CN115948296A - Lactobacillus plantarum for improving muscle and bone health and/or improving athletic ability, and composition and application thereof - Google Patents
Lactobacillus plantarum for improving muscle and bone health and/or improving athletic ability, and composition and application thereof Download PDFInfo
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- CN115948296A CN115948296A CN202211672215.9A CN202211672215A CN115948296A CN 115948296 A CN115948296 A CN 115948296A CN 202211672215 A CN202211672215 A CN 202211672215A CN 115948296 A CN115948296 A CN 115948296A
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Abstract
Lactobacillus plantarum LP550 is preserved in China Wuhan's China center for type culture collection in 2021 year 4 month 23 day, and the preservation number is CCTCC NO: M2021437. The composition comprises lactobacillus plantarum LP550 metazoa, or the composition also comprises lactobacillus plantarum LP550 live strain and inactivated strain thereof, strain metabolite, and vitamin D 2 Vitamin D 3 One or more of lactobacillus rhamnosus and lactobacillus fermentum. The invention also discloses application of the composition containing the lactobacillus plantarum and the metazoan thereof in improving the muscle and bone health and/or improving the athletic ability. Lactobacillus plantarum LP550 of the invention is vitamin D 2 And vitamin D 3 Has strong metabolic capability, can obviously improve skeletal muscle quality index, limb skeletal quality index, whole body bone density, lumbar vertebra density, forelimb holding power and athletic ability, and can improve muscle bone health and athletic ability through various ways.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum for improving muscle and bone health and improving athletic ability, and a composition and application thereof.
Background
Vitamin D is a fat-soluble vitamin and exists in two forms in nature, namely vitamin D 2 (ergocalciferol) and vitamin D 3 (cholecalciferol). Vitamin D of plant origin 2 And vitamin D 3 It cannot be synthesized in human body, and can be supplemented only from diet, such as marine product, yeast, plant extract, etc. With the continuous research on the field of vitamin DTo date, vitamin D metabolites have been found to play an important role in the human body, and the main function of vitamin D is to promote the absorption of calcium and phosphorus by small intestinal mucosal cells. A cross-sectional study published in Nature Communications, conducted in-depth analysis of serum vitamin D Metabolites in 567 elderly Men, revealed that 8 specific intestinal bacterial taxa were associated with the level of vitamin D active form, 1, 25-dihydroxyvitamin D, and higher levels of 1, 25-dihydroxyvitamin D were associated with intestinal microbial diversity, indicating that intestinal flora alters intestinal vitamin D metabolism (Kado D, et al., vitamin D metabolism and the Gut Microbiome in Older Men [ J ] A].Innovation in Aging,2020.)。
In 2009, binkley et al proposed the concept of Osteopenia (OS) based on a similar pathophysiological basis and close correlation between sarcopenia and osteoporosis, mainly referring to patients who met the diagnostic criteria for osteoporosis and had a concurrent decline 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 risk of patient weakness, falls, fractures, hospitalization, disability, and mortality.
According to different diagnosis standards of the sarcopenia, the incidence rate of the sarcopenia in the population over 60 years old ranges from 5 percent to 37 percent, and even has a youthful trend. At present, no targeted medicine for preventing and treating the muscular-skeletal co-reduction syndrome exists, the effective prevention and treatment means at present comprise 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. US15385786 discloses that lactobacillus plantarum compositions are capable of improving muscle mass and endurance, reducing blood lactic acid, improving exercise capacity.
In the prior art, vitamin D is directly compounded with probiotics to intervene related diseases, such as vitamin D released by Chendan 3 Combined lactobacillus rhamnosus and P40 junctionThe protective action and mechanism research of enteritis are not shown, but the research report of the interaction of specific strains, vitamin D and metabolites thereof on musculoskeletal reduction syndrome and the improvement of motor ability is not shown, so that the development of new strains and compositions thereof for preparing a convenient and efficient composition for improving musculoskeletal health and improving motor ability has important social significance and market value.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art, and provides vitamin D which has high biological activity, wide carbon source utilization and high-efficiency metabolism 2 Or and vitamin D 3 The invention also provides a composition consisting of the lactobacillus plantarum and metazoans thereof, and provides a new application of the lactobacillus plantarum and the composition consisting of the lactobacillus plantarum in improving the muscle bone health and the motion ability.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a Lactobacillus plantarum is named Lactobacillus plantarum LP550, and is preserved in China center for type culture Collection in Wuhan, china in 2021, 4 months and 23 days, with the preservation number of CCTCCNO: M2021437.
Biological preservation description: lactobacillus plantarum LP550, deposited in the China center for type culture Collection, accession number: eight-way 299 in Wuchang district, wuhan city, hubei province, the preservation organization is abbreviated as: CCTCC, preservation date: 23/4/2021 (registration was received at 23/4/2021, and survival was detected and was deposited at 30/4), and the biological collection number is CCTCCNO: m2021437, strain number: lactobacillus plantarum LP550.
The lactobacillus plantarum LP550 is isolated from a plant source, and specifically, the lactobacillus plantarum LP550 is obtained by screening and isolating from Pixianchandou farmhouse Pixian bean paste.
The biological properties of the lactobacillus plantarum LP550 of the invention are as follows:
1) Morphological characteristics: the bacterial colony grows well on an MRS agar culture medium, is round, medium in size, milky white, upward convex and glossy, has relatively neat edges, is easy to pick, is rod-shaped under a microscope, and is positive after gram staining.
2) Biological identification: the Lactobacillus plantarum LP550 is sent to China center for type culture collection for 16SrRNA identification, the 16S rRNA gene sequence is shown as SEQ ID NO:1, the 16SrRNA sequence of the Lactobacillus plantarum LP550 is subjected to NCBI BLAST comparison, based on the 16S rRNA gene sequence comparison result, a Neighbor-Joining phylogenetic tree which is constructed by taking Holzapfelia floricola Ryu1-2 (AB 523780) as an outer branch has similarity of more than 99% with Lactobacillus plantarum (Lactobacillus plantarum) in Genebank as shown in FIG. 2, and the strain is identified as Lactobacillus plantarum LP550.
