CN117586908A - Lactobacillus plantarum LP15 strain capable of improving bone mineral density and preparation method and application thereof - Google Patents

Lactobacillus plantarum LP15 strain capable of improving bone mineral density and preparation method and application thereof Download PDF

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CN117586908A
CN117586908A CN202311367913.2A CN202311367913A CN117586908A CN 117586908 A CN117586908 A CN 117586908A CN 202311367913 A CN202311367913 A CN 202311367913A CN 117586908 A CN117586908 A CN 117586908A
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lactobacillus plantarum
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bones
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周金龙
温宇旗
李云芳
徐杨
何晓婷
王小龙
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Inner Mongolia Mengtai Biological Engineering Co ltd
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Abstract

The invention provides a lactobacillus plantarum LP15 strain capable of improving bone mineral density, which belongs to the technical field of microorganisms, and is separated from weathered ox and sheep bones in a Mongolian Utility pasture in Erdos, inner Mongolia, and the preparation method comprises the following steps: s1, taking 8-12g of weathered ox and sheep bones of a Hemsleya paliurus pasture of Erdos of inner Mongolia and soil samples at 5-15cm near the bones, and filling the weathered ox and sheep bones into a clean plastic bag to serve as a sample; s2, primarily screening out strains; s3, carrying out streak separation and purification on the strain obtained by the primary screening, selecting a required strain according to the form and the size of a colony, carrying out 7-24h fermentation culture on the single colony obtained by the selection by using an MRS culture medium, and preserving at-80 ℃ by using glycerin with the concentration of 50 percent.

Description

Lactobacillus plantarum LP15 strain capable of improving bone mineral density and preparation method and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus plantarum LP15 strain capable of improving bone mineral density, and a preparation method and application thereof.
Background
As society is aged, osteoporosis patients are increasingly increased, the osteoporosis is deeply researched, the osteoporosis is taken as a metabolic bone disease and becomes an important cause for influencing the life quality of middle-aged and old people in China, the prevalence rate of the old people over 60 years reaches 36 percent, and the patients are easy to generate systemic metabolic bone disease due to the reduction of bone mass and the destruction of bone tissue microstructure; especially, senile humanized hormone is reduced, osteoclast is stimulated, osteoblast is inhibited, and bone mass is reduced. And with the increase of age, the nutrition absorption capacity is reduced, and the phenomena such as organ failure can occur. Secondary osteoporosis may also result from several common diseases such as endocrine disorders (hyperthyroidism, insulin dependent diabetes mellitus), digestive system disorders (hepatobiliary disorders, post-gastrectomy), connective tissue disorders (gout, rheumatoid arthritis), drugs (cyclosporin, methotrexate), etc.
Research shows that in the next decades, osteoporosis and the incidence of fracture caused by osteoporosis can rise year by year, so that the lactobacillus plantarum is provided, and a microbial preparation for improving bone density and enhancing bone mass is developed by using the lactobacillus plantarum, and the lactobacillus plantarum has very important significance.
Disclosure of Invention
In order to solve the technical problems, the invention provides a lactobacillus plantarum LP15 strain capable of improving bone mineral density, wherein the lactobacillus plantarum LP15 strain is preserved in the China general microbiological culture collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.28353, the preservation time is 2023, 9 months and 5 days, and the 16SrDNA sequence is shown as SEQ ID NO. 1; the sequences obtained by sequencing were aligned in NCBI and the result showed to be Lactobacillus plantarum, designated Lactobacillus plantarum LP15 strain.
Moreover, the lactobacillus plantarum LP15 strain is separated from weathered ox and sheep bones in the pasture of the Ubbelopsis in Erdos of the inner Mongolia.
Moreover, the morphological characteristics of the Lactobacillus plantarum LP15 strain are gram positive bacteria, and the strain is a rod-shaped bacteria with round ends, the width is 0.9-1.2 μm, the length is 3-8 μm, and the strain exists singly, in pairs or in short chains.
