CN111718873B - Lactobacillus fermentum with effect of relieving osteoporosis and application thereof - Google Patents
Lactobacillus fermentum with effect of relieving osteoporosis and application thereof Download PDFInfo
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- CN111718873B CN111718873B CN202010565603.1A CN202010565603A CN111718873B CN 111718873 B CN111718873 B CN 111718873B CN 202010565603 A CN202010565603 A CN 202010565603A CN 111718873 B CN111718873 B CN 111718873B
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Abstract
The invention discloses lactobacillus fermentum with an effect of relieving osteoporosis and application thereof, and belongs to the technical field of microorganisms and medicines. The invention provides a lactobacillus fermentum CCFM1126, the lactobacillus fermentum CCFM1126 can prevent and/or treat osteoporosis, and is specifically represented by the following components: increasing uterine coefficients in ovariectomized rats; the bone density of the ovariectomized rat is obviously improved; the cortical volume of the ovariectomized rat is obviously improved; the content of tartaric acid phosphatase in the serum of the ovariectomized rat is obviously reduced; the content of the type I collagen cross-linked carboxyl terminal peptide in the serum of the ovariectomized rat is obviously reduced; the content of tumor necrosis factor-alpha in the serum of the ovariectomized rat is obviously reduced; reducing the content of interleukin-1 beta in the serum of an ovariectomized rat; obviously improves the content of estradiol in the serum of the ovariectomized rat.
Description
Technical Field
The invention relates to a lactobacillus fermentum strain with an effect of relieving osteoporosis and application thereof, and belongs to the technical field of microorganisms and medicines.
Background
Osteoporosis (osteoporotis) is a systemic skeletal disease characterized by a decrease in bone mass and a deterioration in the microstructure of the bone, leading to increased fragility of the bone. Osteoporosis is often diagnosed by several clinical symptoms, including: pain in the waist and back, pain in the limbs, deformity of the spine, fracture, etc.
According to the morbidity and the demographic data, the osteoporosis is one of the diseases with high morbidity, mortality and health care cost in the world at present. In our country alone, over 7000 million patients with osteoporosis are suffering from the disease, with about 80% of postmenopausal osteoporosis patients.
Compared with the normal population, the mortality rate of the osteoporosis patients with femoral neck or spine fracture is obviously increased within one year after the fracture occurs, in addition, the osteoporosis can cause pain and seriously reduce the life quality of the osteoporosis patients, in addition, the osteoporosis can cause the residue under the serious condition, the activity of the patients is limited, the life cannot be self-managed, and the heavy mental and economic burden is brought to individuals, families and society.
At present, the osteoporosis is usually treated by selecting western medicines with quick response and strong effect clinically, and the more common medicines comprise calcitonin, diphosphate, ipriflavone, fluoride, synthetic steroids and the like. The medicines can effectively promote the formation of osteoblasts and inhibit the formation of osteoclasts, thereby achieving the aim of treating osteoporosis. However, these drugs have their own metabolic characteristics and are suitable for people with poor universality (for example, zoledronic acid is only suitable for people with postmenopausal osteoporosis), some drugs have special adverse reactions after long-term use (for example, the selective estrogen receptor modulator raloxifene can cause venous thromboembolism and stroke), some drugs can cause dependence and tolerance of patients after long-term use, and various withdrawal symptoms can be caused once the drugs are stopped.
Therefore, a medicine is still needed, which can effectively relieve osteoporosis, does not cause adverse reaction, dependence and tolerance of patients after long-term use, and has strong universality and wide applicable population.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing a Lactobacillus fermentum which can relieve osteoporosis, does not cause adverse reaction, dependence and tolerance of patients after long-term use, and has strong universality and wide application range.
[ solution ]
In order to solve the problems, the invention provides a Lactobacillus fermentum (CCFM 1126), wherein the Lactobacillus fermentum CCFM1126 is preserved in Guangdong province microbial strain collection center with the preservation number of GDMCC No.61020 and the preservation date of 2020, 05 and 06 days.
The lactobacillus fermentum CCFM1126 is derived from fresh excrement samples of healthy people in Enshi areas of Hubei, the 16S rDNA sequence of the strain is shown as SEQ ID No.1 through sequencing analysis, the sequence obtained through sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is the lactobacillus fermentum and is named as the lactobacillus fermentum CCFM 1126.
The lactobacillus fermentum CCFM1126 has the morphological characteristics that: gram-positive bacteria; the microscopic examination is in a short rod shape, and the short rod shape is arranged in pairs without flagella and spores;
the colony characteristics of the lactobacillus fermentum CCFM1126 are as follows: round, white, smooth, no pigment production (colony features can be seen in fig. 1 in particular);
the fermented milkThe physiological and biochemical characteristics of bacillus CCFM1126 are as follows: arabinose, fructose, glucose, maltose, lactose, melibiose, ribose and sucrose can be utilized; esculin, salicin, raffinose, sorbitol, inositol, dulcitol, rhamnose, and galactose are not used; catalase, H 2 S production, urea decomposition, nitrate reduction, etc. were all negative.
The invention also provides application of the lactobacillus fermentum in preparing products for preventing and/or treating osteoporosis.
In one embodiment of the present invention, the viable count of the lactobacillus fermentum CCFM1126 in the product is not less than 1 × 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the product comprises a food or pharmaceutical product.
In one embodiment of the present invention, the pharmaceutical product contains lactobacillus fermentum CCFM1126, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipient comprises an excipient and/or an additive.
In one embodiment of the invention, the excipient comprises a colorant, an absorbent, a diluent, a flocculating agent and a deflocculating agent.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
In one embodiment of the present invention, the pharmaceutical composition is in the form of powder, granule, capsule, tablet, pill or oral liquid.