The culture medium for lactobacillus plantarum LP550 fermentation is MRS and vitamin D 2 + MRS Medium or vitamin D 3 + MRS Medium, said vitamin D 2 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 O2.0 g/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 2 (15 g/L of agar powder is added to be a solid culture medium); the vitamin D 3 + MRS culture medium comprises peptone 10.0g/L, beef powder 5.0g/L, yeast powder 4.0g/L, glucose 20.0g/L, tween 80.0 g/L, K 2 HPO 4 ·7H 2 O2.0 g/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 The growth of lactobacillus plantarum LP550 is remarkably promoted, the acid production level is equivalent to that of the acid production level in a conventional MRS culture medium, and meanwhile, the increase of short-chain fatty acids can be promoted, and particularly, the content of butyric acid playing an important role in the intestinal muscle axis and the intestinal bone axis is remarkably increased; in addition, vitamin D 3 Can promote and improve the bacteriostatic ability of Lactobacillus plantarum LP550 to pathogenic microorganisms, and can also improve the bacteriostatic ability of helicobacter pylori ATCC26695 and variant chainBacteriostatic effects of coccus 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 Lactobacillus plantarum LP550 metazoan, which is MRS medium or vitamin D 2 + MRS Medium or vitamin D 3 + MRS culture medium fermenting and culturing Lactobacillus plantarum to obtain fermentation liquid, concentrating, spray drying solid powder containing thallus and its metabolite, optionally vitamin D 2 Vitamin D 3 。
Further, the composition also comprises the viable lactobacillus plantarum LP550 strain, the inactivated lactobacillus plantarum LP550 strain, a strain metabolite and vitamin D 2 Vitamin D 3 A mixture of one or more of lactobacillus rhamnosus and lactobacillus fermentum, wherein lactobacillus rhamnosus is preferably lactobacillus rhamnosus S24 selected by the applicant and lactobacillus fermentum is preferably lactobacillus fermentum GF1800 selected by the applicant.
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 used for improving muscle and bone health and improving motor ability, 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 plantarum LP550 and the LP550 metazoans thereof can obviously improve the whole body bone density and the lumbar vertebrae density of an OS rat, thereby improving the musculoskeletal health or preventing the formation of musculoskeletal co-reduction disease (OS).
The lactobacillus plantarum LP550 and the LP550 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 the improvement of muscle and bone health or the effective prevention and treatment of Sarcopenia (SP).
The lactobacillus plantarum LP550 can also obviously prolong the running time and running distance of rats, so that the motor ability of the body is improved, and further the muscular-skeletal syndrome OS is prevented and treated.
The lactobacillus plantarum LP550 can also promote the increase of butyric acid content in excrement of OS rats, so that the effect of preventing and treating the muscular-skeletal syndrome OS is achieved.
Based on the above characteristics of the lactobacillus plantarum LP550 of the invention, one of the applications of the lactobacillus plantarum of the invention and its metazoans:
use of lactobacillus plantarum LP550 metazoans for the preparation of a functional food or medicament for improving musculoskeletal health and increasing exercise capacity; or, the application of the composition formed by the lactobacillus plantarum LP550 metazoa and one or more of lactobacillus plantarum LP550, lactobacillus plantarum LP550 inactivated strains and strain metabolites in the preparation of functional food or medicines for improving muscle and bone health and improving exercise capacity.
In an exemplary embodiment, the amount of Lactobacillus plantarum LP550 is ≥ 6X 10 8 The dosage of the inactivated strain of the CFU/per mouse/day lactobacillus plantarum LP550 is 3-8 multiplied by 10 10 CFU/day, the dosage of the lactobacillus plantarum LP550 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: the lactobacillus plantarum LP550 has extensive bacteriostatic activity on pathogenic bacteria. Specifically, the medicament is used for treating helicobacter pylori ATCC26695, streptococcus mutans CGMCC1.2499 and staphylococcus aureusCMCC26003, clostridium perfringens ATCC13124 and Candida albicans ATCC10231 all have good inhibition effects, wherein the inhibition effect on streptococcus mutans CGMCC1.2499 is strongest, and vitamin D is adopted 2 MRS and vitamin D 3 The bacteriostatic performance of lactobacillus plantarum LP550 cultured by the MRS culture medium on pathogenic bacteria is stronger than that of lactobacillus plantarum LP550 cultured by the conventional MRS culture medium.
Based on the above characteristics of Lactobacillus plantarum LP550, another application of Lactobacillus plantarum and its metazoans of the invention:
the lactobacillus plantarum LP550 and the metazoan thereof are applied to the preparation of functional food or medicines for inhibiting pathogenic bacteria.
The lactobacillus plantarum LP550 disclosed by the invention has very good tolerance to gastric acid and bile salt, and is suitable for oral administration.
Lactobacillus plantarum LP550 has a broad-spectrum carbon source utilization capacity and is vitamin D-tolerant 2 And vitamin D 3 Has strong metabolic capability and acid production capability, and the acid production capacity is rapid in the initial stage of fermentation and large and tends to be stable along with the increase of the fermentation time. In particular, lactobacillus plantarum LP550 is found in vitamin D 2 The growth performance and acid production capability of the MRS culture medium are greatly improved compared with those of the conventional MRS culture medium, and the vitamin D 3 The MRS culture medium only has a promoting effect on the growth of the lactobacillus plantarum LP550, and the acid production capacity of the MRS culture medium is equivalent to that of the MRS culture medium, and based on the characteristics, the lactobacillus plantarum and the postnatal thereof have another application:
the lactobacillus plantarum LP550 and the metazoan thereof are used as a leavening agent in the preparation of fermented foods, health-care foods or dietary supplements.
The lactobacillus plantarum has the beneficial effects that:
the Lactobacillus plantarum LP550 is cultured in MRS agar and vitamin D 2 + MRS Medium and vitamin D 3 The MRS culture medium has good growth, high biological activity, wide carbon source utilization capacity, good acid production characteristic and good tolerance to artificial gastric juice, intestinal juice and bile salt.
The Lactobacillus plantarum LP550 can be metabolized and utilizedVitamin D 2、 Vitamin D 3 Further improving growth performance and antibacterial effect, vitamin D 2 Can also promote acid production of lactobacillus plantarum LP550.