Moreover, the growth pH value of the lactobacillus plantarum LP15 strain is 3.4-8.8, and the growth temperature is 12-40 ℃; the viable count of Lactobacillus plantarum stored under cold storage at 4deg.C is still high, and can reach 1×10 6 CFU/ml or more.
On the other hand, the invention also provides an application of lactobacillus plantarum or lactobacillus plantarum-containing preparation prepared by fermenting lactobacillus plantarum LP15 strain capable of improving bone mineral density, which is used for improving bone mineral density and preventing osteoporosis.
Moreover, the viable count of the lactobacillus plantarum is not less than 1 multiplied by 10 8 CFU/ml or 1X 10 8 CFU/g。
In another aspect, the invention also provides an application of the lactobacillus plantarum LP15 strain capable of improving bone mineral density in foods, feeds or medicines.
In another aspect, the invention also provides a method for preparing the lactobacillus plantarum LP15 strain capable of improving bone mineral density, which comprises the following steps:
s1, taking 8-12g of weathered ox and sheep bones and soil samples at 5-15cm near the bones of a Mongolian Wu-trial hercynomorium grassland in Erdos, inner Mongolia, and filling the bones and the soil samples into a clean plastic bag to be used as a sample;
s2, preparing an MRS solid culture medium, adding calcium carbonate into the MRS solid culture medium, uniformly mixing the MRS solid culture medium and the calcium carbonate, and pouring the mixture into a flat plate; adding water into the sample to prepare suspension, smearing the suspension on a flat plate for culture, hydrolyzing calcium carbonate around bacterial colonies by using the lactobacillus plantarum LP15 strain to form clear transparent circles, identifying the lactobacillus plantarum LP15 strain, and primarily screening out the strain;
s3, carrying out streak separation and purification on the strain obtained by the primary screening, selecting a required strain according to the form and the size of a colony, carrying out fermentation culture on a single colony obtained by picking by utilizing an MRS culture medium, wherein the fermentation culture time is 7-24h, and preserving at-80 ℃ by using 50% glycerol;
wherein the formulation of the MRS medium comprises: 10.0g of casein peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 1.0mL of tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 15.0g of agar, 2.0g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 6.2+/-0.2 of final pH and 15min of sterilization at 115 ℃.
The beneficial effects of the invention are as follows:
1. the invention provides a lactobacillus plantarum LP15 strain capable of improving bone mineral density and a preparation method thereof, and the viable count of the lactobacillus plantarum LP15 strain prepared by the invention is not less than 1 multiplied by 10 8 CFU/ml or 1X 10 8 CFU/g, the application of the lactobacillus plantarum or lactobacillus plantarum-containing preparation can improve bone density of middle-aged and elderly people and prevent osteoporosis, and the lactobacillus plantarum or lactobacillus plantarum-containing preparation does not have adverse effect on individuals after long-term use and has strong universality.
2. The lactobacillus plantarum LP15 strain provided by the invention is salt-tolerant and can survive for a long time at low pH, and toxicity experiments prove that the strain is a nontoxic substance and can be used as a starter or starter component or as probiotics in dairy products and other food fermentation.
Drawings
Fig. 1: colony morphology of lactobacillus plantarum LP15 strain.
Fig. 2: glucose metabolism rate of lactobacillus plantarum and non-acid tolerant strains at low pH.
Fig. 3: effect of lactobacillus plantarum on GSH-Px activity in liver, spleen and serum of mice of different treatment groups.
Fig. 4: and (5) a strain phylogenetic tree.
Detailed Description
Example 1
The strain of lactobacillus plantarum LP15 capable of improving bone mineral density, wherein the strain of lactobacillus plantarum LP15 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.28353, the preservation time is 2023, 9 months and 5 days, and the 16SrDNA sequence is shown as SEQ ID NO. 1; the sequences obtained by sequencing were aligned in NCBI and the result showed to be Lactobacillus plantarum, designated Lactobacillus plantarum LP15 strain.
