 6 x 10 or more 8 CFU/per mouse/day; the dosage of the inactivated strain of the lactobacillus rhamnosus S24 is 3-8 multiplied by 10 10 The dosage of CFU/day and the number of the retrobiotics of the lactobacillus rhamnosus S24 is more than or equal to 6 multiplied by 10 8 Per mouse/day, free amino acids > 1mg/g.
9. The use of Lactobacillus rhamnosus S24 and its metagens according to any of claims 1 to 3 for the preparation of a functional food or medicament for the suppression of pathogenic bacteria.
10. Use of lactobacillus rhamnosus S24 according to any of claims 1 to 3 and/or the composition according to claim 4 or 5 as a starter in the preparation of fermented foods, nutraceutical products or dietary supplements.
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