The lactobacillus plantarum LP550 and the metazoan group can obviously improve the skeletal muscle mass index, the limb skeletal mass index, the whole body bone density, the lumbar vertebra density, the forelimb holding power and the exercise capacity, increase the butyric acid content in excrement, improve the muscle bone health and the exercise capacity through various ways, and further delay or improve the muscle bone reduction syndrome.
The lactobacillus plantarum LP550 has good safety, and the lactobacillus plantarum LP550 has wide application, can be used for preparing functional food or medicines for improving muscle and bone health and improving athletic ability, or inhibiting pathogenic bacteria, or can be used as a leaven for preparing fermented food and health-care food.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum LP550 on MRS agar medium, wherein (A) plate colony morphology, (B) strain morphology under microscope;
FIG. 2 shows a Lactobacillus plantarum LP550 phylogenetic tree;
FIG. 3 shows the vitamin D pair of Lactobacillus plantarum LP550 and other strains 2 Vitamin D 3 Comparative analysis of metabolic capacity of (a);
FIG. 4 shows vitamin D 2 Vitamin D 3 The effect on the growth performance of lactobacillus plantarum LP550;
FIG. 5 shows vitamin D 2 Vitamin D 3 Influence on acid production performance of lactobacillus plantarum LP550;
FIG. 6 is a graph showing the comparative analysis of the 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. 7 is a graph showing comparative analysis of the front limb grasping force of rats in different experimental groups;
FIG. 8 is a graph of SMI (A) and RSMI (B) comparative analyses of rats in different experimental groups;
FIG. 9 is a graph showing comparative analysis of the running time (A) and distance (B) of rats in different experimental groups;
FIG. 10 is a graph comparing the content of butyric acid in feces of rats in 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 plantarum LP 550): 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 O2.0 g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L (agar powder 15g/L as solid medium).
Vitamin D 2 + MRS culture medium (Lactobacillus plantarum LP 550) (abbreviation: VD) 2 + 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 O2.0 g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L,4000IU vitamin D 2 (15 g/L of agar powder is added as a solid culture medium).
Vitamin D 3 + MRS culture medium (Lactobacillus plantarum LP 550) (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 O2.0 g/L, anhydrous sodium acetate 5g/L, citric acid diammine 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L,4000IU vitamin D 3 (15 g/L of agar powder is added as a solid culture medium).
Example 1
The Lactobacillus plantarum LP550 (Lactobacillus plantarum LP 550) of the invention is preserved in China center for type culture Collection at 23.4.2021 with the preservation number: CCTCC NO: M2021437.
1) Separation and screening of lactobacillus plantarum LP550 and determination of gastric acid and bile salt resistance
Collecting a microorganism screening sample from Pixianchengdu Pixian broad bean paste, cutting the collected sample into pieces, weighing 1g, putting into 9mL sterile physiological saline, fully shaking and uniformly mixing, diluting by 10 times, coating in an MRS solid culture medium, and culturing for 48h at 37 ℃. Observing with naked eyes, picking single colonies with different shapes and sizes in a 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 ℃ by using 45% glycerol for later use.
a) Morphological observation
The purified lactobacillus plantarum LP550 is streaked on an MRS agar culture medium, and after the lactobacillus plantarum LP550 is subjected to inverted culture at 37 ℃ for 48 hours, the colony morphology of the strain is observed by an electron microscope, and the result is shown in the attached figure 1: the bacterial strain grows well on an MRS agar culture medium, the bacterial colony is circular, medium in size, milky white, upward convex and glossy, the edge of the bacterial colony is relatively neat and easy to pick, the bacterial colony is rod-shaped under a microscope, and the bacterial colony is positive after gram staining.
b) Molecular biological identification of strains
The purified strain is sent to China center for type culture collection to carry out 16S rRNA identification, the measured 16S rRNA sequence is compared with NCBI BLAST, the similarity with Lactobacillus plantarum in Genebank is more than 99 percent, and the strain can be preliminarily identified as Lactobacillus plantarum. The 16S rRNA identification sequence of the strain is shown as SEQ ID NO:1 and is named as Lactobacillus plantarum LP550 (Lactobacillus plantaumLP 550), and the Lactobacillus plantarum LP550 is a Neighbor-Joining phylogenetic tree which is constructed by taking Holzapfelia floricola Ryu1-2 (AB 523780) as an epistyle based on the 16S rRNA gene sequence comparison result and is shown as figure 2.
2) Comparative test of strain metabolism vitamin D
After candidate strains of lactobacillus plantarum LP550, lactobacillus plantarum 360, lactobacillus plantarum 220, lactobacillus plantarum M71, M72, lactobacillus fermentum GF1800 and lactobacillus paracasei S6 are activated twice in MRS culture medium, activated lactobacillus plantarum LP550 and bacterial liquid of other strains are inoculated to vitamin D according to the inoculation amount of 5 percent 2 + MRS medium, vitamin D 3 +MMixing with RS culture medium, and packaging into sterile test tubes (18 mm × 180 mm) at a volume of 8 ml/tube; standing and culturing the subpackaged candidate strain liquid in a constant-temperature incubator at 37 ℃ for 24 times, and then measuring vitamin D by HPLC 2 With vitamin D 3 In an amount (in terms of M) 1 ) Vitamin D is added into the culture medium 2 With vitamin D 3 The content is taken as the initial content (calculated as M) 0 ) Wherein the culture medium is not inoculated as a control, and the strain metabolizes vitamin D with the calculation formula (%) = (M) 0 -M 1 )/M 0 *100％。
The results of the experiment are shown in FIG. 3, and the vitamin D pair of Lactobacillus plantarum LP550 in the candidate strains 2 With vitamin D 3 Has a metabolic capacity of 32.15% and 42.57%, respectively, and Lactobacillus plantarum LP550 has vitamin D pair activity 2 With vitamin D 3 Is significantly more potent than other candidate strains.
Based on the strong metabolic capability of lactobacillus plantarum LP550 on vitamin D, the influence of vitamin D on the growth and metabolism of lactobacillus plantarum LP550 is further analyzed, and a new application of the vitamin D is further explored.