Moreover, the lactobacillus plantarum LP15 strain is separated from weathered ox and sheep bones in the pasture of the Ubbelopsis in Erdos of the inner Mongolia.
Moreover, the morphological characteristics of the Lactobacillus plantarum LP15 strain are gram positive bacteria, and the strain is a rod-shaped bacteria with round ends, the width is 0.9-1.2 μm, the length is 3-8 μm, and the strain exists singly, in pairs or in short chains.
Moreover, the growth pH value of the lactobacillus plantarum LP15 strain is 3.4-8.8, and the growth temperature is 12-40 ℃; the viable count of Lactobacillus plantarum stored under cold storage at 4deg.C is still high, and can reach 1×10 6 CFU/ml or more.
Moreover, the preparation method of the lactobacillus plantarum LP15 strain capable of improving bone mineral density comprises the following steps:
s1, taking 8-12g of weathered ox and sheep bones and soil samples at 5-15cm near the bones of a Mongolian Wu-trial hercynomorium grassland in Erdos, inner Mongolia, and filling the bones and the soil samples into a clean plastic bag to be used as a sample;
s2, preparing an MRS solid culture medium, adding calcium carbonate into the MRS solid culture medium, uniformly mixing the MRS solid culture medium and the calcium carbonate, and pouring the mixture into a flat plate; adding water into the sample to prepare suspension, smearing the suspension on a flat plate for culture, hydrolyzing calcium carbonate around bacterial colonies by using the lactobacillus plantarum LP15 strain to form clear transparent circles, identifying the lactobacillus plantarum LP15 strain, and primarily screening out the strain;
s3, carrying out streak separation and purification on the strain obtained by the primary screening, selecting a required strain according to the form and the size of a colony, carrying out fermentation culture on a single colony obtained by picking by utilizing an MRS culture medium, wherein the fermentation culture time is 7-24h, and preserving at-80 ℃ by using 50% glycerol;
wherein the formulation of the MRS medium comprises: 10.0g of casein peptone, 5.0g of beef extract powder, 4.0g of yeast extract powder, 20.0g of glucose, 1.0mL of tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 15.0g of agar, 2.0g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 6.2+/-0.2 of final pH and 15min of sterilization at 115 ℃.
The isolated bacteria were subjected to strain identification and phylogenetic studies using 16SrDNA having good clock properties and high conservation in structure and function.
The sequence of the 16SrDNA is shown as SEQ ID NO.1 after sequencing analysis.
And constructing a phylogenetic tree by adopting a adjacency method by adopting MEGA7.0 software, wherein the strain phylogenetic tree is shown in figure 4.
As can be seen from the phylogenetic tree of the strains in FIG. 4, all the sequences are clustered into two clusters, wherein the LP15 strain has extremely high similarity with the sequences of Lactiplantibacillus plantarum strain CIP 103151, lactiplantibacillus plantarum strain NBRC 15891, lactiplantibacillus plantarum strain NRRL B-14768, lactiplantibacillus plantarum strain JCM 1149 and the like, and no genetic distance exists.
Example 2
The strains obtained by screening in example 1 were subjected to the characterization of physiological and biochemical characteristics according to the API50CH carbohydrate identification kit, a bacterial suspension of SZD strains corresponding to 2 Maillard turbidity was prepared, the bacterial suspension was added to 50 biochemical tubes on the kit, and finally, the kit was incubated at 35℃with sterile liquid paraffin, and the results were observed for 1 time in 24, h and 48 h, respectively, the kit turned yellow to positive, turned green to weak positive, and not turned negative, and the results observed for 48 h were used as the final results, and the physicochemical test results are shown in Table 1 below:
TABLE 1 API50CH test strip experiment
result result result
control 0 - inositol 17 - D-melezitose 34 -
glycerol 1 - mannitol 18 - D-raffinose 35 -
erythritol 2 - sorbitol 19 - methyl-α-D-mannopyranoside 36 -
D-arabinose 3 - glycogene 20 - methyl-α-D-Glucopyranoside 37 -
L-arabinose 4 - xylitol 21 - methyl-β-D-xylopyranoside 38 -
D-ribose 5 - N-acetylglucosamine 22 - gentiobiose 39 -
D-xylose 6 - amygdaline 23 - D-turanose 40 -
L-xylose 7 - arbutine 24 - D-lyxose 41 -
D-adonitol 8 - esculine 25 - D-tagatose 42 -
starch 9 - salicine 26 - D-fucose 43 -
galactose 10 - D-cellobiose 27 - L-fucose 44 -
glucose 11 + D-maltose 28 - D-arabitol 45 -
fructose 12 - D-lactose 29 + L-arabitol 46 -
mannose 13 - D-melibiose 30 - potassium gluconate 47 -
sorbose 14 - D-saccharose 31 + potassium -cetogluconate 48 -
rhamnose 15 - D-trehalose 32 - potassium -cetogluconate 49 -
dulcitol 16 - inuline 33 -
The tested lactobacillus plantarum LP15 strain was tested for its ability to utilize carbon sources by performing a sugar fermentation reaction using an API50CH test strip according to API bacterial identification standards, and as a result, the strain LP15 strain was able to utilize glucose, sucrose and lactose.
Example 3
The lactobacillus plantarum LP15 strain is subjected to acid resistance and cholate resistance tests, and the specific steps are as follows:
acid resistance experiment: after the activated lactobacillus plantarum is cultured for 12-16 hours at 37 ℃ in MRS liquid culture medium, the thalli are collected by centrifugation at 4 ℃, the collected thalli are added into artificial gastric juice (pH 2.0) for culture, the result of 0 hour is taken as a control, 1 hour/2 hour/4 hour/8 hour is taken for living bacteria number analysis, the acid resistance of the strain is evaluated according to the survival rate (the colony number after treatment/0 hour colony number), each group of three bacteria are parallel, and the specific results are shown in table 2:
TABLE 2 survival rates of colonies at different times
Time（h) Colony Forming Units(CFU) Survival Rate
0 （3.57±0.24）×10 8 100%
1 （3.47±0.19）×10 8 97.20%
2 （3.26±0.20）×10 8 91.32%
4 （3.16±0.11）×10 8 88.52%
8 （2.98±0.42）×10 8 83.47%
The survival rate of the strain in artificial gastric juice is shown in table 2, the survival rate of the strain in the artificial gastric juice can reach 91.32% after 2 hours of treatment, the survival rate of the strain in the artificial gastric juice can reach 88.52% after 4 hours of treatment, and the survival rate of the strain in the artificial gastric juice can reach 83.47% after 8 hours of treatment, so that the strain has good acid resistance and can reach the stomach to play a probiotic role.
Bile salt resistance experiment: the activated lactobacillus plantarum is inoculated into MRS culture media containing sodium taurocholate with different concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) at an inoculation amount of 5%. Taking the 0h result as a control, taking 0h/2h/4h, carrying out viable bacteria count analysis, and evaluating the bile salt resistance of the strain according to the survival rate (the number of bacterial colonies after treatment/the number of bacterial colonies after 0 h), wherein each group is three in parallel, and the specific results are shown in Table 3:
TABLE 3 survival rates of bacterial Living in sodium taurocholate at different concentrations
The survival rate of the strain in bile salts with different concentrations is shown in Table 3, the concentration of the additive is in the range of 0.0% -1.0%, the growth of the strain is basically not influenced, when the additive reaches 1.5% -2.0%, the viable count is gradually reduced along with time, but the survival rate in 4 hours can still reach more than 88.4%, which indicates that the strain has good salt tolerance and can stably survive in intestinal tracts.