3) Physiological and biochemical characteristics of vitamin D on lactobacillus plantarum LP550
(1) Growth Performance of Lactobacillus plantarum LP550
Inoculating lactobacillus plantarum LP550 strain strains to an MRS culture medium according to the inoculation amount of 5 percent for culturing for 24 hours, and continuously activating twice. Inoculating the activated lactobacillus plantarum LP550 bacterial liquid to MRS and vitamin D according to the inoculation amount of 5 percent 2 + MRS, vitamin D 3 + MRS liquid culture medium, mixing well, subpackaging into sterile test tubes (18 mm × 180 mm) according to 8 ml/piece; standing and culturing the subpackaged lactobacillus plantarum LP550 bacterial liquid in a constant-temperature incubator at 37 ℃ for 24h, and measuring the absorbance values OD of the bacterial liquid for 15h and 24h by taking 3 test tubes 600 Calculating an average value; absorbance value OD with time as abscissa 600 The growth curve is plotted for the ordinate.
The results of the growth experiment of Lactobacillus plantarum LP550 are shown in FIG. 4, wherein Lactobacillus plantarum LP550 is vitamin D 2 + MRS, vitamin D 3 The growth in the MRS liquid culture medium is respectively improved by 22.99 percent and 33.70 percent, and the growth is obviously excellentDemonstration of vitamin D in MRS Medium 2 With vitamin D 3 And the metabolite thereof not only can not influence the growth of the lactobacillus plantarum LP550, but also can promote the growth of the lactobacillus plantarum LP550.
(2) Acid production performance test of lactobacillus plantarum LP550
Inoculating lactobacillus plantarum LP550 strain strains to an MRS culture medium according to the inoculation amount of 5 percent for culturing for 24 hours, and continuously activating twice. Inoculating the activated lactobacillus plantarum LP550 bacterial liquid to MRS and vitamin D according to the inoculation amount of 5 percent 2 + MRS, vitamin D 3 + MRS liquid medium, mixed well and dispensed into sterile test tubes (18 mm × 180 mm) at an amount of 8 ml/tube. Placing the subpackaged lactobacillus plantarum LP550 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 bacteria liquid, and an acid production curve is drawn by taking the time as an abscissa and the acid production amount as an ordinate, as shown in fig. 5.
Referring to FIG. 5, lactobacillus plantarum LP550 is shown in vitamin D 2 + MRS and vitamin D 3 The total acid level in the MRS liquid culture medium has no significant difference relative to the acid production level in the MRS liquid culture medium, and further analysis by HPLC shows that the short-chain fatty acid is significantly improved, particularly the content of butyric acid is significantly increased (Table 1), and the results show that: vitamin D 2 Or vitamin D 3 Has little influence on the production of lactobacillus plantarum LP550 total acid, but can increase the content of short-chain fatty acid, vitamin D 2 The MRS is improved by 16.4 percent compared with the MRS, and the vitamin D 3 The MRS is improved by 27 percent compared with the MRS, and particularly, the content of butyric acid and vitamin D which play important roles in the enteromyo-axial and the entero-osseous axial are obviously improved 2 + MRS, vitamin D 3 And the + MRS is respectively improved by 4.7 times and 7.4 times compared with the MRS.
TABLE 1 short-chain fatty acid test results
And (3) annotation: the unit of short chain fatty acid is mug/ml.
(3) Bacteriostatic ability test of lactobacillus plantarum LP550 on pathogenic bacteria
Bacteriostatic experiments on pathogenic bacteria: pouring 10mL of water agar culture medium in a sterile plate, placing an Oxford cup after cooling and solidifying, respectively adding indicator bacterium suspensions (helicobacter pylori ATCC26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC 10231) into the agar culture medium which is cooled to 50 ℃ and is correspondingly grown by the indicator bacterium, and enabling the concentration of the indicator bacterium to be 10 6 Mixing CFU/mL, pouring onto water agar, solidifying, taking out Oxford cup with forceps to form holes, adding 200 μ L of sample to be tested (MRS culture medium, vitamin D) into each hole 2 + MRS Medium, vitamin D 3 + MRS culture medium), spreading for 30min, and culturing at 37 deg.C 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 Lactobacillus plantarum LP550 fermentation broth cultured in different media
Streptococcus mutans CGMCC 1.2499.17 + -0.68 23.32 + -0.67.22.08 + -0.63
Staphylococcus aureus CMCC 26003.81 plus or minus 0.75 plus or minus 0.18 plus or minus 0.63.75 plus or minus 0.42
Clostridium perfringens ATCC 13124.72 + -0.44.22.57 + -0.88 20.98 + -0.45
The results are shown in Table 2, relative to the plants obtained by fermentation on MRS mediumAdding vitamin D into Lactobacillus plantarum LP550, MRS culture medium 2 Or vitamin D 3 The inhibition zones of the lactobacillus plantarum LP550 obtained by post-fermentation on helicobacter pylori ATCC26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231 reach more than 20mm, which shows that: vitamin D 2 And vitamin D 3 Synergistically promotes the bacteriostatic ability of the lactobacillus plantarum LP550 to pathogenic microorganisms, and the bacteriostatic ability of the lactobacillus plantarum LP550 is improved by more than 11 percent on the basis of a conventional MRS culture medium, especially vitamin D 2 The synergistic effect of the lactobacillus plantarum LP550 on the bacteriostasis capacity of the candida albicans ATCC10231 is improved by 49 percent, and the vitamin D 3 The antibacterial ability of the lactobacillus plantarum LP550 to the helicobacter pylori ATCC26695 is synergistically improved by as much as 42 percent; incorporation of Lactobacillus plantarum in vitamin D 2 + MRS Medium, vitamin D 3 + MRS medium, the number of bacteria increased, but the total acid change was not significant, indicating that Lactobacillus plantarum LP550 was vitamin D 2 + MRS Medium, vitamin D 3 The content of antibacterial substances such as antibacterial peptide in the MRS culture medium is improved, so that the antibacterial performance of the lactobacillus plantarum LP550 is remarkably improved.