Based on the experimental result, preparing a pH2.0MRS liquid culture medium, additionally adding 1.5% sodium taurocholate and 1g/L glucose, and monitoring the change of glucose content, wherein the result is that lactobacillus plantarum can generate a metabolite by utilizing glucose under the condition of low pH, so that the pH in a bacterial suspension is increased, but the reaction cannot be realized by acid-resistant strains.
Example 4
Use of lactobacillus plantarum LP15 strain for improving bone health in mice.
1. Experimental animal
The study selected the most commonly used female mice of the Kunming species in China, 15 animals at 12 weeks of age, at which time the animals grew most.
2. Experimental method
After 7 days of adaptive feeding, the mice were randomly divided into a control group, a blank group and an experimental group, and 5 mice were in each group.
The attenuation and transmission speed of QUS quantitative ultrasound in bones are not only related to BMD, but also influenced by bone structures (including trabecular number, connection mode, trabecular interval, trend and the like), so that the QUS examination can know the changes in two aspects of bone quantity and quality, and the QUS measurement is the only current noninvasive and non-radiative diagnostic method capable of comprehensively reflecting bone quantity, bone structure and bone quality of bones.
The study examined mice with pre-established pQcT, and selected tomographic determinations at 1.8mm and 3.5mm of proximal femur epiphyseal line, as the former was predominantly cancellous bone, while the latter had relatively more cortical bone.
All experimental data are expressed as mean ± standard deviation, plotted using Excel calculation analysis, graphPad, and judged as significant differences as P < 0.05.
The gonadotropin releasing hormone leuprorelin produced by inhibiting sex hormone is injected into normal mice, obvious bone mass loss can occur, the bone density is reduced, and the effect of ovariectomy of the mice can be simulated.
3. Grouping and administration of animals
The control group was continuously injected with gonadotropin releasing hormone agonist (GnRH-a) for 4 weeks; blank group was not modeled, and normal saline was administered daily by lavage at 0.02mL/g at the same time; the experimental group was continuously injected with gonadotropin releasing hormone agonist (GnRH-a) for 4 weeks and was given Lactobacillus plantarum dietary supplements at 0.02mL/g per day by gavage.
4. Sample measurement
The femoral bone densities of the mice in each group are shown in tables 4 and 5:
table 4 pre-experiment mouse femoral bone density
Table 5 4 after week the femoral bone density of the mice
After the experiment is finished, the bone density is different, the experimental group is higher than the other two groups, and the experimental modeling is proved to be successful and the effect is obvious.
Example 5
The test method comprises the steps of carrying out subacute toxicity and oxidation resistance index detection experiments on mice, wherein the physiological state and metabolism of organism tissues of a tested animal are easy to change due to aging phenomenon, so that repeated contamination observation is needed on the tested animal, subacute toxicity refers to poisoning reaction caused by the fact that the tested animal is continuously contacted with a tested object for a long time and in a large dosage, the experiment period is generally about 1 month, and the index detection of the whole system is carried out on the tested animal after the experiment is finished, so that indexes of a dosage reaction relation are found, wherein main indexes comprise: appearance characteristics, behavioral activity status, weight changes, and the like. General clinical indicators include: blood routine, urine routine, blood biochemical indexes and the like, experimental animals can be dissected, and organ indexes, histopathological observations and the like of the experimental animals are calculated by weighing.
1. Experimental animal
SPF KM female mice (18-22 g) purchased from Si Bei Fu (Beijing) biotechnology Co., ltd, which were fed with food and water at 26+ -1deg.C without pathogen, were used in the present invention, and the light/dark cycle was 12h.
2. Experimental grouping and dosing.
20 SPF-grade KM female mice (18-22 g) were randomly divided into 4 groups: each group of 5. Of these, 3 groups were administered (gavage doses of 1, 5, 10g/kg+bw, respectively) and the remaining 1 group was blank (equal volume of distilled water was administered), mice were first fed adaptively, and then gavage administration was started.
The lactobacillus plantarum preparation is prepared into an aqueous solution with the concentration, and is administrated by gavage according to 0.1ml/10g+bw, and the mice are fed with food and water freely after gavage for 7 days continuously, and the states of the mice, including mental states, behavioral states, appearance changes, poisoning conditions, death conditions and the like, are observed and recorded every day during the gavage.
After 28d of feeding, the relevant organ indexes of the mice are calculated, the peripheral blood of the experimental mice is subjected to blood routine detection by using a full-automatic animal blood cell analyzer, the blood physiological indexes of the mice are quantitatively analyzed, a 0.5ml EP tube is prepared before blood taking, the group marks are reserved, and distilled water is used for preparing K2EDTA with the concentration of 400mg/ml as a blood anticoagulant.
After the gastric lavage experiment is finished, all mouse carcasses are dissected, main organ tissues of the mice are obtained, 75% alcohol is used for sterilization, and whether the color and the shape of main organs such as the liver, spleen, kidney and heart of the mice are abnormal or not is observed. Then, main organs such as the liver and the spleen of the mice are put into a plate containing normal saline to lightly wash off blood stains, the excessive water is lightly dipped with filter paper, and then the weights of the liver and the spleen of the mice are accurately weighed by using an analytical balance and stored in a sealed bag and stored in a refrigerator at the temperature of minus 80 ℃.
The organ index calculation formula is as follows: organ index = average weight of mouse liver or spleen/average weight of mice in each group.
Taking 0.1g of liver and spleen tissues of a mouse respectively, adding 1.0ml of precooled extracting solution for homogenization, putting into a centrifuge tube, centrifuging at 4 ℃ and 6000rpm for 10min, collecting supernatant for later use, taking a proper amount of fresh peripheral blood of the mouse into an EP tube, standing for 2h, centrifuging at 4 ℃ and 6000rpm in a low-temperature centrifuge for 10min, and taking supernatant for later use.
Taking the prepared liver tissue, spleen tissue homogenate supernatant and serum supernatant, and respectively detecting GSH-Px activity in the liver, spleen and serum by using a GSH-Px detection kit.
3. Sample measurement
Subacute toxicity tests are key steps in drug safety evaluation and drug entry from pharmacodynamics to clinical stages. The experimental period of 28 days can be used as a good basis for the next clinical experiment of subacute toxicity experiments.
Experiment sets I, II, III, IV, V and blank groups, the stomach-filling doses were 0.5 g/kg.bw, 1.0 g/kg.bw, 2.0 g/kg.bw, 5.0 g/kg.bw, 10.0 g/kg.bw and distilled water, and the mice related to subacute toxicity were observed and analyzed after continuous stomach filling for 28 days, and the specific results are shown in Table 6:
TABLE 6 clinical observations of sub-acute toxicity experiments in mice
After 28d subacute toxic lavage was completed, the mice were autopsy, the weights of liver, spleen, heart and thymus organs of each mouse were weighed, calculated according to the formula, and the wells were statistically analyzed, and as shown in table 7, the liver index of the 5 dose group was not linearly related to the corresponding lavage dose size by comparing the I-V group with each organ index of the blank group. The other organ indexes have no significant difference compared with the corresponding organ indexes of the blank group.
TABLE 7 mouse immune organ index results
Group of Dosage (g/kg bw) Liver (mg/g) Spleen (mg/g) Heart (mg/g) Thymus (mg/g)
Blank group 0 37.72±0.56 2.77±0.24 6.12±0.27 3.75±0.11
Group I 0.5 42.22±0.07 2.97±0.25 4.83±0.37 4.56±0.38
Group II 1 45.72±0.97 2.79±0.38 6.43±0.18 3.88±0.83
Group III 2.5 48.28±0.46 3.35±0.77 5.58±1.42 5.35±0.98
Group IV 5 49.07±0.86 3.30±0.25 6.77±0.32 4.88±0.89
V group 10 51.53±1.50 3.23±0.17 7.14±0.07 5.12±0.12
The results of the blood indexes of the mice are shown in Table 8, and the blood physiological indexes are not significantly different from the blood physiological indexes of the mice in the blank group and are all in the normal reference value range.