4) Tolerance test of lactobacillus plantarum LP550 to human gastric acid and bile salt
(1) Determination of gastric acid resistance of bacterial strains
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 the LP550 strain preserved by the glycerin tube into an MRS culture medium at the temperature of 37 ℃ for activation for 24 hours in an inoculation amount of 10 percent; adding an equal amount of LP550 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 of the normal gastric acid secreted by the stomach of a human 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. The experiment result shows that the survival rate of LP550 is 49.52% when the pH value is 2.0; the survival rate of LP550 is 74.37% when the pH is 2.50; at pH3.0, the survival rate of LP550 was 95.43%, indicating that the LP550 strain had good gastric acid tolerance.
(2) Determination of bile salt resistance of strain
The strain LP550 is inoculated into MRS liquid culture medium according to the inoculation amount of 5 percent, activated and cultured for 24 hours at 37 ℃, and activated twice continuously. Inoculating the activated LP550 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 bacteria 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%.
LP550 bile salt tolerance data indicate: when the concentration of the bile salts is 1.0g/L and 2.0g/L, the survival rate of the strain is 100 percent and 98.13 percent respectively, but when the concentration of the bile salts reaches 3.0g/L, the survival rate of the strain still reaches 95.76 percent. The concentration of bile salt in intestinal canal is not more than 3.0g/L, which indicates that the LP550 strain has better bile salt tolerance.
In conclusion, lactobacillus plantarum LP550 can not only metabolize vitamin D in the medium 2 Or vitamin D 3 At the same time, vitamin D 2 And vitamin D 3 Can also promote the growth of lactobacillus plantarum LP550, the formation of metabolites such as antibacterial peptide and the like, and promote the growth of lactobacillus plantarum LP550The lactobacillus plantarum LP550 has good tolerance on gastric juice, intestinal juice and bile salts, and has the main functions of promoting the absorption of calcium and phosphorus by small intestinal mucosa cells, improving the concentration of calcium and phosphorus in blood, facilitating the generation and calcification of new bones and providing a new direction for the development and research of new application of the lactobacillus plantarum LP550 based on vitamin D.
Example 2
A composition comprising Lactobacillus plantarum LP550 metazoans containing 100 hundred million cells/g of the Lactobacillus plantarum strain of example 1, vitamin D 3 The content is 400IU/g, the free amino acid is more than 1mg/g, and the preparation method of the metazoan comprises the following steps: the Lactobacillus plantarum LP550 of example 1 was treated with vitamin D 3 + MRS liquid culture medium to ferment and concentrate the fermentation liquor, and spray-drying the fermentation liquor containing thallus and vitamin D 3 And solid powders of metabolites.
Example 3
Use of Lactobacillus plantarum LP550 and its metazoan for the preparation of a functional food or medicament for improving muscle bone health and increasing exercise capacity.
And (3) testing a sample: (1) metaplasia lactobacillus plantarum LP550 (abbreviation: metaplasia LP 550): lactobacillus plantarum LP550 metazoan (number of cells 100 hundred million/g, D) in example 2 3 The content is 400IU/g, the free amino acid is more than 1 mg/g), and the preparation method comprises the following steps: mixing Lactobacillus plantarum LP550 in vitamin D 3 + MRS liquid culture medium to ferment and concentrate, spray drying the fermented liquid containing thallus and vitamin D 3 And solid powders of metabolites. (2) Compound vitamin D 3 And a post-growth LP550 (for short, compound VD) 3 ): in example 1, fermentation culture was performed in MRS medium to obtain Lactobacillus plantarum LP550 broth, which was concentrated, spray-dried to obtain solid powder containing bacterial cells and metabolites, and then mixed with vitamin D 3 Mixing the finished products, and compounding to obtain the vitamin D product 3 The content is 400, and the number of the lactobacillus plantarum LP550 thalli is 100 hundred million/g.
Research test of lactobacillus plantarum LP550 on improvement of muscle and bone health and improvement of athletic ability
Test pointGroup (2): 30 SPF SD rats purchased from Doudou company were randomly divided into 5 groups according to their physical qualities, namely, normal group (CON group), sham group (SHM group), OS group, and vitamin D complex 3 And a post-growth LP550 group (OS + compound VD) 3 Group) and OS metaplasia lactobacillus plantarum LP550 (OS + metaplasia LP550 group). Wherein OS group, OS + compound VD 3 Rats in the group and the OS + metazoan LP550 group are subjected to castration method modeling, rats in the SHM group are subjected to pseudo-operation modeling, and except the CON group, rats in other groups are subjected to postoperative intramuscular injection of 8 ten thousand units of penicillin sodium per day for 3 days continuously. After the surgery is recovered to 7d, OS + is added into VD 3 Rats in groups and OS + metazoan LP550 were annotated with dexamethasone sodium phosphate injection (DXM) 1mg (/ kg. D) in the abdominal cavity for 2 weeks. After the molding is finished, starting to perform medicine intervention: rats of CON group, SHM group and OS group were gavaged with 10mL/kg physiological saline daily, and OS + Compound VD 3 Compound vitamin D for continuous intragastric administration for 12 weeks 3 And metazoan LP550 (1X 10) 8 Per mouse/day), OS + metazoan LP550 group for 12 consecutive weeks intragastric administration of metazoan Lactobacillus plantarum LP550 (1X 10) of example 2 8 Per mouse/day), the data was analyzed after the experimental results were completed, 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; is compounded with OS + VD 3 Compared with the group, the group comparison method has the advantages that, 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 of rats was measured periodically weekly using an electronic scale.
During the experiment, no mice died. The OS group rats had dry fur, disordered hair, and reduced glossiness compared to the other groups. OS + compound VD 3 In the group, OS + metazoan LP550 group rats had a more shiny, softer, more psychologically and more motile fur than the OS group.
The body weight changes of the rats in the different test groups are shown in Table 3. No significant difference (P) was observed between each dose group and the control group and SHM group>0.05 ); the weight gain of OS group was small compared to CON group and SHM group, which is associated with musculoskeletal regionConsistent weight loss, one of the clinical manifestations of co-decreasing patients; OS + compound VD 3 Compared with the group OS, the group OS + metazoan LP550 group has a trend of weight increase but has an insignificant increase range, which indicates that the lactobacillus plantarum LP550 and LP550 metazoan have a trend of relieving weight decrease of the OS rats, and the vitamin D is orally taken for a long time 3 And the lactobacillus plantarum LP550 viable strain or metazoan thereof can effectively prevent the animal muscular-bone reduction syndrome and has good growth safety.