TABLE 8 results of blood routine index detection for mice in the same dose group
Blood index Number of red blood cells Hemoglobin concentration Platelet count
Unit (B) 10 9 /L 10 9 /L 10 9 /L
Blank group 8.62±0.46 132.66±10.24 623.12±10.26
Group I 9.64±0.11 134.46±16.38 633.88±6.83
Group II 10.22±0.06 142.96±4.24 646.83±9.36
Group III 14.62±0.96 144.69±10.38 662.43±11.18
Group IV 19.06±0.86 148.30±9.24 686.66±6.32
V group 21.43±1.40 143.23±8.16 714.14±10.06
The experimental results show that the conventional indexes of blood are not abnormal, and all mice in each dosage group have no anemia or the immune system is damaged, namely the bacteria have no toxicity to the physiological indexes of the blood of the mice.
The results of the biochemical indexes of the blood of the mice in different dosage groups are shown in the table 9, and each index of each dosage group has no obvious regular change trend compared with the blank group, is irrelevant to the size of the gastric lavage dosage of the strain, and is in a normal reference value range although individual numerical values in the table are remarkably different compared with the blank group, so that the strain has no adverse effect on the biochemical indexes of the blood of the mice.
TABLE 9 results of differential dose group mice leukocyte classification
White blood cell index Absolute value of neutrophils Absolute value of lymphocyte Absolute value of eosinophils Absolute value of basophils Percentage of monocytes
Unit (B) 10 9 /L 10 9 /L 10 9 /L 10 9 /L %
Blank group 2.32±0.57 82.66±1.24 0.12±0.26 6.42±0.56 42.77±1.24
Group I 3.35±0.22 84.46±0.38 0.44±0.43 6.55±0.11 44.56±1.38
Group II 3.52±0.03 86.96±0.24 0.63±0.36 6.62±0.05 42.97±0.25
Group III 3.72±0.93 94.69±0.38 0.73±0.14 6.62±0.95 45.79±0.38
Group IV 3.93±0.87 98.30±0.24 0.96±0.32 6.65±0.86 48.30±0.25
V group 4.53±2.50 103.23±0.16 1.14±10.06 6.83±1.50 53.23±0.17
After the gastric lavage experiment is finished, main organ tissues of the mice are obtained, compared with a blank group, the color, the size, the morphology and the like of livers and spleens of the mice are observed visually, no obvious abnormal results are shown in figure 3, in the liver tissues of the mice, the activity of GSH-Px is obviously improved compared with the blank group, and the activity of GSH-Px is integrally increased along with the increase of gastric lavage concentration, so that the strain can improve the activity of GSH-Px in livers. And compared with a blank group, the activity value of the spleen tissue GSH-Px of the mice has no significant difference. Indicating that it has no toxicity to the spleen of mice and no influence on antioxidant properties.
In summary, the invention obtains the lactobacillus plantarum LP15 strain capable of improving bone density through separation and purification, cultures and analyzes the lactobacillus plantarum LP15 strain, researches in many aspects from acid resistance and bile salt resistance tests, determination of femur bone density of mice to safe toxicity evaluation, proves that the lactobacillus plantarum LP15 strain is nontoxic substances, combines GSH-Px activity indexes in livers, spleens and serum of the mice, and simulates mice ovariectomy experiments, and further discovers that the femur density can be improved by taking a certain amount of lactobacillus plantarum, the activity of antioxidant enzymes of liver tissues and serum of the bodies of the mice can be improved, and the antioxidant capacity of the bodies of the mice can be improved.