TABLE 3 weight change of rats in each experimental group
And (3) annotation: 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; is compounded with OS + VD 3 Compared with the group, the group comparison method has the advantages that, 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. 6, the test results show that: in the OS group, the rats had a significant decrease in systemic BMD and lumbar BMD (P) compared with the CON group and the SHM group<0.05 Indicating that the model is successfully made and OS has significant influence on the musculoskeletal health of rats; compared with OS group, OS + compound VD 3 Group, OS + metazoan LP550 group rats had a significant increase in systemic BMD and lumbar BMD (P)<0.05 Indicated that: using MRS Medium and vitamin D 3 The lactobacillus plantarum LP550 metaplast obtained from the MRS culture medium can obviously improve the bone density of rats with muscular-skeletal co-reduction disease (OS group), and further effectively improve the muscular-skeletal health of the OS rats.
Is compounded with OS + VD 3 Group comparison, OS + metazoan LP550 group Total BMD and lumbarSignificant increase in BMD (P)<0.05 To explain: compared with the conventional MRS culture medium, the lactobacillus plantarum LP550 fermentation liquid obtained by fermentation culture forms anagen and vitamin D 3 Formulated composition for metabolizing vitamin D 3 The metaplast of Lactobacillus plantarum LP550 has a better effect on improving bone density of OS rats, and vitamin D is probably utilized together with Lactobacillus plantarum LP550 3 Except for using the conventional MRS culture medium to grow metabolites, the metabolite capable of promoting the bone density of the OS rat to be improved is synthesized in the growth and metabolism process, so that the promotion effect of the metabolite on the bone density of the OS rat is more obvious.
(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 of the rats (forelimib grip strength) was measured using a grip tester for a fixed time per week. The rats were allowed to acclimate on the grab-dynamometer for 5 minutes before testing, then the tail was grasped and pulled back slowly horizontally until the forelimbs of the rat failed to grasp the rail and the maximum grab reading on the instrument was recorded. Three times are measured and recorded, and the average value of the three grasping powers is used as an index reflecting the strength of the 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 a rat skeletal muscle mass index (SMI = LM/BW) and a limb skeletal muscle mass index (RSMI = ALM/BW) were calculated.
Referring to FIGS. 7 and 8, the front limb holding power of the OS group rats was significantly decreased compared to the CON group and the SHM group (P)<0.05 SMI and RSMI also decreased significantly (P)<0.05 ); OS + Complex VD compared with OS group 3 Group and OS + metazoan LP550 group rats showed significant increases in forelimb grasping power, SMI and RSMI (P<0.05 LP550 group of OS + metagens is obviously superior to OS + compound VD 3 Indicating OS + compound VD 3 The group and OS + metazoan LP550 group significantly improved OS rat forelimb grip and skeletal muscle mass, thereby improving musculoskeletal health.
Compared to the CON group, the OS + metazoan LP550 group rats had significantly increased forelimb grasping power, SMI and RSMI (P < 0.05), indicating: the metazoan LP550 can not only promote the recovery of the forelimb holding power, SMI and RSMI of the OS rat, but also promote the further improvement of the holding power of the forelimb and the skeletal muscle quantity of the rat, namely, the metazoan LP550 has the functions of improving the muscle-bone health and improving the movement capacity of the OS rat or the healthy rat, and can effectively prevent and treat sarcopenia or osteoporosis or sarcopenia syndrome.
(4) Running experiment of rats in each group
The test method comprises the following steps: the endurance performance of rats was tested 12 weeks after continuous feeding of the rats, and the rats were acclimatized to treadmill training 30 minutes after each feeding 1 week before self-testing. The rat is placed in a small chamber of a running machine, the set rotating speed is 20r/min, the current intensity of an electric stimulation area is 1.2A, the time and the movement distance from the rat to exhaustive running are recorded, and the standard of exhaustive judgment is that the rat is static for more than 10S in an electric shock area.
As shown in FIG. 9, the running time and running distance of the rats in the OS group were significantly shortened as compared with those in the control group (P)<0.05 Indicating that OS had a significant effect on running ability in rats; compared with running time and running distance of rats in the OS group, the OS + compound VD 3 Both the group and the OS + metazoan LP550 group can obviously prolong the running time and the running distance of the rats (P)<0.05 And the running time and running distance of the rats in the OS + metazoan LP550 group are also obviously superior to those of the OS + compound VD 3 Group, it is demonstrated that metazoan LP550 contributes to the improvement of the body motor capacity of rats.
Running time and running distance were significantly prolonged in OS + metazoan LP550 group rats compared to CON group (P < 0.05), indicating: the metazoan LP550 can not only promote the recovery of the motor ability of the OS rat, but also promote the further improvement of the motor ability of the rat, namely, the metazoan LP550 has the function of improving the motor ability of the organism to the OS rat or a healthy rat, thereby effectively preventing and treating sarcopenia or osteoporosis or sarcopenia syndrome.
(5) Detection and analysis of butyric acid in feces of rats in each group
After feeding rats continuously for 12 weeks, fresh feces of each group of rats are collected, and samples are prepared and detected according to the HPLC on-board requirement. The test results are shown in FIG. 10, and the content of butyric acid in rats in the model group is significantly reduced (P) compared with that in CON group<0.01). After 12 weeks of intervention, OS + compound VD 3 The content of butyric acid in the feces of rats in the group and the group of OS + metazoan LP550 is obviously increasedHigh, simultaneously, OS + metazoan LP550 group is obviously superior to OS + compound VD 3 Group (P)<0.01). The lactobacillus plantarum LP550 metagenesis can improve the content of butyric acid required by meeting the energy supply of colon mucosal epithelial cells in rat intestinal tracts, further improve the rat intestinal flora, further promote the absorption of vitamin D and lactobacillus plantarum LP550 metabolites by the intestinal tracts, and achieve the purpose of effectively preventing and treating the muscular-bone reduction syndrome (OS).
In conclusion, the total BMD, lumbar BMD, SMI and RSMI in the OS group rats were significantly reduced compared to the CON group and the SHM group, indicating that castration in combination with DXM resulted in the development of sarcopenia (OS for short) in the rats. Compared with OS group, OS + compound VD 3 The rat whole body BMD, lumbar vertebra BMD, forelimb holding power, SMI, RSMI, running time and running distance of the group of OS + metazoan LP550 are obviously increased, the content of butyric acid in excrement is obviously increased, and the corresponding performance of the metazoan LP550 group is superior to that of compound VD 3 Group, indicated: the lactobacillus plantarum LP550 and the metazoan thereof can obviously improve the bone health of OS rats and improve the motor ability, thereby effectively preventing and treating the muscular-skeletal syndrome.
Example 4
A composition of this example comprises Lactobacillus plantarum LP550 metazoans containing 100 hundred million cells/g of the Lactobacillus plantarum strain of example 1, vitamin D 2 The content is 400IU/g, the free amino acid is more than 1mg/g, and the preparation method of the metazoan comprises the following steps: the Lactobacillus plantarum LP550 of example 1 was treated with vitamin D 2 + MRS liquid culture medium to ferment and concentrate, spray drying the fermented liquid containing thallus and vitamin D 2 And solid powders of metabolites.
The composition of this example was used for the treatment of OS rats, and the weight loss inhibitory effect and the elevating effect on the whole body BMD, lumbar BMD, forelimb grip, SMI, RSMI, running time and running distance of OS rats were slightly superior to those of the composition of example 2.
Example 5
The composition of this example comprises a live strain of Lactobacillus plantarum LP550 and an inactivated strain of Lactobacillus plantarum LP550, wherein the milk of the plant isLive strain of Bacillus LP550 is vitamin D in example 1 2 + lactobacillus plantarum LP550 obtained by culturing in MRS liquid medium; the inactivated strain of Lactobacillus plantarum LP550 is vitamin D 2 Culturing in MRS liquid culture medium at 37 deg.C for 48 hr to obtain Lactobacillus plantarum LP550 bacterial liquid, sterilizing at 125 deg.C for 5 min; centrifuging the inactivated bacterial liquid for 5min at 8000g to obtain an inactivated strain of lactobacillus plantarum LP550; the weight ratio of the live strain of lactobacillus plantarum LP550 to the inactivated strain of lactobacillus plantarum LP550 is 1.
The use of a composition of this example for the manufacture of a medicament for improving muscle bone health and increasing exercise capacity, wherein the amount of Lactobacillus plantarum LP550 used in the composition is 1.0 x 10 10 The dosage of CFU/day and the inactivated lactobacillus plantarum LP550 is 6.0 x 10 10 CFU/day。
Example 6
Application of lactobacillus plantarum LP550 in preparation of functional food or medicine for preventing and treating OS (oxygen-dependent System), wherein the dosage of the lactobacillus plantarum LP550 is 1.5 multiplied by 10 10 CFU/day。
Wherein, the lactobacillus plantarum LP550 is obtained by adopting MRS culture medium to culture as described in example 1.
Example 7
Application of lactobacillus plantarum LP550 for postnatal life in functional food or medicine for improving muscle and bone health and improving exercise capacity, wherein the lactobacillus plantarum LP550 postnatal life is 500mg/day.
The preparation method of the metaplasia lactobacillus plantarum LP550 comprises the following steps: the lactobacillus plantarum LP550 of example 1 was fermented in MRS broth to obtain a fermentation broth, which was concentrated and spray-dried to obtain a solid powder containing bacterial cells and metabolites.
Example 8
The composition of the embodiment comprises a viable lactobacillus plantarum LP550 strain and a post-growth lactobacillus plantarum LP550 strain in a mass ratio of 1.
As shown in the experiments of the bacteriostatic ability of the Lactobacillus plantarum LP550 to pathogenic bacteria in the embodiments 3-3 of example 1, the live Lactobacillus plantarum LP550 strain has good inhibitory effect on common pathogenic bacteria such as helicobacter pylori ATCC26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124, candida albicans ATCC10231 and the like.
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 ATCC26695, streptococcus mutans CGMCC1.2499, staphylococcus aureus CMCC26003, clostridium perfringens ATCC13124 and candida albicans ATCC10231. When in use, the dosage of the lactobacillus plantarum LP550 viable strain is 1 multiplied by 10 10 CFU/day, 2X 10 inactivated strain of Lactobacillus plantarum LP550 10 CFU/day, the dosage of metazoan is 100mg/day.
Example 9
The composition of this example includes live Lactobacillus plantarum LP550 strain and Lactobacillus plantarum LP550 metazoa, and the preparation method of Lactobacillus plantarum LP550 metazoa is the same as in example 4.
According to example 1, vitamin D 2 + the live Lactobacillus plantarum LP550 strain cultured on MRS culture medium has better acid production and bacteriostasis capacity, and the composition of the embodiment is used as a leavening agent in the preparation of fermented food and health food.
The fermented food is pickle, and the application method of the composition serving as the leavening agent in preparing the pickle is as follows:
cleaning fresh vegetables, adding into 4-5 times of drinking water, adding edible glucose 1% of total volume and edible sodium chloride 0.5-2.0% of total volume, inoculating into Lactobacillus plantarum LP550 prepared in the embodiment 1 of the invention, and making its concentration reach 10 7 Fermenting at room temperature for 5-24 hr at CFU/mL or above to obtain fermented sauerkraut containing Lactobacillus plantarum LP550 and composition of Lactobacillus plantarum LP550 metazoan. The fermented sauerkraut has crisp and tasty taste, unique flavor, contains Lactobacillus plantarum LP550 thallus and metabolite, and has good safety and probiotic function.
Example 10
Use of Lactobacillus plantarum LP550 and metazoan in functional food or medicine for improving bone health, wherein Lactobacillus plantarumThe strain LP550 is used in an amount of 5 × 10 10 CFU/day, and 5 x 10 dosage of postbiotic lactobacillus plantarum LP550 10 Person/day.