The survival rate of the lactobacillus plantarum is still relatively high under the low pH condition, and when glucose exists, a carbon source can be utilized to improve the survival rate, so that favorable conditions are provided for the subsequent industrialized production of the lactobacillus plantarum.

Claims (10)

1. The lactobacillus plantarum LP15 strain capable of improving bone mineral density is characterized in that the lactobacillus plantarum LP15 strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.28353 and the preservation time of 2023, 9 months and 5 days, and the 16SrDNA sequence is shown as SEQ ID NO. 1.
2. The lactobacillus plantarum LP15 strain for increasing bone mineral density of claim 1, wherein the lactobacillus plantarum LP15 strain is isolated from weathered cattle and sheep bones in a pasture of a flag herd in erdos, an inner Mongolian autonomous region.
3. A lactobacillus plantarum LP15 strain with increased bone mineral density as claimed in claim 1, characterized in that the lactobacillus plantarum LP15 strain is characterized by gram-positive bacteria, rod-shaped bacteria with rounded ends, 0.9-1.2 μm wide and 3-8 μm long, singly, in pairs or in short chains.
4. The lactobacillus plantarum LP15 strain of claim 1, wherein the lactobacillus plantarum LP15 strain has a growth pH of 3.4-8.8 and a growth temperature of 12-40 ℃.
5. A method for preparing lactobacillus plantarum bacterial liquid by using the lactobacillus plantarum LP15 strain capable of improving bone mineral density as claimed in any one of claims 1-4, which is characterized by being used for improving bone mineral density and preventing osteoporosis.
6. The Lactobacillus plantarum strain LP15 strain with increased bone mineral density as recited in claim 5, wherein the viable count of Lactobacillus plantarum is no less than 1X 10 8 CFU/ml。
7. Preparation of a preparation containing Lactobacillus plantarum by using a Lactobacillus plantarum LP15 strain capable of increasing bone mineral density according to any of claims 1-4, characterized in that it is used for increasing bone mineral density and preventing osteoporosis.
8. A preparation comprising Lactobacillus plantarum LP15 strain for increasing bone mineral density as recited in claim 7, wherein the viable count of Lactobacillus plantarum is no less than 1X 10 8 CFU/g。
9. Use of a lactobacillus plantarum LP15 strain as claimed in any one of claims 1-4 to increase bone mineral density in food, feed or pharmaceutical products.
10. The method for preparing the lactobacillus plantarum LP15 strain capable of improving bone mineral density according to claim 1, which comprises the following steps:
s1, taking 8-12g of weathered ox and sheep bones and soil samples at 5-15cm near the bones of a Mongolian Wu-trial hercynomorium grassland in Erdos, inner Mongolia, and filling the bones and the soil samples into a clean plastic bag to be used as a sample;
s2, preparing an MRS solid culture medium, adding calcium carbonate into the MRS solid culture medium, uniformly mixing the MRS solid culture medium and the calcium carbonate, and pouring the mixture into a flat plate; adding water into the sample to prepare suspension, smearing the suspension on a flat plate for culture, hydrolyzing calcium carbonate around bacterial colonies by using the lactobacillus plantarum LP15 strain to form clear transparent circles, identifying the lactobacillus plantarum LP15 strain, and primarily screening out the strain;
s3, carrying out streak separation and purification on the strain obtained by the primary screening, selecting a required strain according to the form and the size of a colony, and carrying out fermentation culture on the single colony obtained by the selection by using an MRS culture medium, wherein the fermentation culture time is 7-24h, and the single colony is preserved at the temperature of minus 80 ℃ by using glycerin with the concentration of 50%.
CN202311367913.2A 2023-10-23 2023-10-23 Lactobacillus plantarum LP15 strain capable of improving bone mineral density and preparation method and application thereof Pending CN117586908A (en)

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