Wherein, the Lactobacillus plantarum LP550 and its metazoans are as vitamin D, respectively, in example 1 3 + MRS medium, obtained as described in example 2.
Example 11
A composition of this example comprises, in parts by weight, lactobacillus plantarum LP550 powder (4.0X 10) 10 CFU/g) 10 parts by weight, lactobacillus rhamnosus S24 (4.0X 10) 10 CFU/g) 8 parts by weight, lactobacillus plantarum LP550 metazoan (cell number 4.0X 10) 10 2 parts by weight of the extract per gram) 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, 10 parts by weight of kiwi fruit extract, 10 parts by weight of apple extract, 5 parts by weight of vitamin C and 3 parts by weight of folic acid.
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, 10 parts by weight of kiwi fruit extract, 10 parts by weight of apple extract, 5 parts by weight of vitamin C and 3 parts by weight of folic acid, uniformly mixing, granulating into wet granules by adopting a 30% alcohol wet method 20-mesh screen, drying at 55 ℃ for 3.5 hours, finishing granules by using a 20-mesh screen, and then adding lactobacillus plantarum LP550 bacterial powder (4.0 multiplied by 10) 10 CFU/g) 10 parts, lactobacillus rhamnosus S24 (4.0X 10) 10 CFU/g) 8 weight parts, lactobacillus plantarum LP550 metazoa (cell number 4.0X 10) 10 Per g) 2 parts, magnesium stearate 2 parts, mixing homogeneously and tabletting in a rotary tablet press to obtain tablets with the lactobacillus plantarum LP550 dietary supplement.
The composition of the present example was used to prepare a functional food for improving bone health and increasing exercise capacity.
Example 12
A composition of this example, in parts by weight, comprises Lactobacillus plantarum LP550 (4.0X 10) 10 CFU/g) 10 weight portions, lactobacillus plantarum LP550 metazoa (4.0X 10) 10 Per g) 2 parts by weight of fermented milk barBacterium GF1800 (4.0X 10) 10 CFU/g) 8 parts by weight, 10 parts by weight of maltodextrin, 10 parts by weight of sorbitol, 10 parts by weight of corn peptide, 10 parts by weight of sodium hyaluronate, 10 parts by weight of anserine, 10 parts by weight of soybean peptide, 4 parts by weight of xylo-oligosaccharide, 4 parts by weight of broccoli seed water extract, 2 parts by weight of selenium-enriched yeast, 2 parts by weight of folic acid, 2 parts by weight of malic acid, 2 parts by weight of glutathione, 2 parts by weight of vitamin E and 2 parts by weight of vitamin C.
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 the solid beverage with 2-10 g/bag and the functions of improving muscle and bone health and improving athletic ability.
Example 13
Application of lactobacillus plantarum LP550 and metazoan thereof in medicines for preventing and treating OS (OS), wherein the dosage of lactobacillus plantarum LP550 is 5 x 10 10 CFU/day, the post-natal dose of Lactobacillus plantarum LP550 was 1.2 x 10 11 Person/day. The Lactobacillus plantarum LP550 was as described in example 1 in vitamin D 3 + MRS medium, said Lactobacillus plantarum metazoa being obtained by culturing as described in example 2.
Claims (10)
1. The Lactobacillus plantarum is named Lactobacillus plantarum LP550 and is preserved in China Wuhan Chinese type culture collection at 23 months 4 in 2021 with the preservation number of CCTCC NO: m2021437.
2. The Lactobacillus plantarum of claim 1, wherein the 16S rRNA gene sequence of Lactobacillus plantarum LP550 is shown in SEQ ID NO 1.
3. The Lactobacillus plantarum of claim 1, wherein the fermentation medium for Lactobacillus plantarum LP550 is MRS, vitamin D 2 + MRS Medium or vitamin D 3 + MRS Medium, said vitamin D 2 + MRS medium, vitamin D 3 The + MRS culture medium is formed by adding 1000-4000 IU of vitamin D on MRS culture medium 2 、1000～4000IU vitamin D 3 。
4. A composition, comprising Lactobacillus plantarum LP550 metazoan, the Lactobacillus plantarum LP550 metazoan being either MRS medium or vitamin D 2 + MRS Medium or vitamin D 3 + MRS Medium fermentation culture the fermentation broth obtained from the Lactobacillus plantarum according to any of claims 1-3, concentrated, spray dried, solid powder containing the bacterial cells and their metabolites, optionally with vitamin D 2 Vitamin D 3 。
5. The composition according to claim 4, further comprising the viable Lactobacillus plantarum LP550 strain, the inactivated strain thereof, the metabolite of the strain, vitamin D, according to any of claims 1-3 2 Vitamin D 3 A mixture of one or more of lactobacillus rhamnosus and lactobacillus fermentum.
6. Use of lactobacillus plantarum LP550 according to any one of claims 1-3 and/or a composition according to claim 4 or 5 for the preparation of a functional food or medicament for improving muscle bone health and/or for improving exercise capacity.
7. The use according to claim 5, wherein Lactobacillus plantarum LP550, or a composition consisting thereof, is used for improving musculoskeletal health and increasing exercise capacity by one or more of alleviating weight loss, increasing systemic bone density and lumbar vertebrae density in OS rats, increasing forelimb grip, systemic skeletal muscle mass and limb skeletal muscle mass in OS rats.
8. The use according to claim 6, wherein the Lactobacillus plantarum LP550 is used in an amount of ≥ 6 x 10 8 CFU/per mouse/day; the dosage of the inactivated strain of the lactobacillus plantarum LP550 is 3-8 multiplied by 10 10 CFU/day, the dosage of the post-growth element of the lactobacillus plantarum LP550 is more than or equal to the number of the cells of the lactobacillus plantarum LP5506×10 8 Per mouse/day, free amino acids > 1mg/g.
9. Use of the Lactobacillus plantarum LP550, and its progeny, according to any of claims 1-3, for the preparation of a functional food or medicament for the suppression of pathogenic bacteria.
10. Use of the Lactobacillus plantarum LP550 according to any of claims 1-3 and/or the composition according to claim 4 or 5 as starter culture in the preparation of fermented food, nutraceutical or dietary supplement.
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