In one embodiment of the invention, the food is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leavening agent containing the lactobacillus fermentum CCFM 1126; or the food is a beverage or a snack containing the lactobacillus fermentum CCFM 1126.
In one embodiment of the invention, the preparation method of the fermentation agent comprises the steps of inoculating the lactobacillus fermentum CCFM1126 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 18h to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the thalli with normal saline for 3 times, and then resuspending the thalli with a freeze-drying protective agent to obtain a resuspension solution; and (4) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the leavening agent.
In one embodiment of the present invention, the mass ratio of the lyoprotectant to the bacterial cells is 2: 1.
In one embodiment of the invention, the lyoprotectant comprises 130g/L of skimmed milk powder.
In one embodiment of the present invention, the culture medium is MRS liquid culture medium.
In one embodiment of the invention, the pH of the medium is 6.8.
The invention also provides a product for preventing and/or treating osteoporosis, which contains the lactobacillus fermentum CCFM 1126.
In one embodiment of the present invention, the viable count of the lactobacillus fermentum CCFM1126 in the product is not less than 1 × 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the product comprises a food or pharmaceutical product.
In one embodiment of the present invention, the pharmaceutical product contains lactobacillus fermentum CCFM1126, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.
In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and/or an additive.
In one embodiment of the invention, the excipient comprises a colorant, an absorbent, a diluent, a flocculating agent and a deflocculating agent.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
In one embodiment of the present invention, the pharmaceutical composition is in the form of powder, granule, capsule, tablet, pill or oral liquid.
In one embodiment of the invention, the food is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leavening agent containing the lactobacillus fermentum CCFM 1126; or the food is a beverage or snack containing the lactobacillus fermentum CCFM 1126.
In one embodiment of the invention, the preparation method of the fermentation agent comprises the steps of inoculating the lactobacillus fermentum CCFM1126 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 18h to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the thalli with normal saline for 3 times, and then resuspending the thalli with a freeze-drying protective agent to obtain a resuspension solution; and (4) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the leavening agent.
In one embodiment of the present invention, the mass ratio of the lyoprotectant to the microbial cells is 2: 1.
In one embodiment of the invention, the lyoprotectant comprises 130g/L of skimmed milk powder.
In one embodiment of the present invention, the culture medium is MRS liquid culture medium.
In one embodiment of the invention, the pH of the medium is 6.8.
Has the advantages that:
1. the invention provides a Lactobacillus fermentum (CCFM 1126), the Lactobacillus fermentum CCFM1126 can prevent and/or treat osteoporosis, and the specific expression is as follows:
(1) increasing uterine coefficients in ovariectomized rats;
(2) the bone density of the ovariectomized rat is obviously improved;
(3) the cortical volume of the ovariectomized rat is obviously improved;
(4) the content of tartrate phosphatase (TRACP) in serum of the ovariectomized rat is obviously reduced;
(5) the content of the type I collagen cross-linked carboxyl terminal peptide (CTX-I) in the serum of the ovariectomized rat is obviously reduced;
(6) the content of tumor necrosis factor-alpha (TNF-alpha) in the serum of the ovariectomized rat is obviously reduced;
(7) reducing the content of interleukin-1 beta (IL-1 beta) in the serum of an ovariectomized rat;
(8) obviously improve the estradiol (E) in the serum of the ovariectomized rat 2 ) The content of (a) in (b),
therefore, the Lactobacillus fermentum (CCFM 1126) has great application prospect in preparing products (such as food or medicine and the like) for preventing and/or treating osteoporosis.
2. Lactobacillus fermentum is one kind of probiotics, and is included in the list of strains available for food issued by the ministry of health at present, so that the Lactobacillus fermentum CCFM1126 and the product with the Lactobacillus fermentum CCFM1126 as the active ingredient can not cause adverse reaction, dependence and tolerance of patients after long-term use, and meanwhile, the Lactobacillus fermentum has strong universality and wide applicable population.
Biological material preservation
A Lactobacillus fermentum (Lactobacillus fermentum) CCFM1126, which is classified and named Lactobacillus fermentum, is deposited in Guangdong province microorganism strain collection center in 2020 and 06 months, with the preservation number of GDMCC No.61020 and the preservation address of Guangzhou Mielizhou No. 100, large yard No. 59, building 5.
Drawings
FIG. 1: the colony characteristics of Lactobacillus fermentum (CCFM 1126).
FIG. 2 is a schematic diagram: uterine coefficients of ovariectomized rats of different groups.
FIG. 3: bone density in ovariectomized rats of different groups.
FIG. 4 is a schematic view of: cortical volumes of ovariectomized rats in different groups.
FIG. 5: the content of tartrate phosphatase (TRACP) in serum of ovariectomized rats of different groups.
FIG. 6: content of type I collagen cross-linked carboxyl terminal peptide (CTX-I) in serum of ovariectomized rats of different groups.
FIG. 7 is a schematic view of: the content of tumor necrosis factor-alpha (TNF-alpha) in the serum of different groups of ovariectomized rats.
FIG. 8: the content of interleukin-1 beta (IL-1 beta) in the serum of the ovariectomized rats of different groups.
FIG. 9: estradiol (E) in serum of ovariectomized rats of different groups 2 ) The content of (a).
In fig. 2-9, p <0.05, p <0.01, p <0.001, p < 0.0001.
Detailed Description
Animal model of postmenopausal osteoporosis was first established in 1969 by Saville in ovariectomized rats and was repeatedly later confirmed to have now become a classic animal model for studying postmenopausal osteoporosis, and therefore, in the following examples, ovariectomized rats were used to simulate osteoporotic rats.
The following examples relate to SPF grade SD female rats purchased from slaick laboratory animals ltd; the skim milk powder referred to in the following examples was purchased from nieuruiz food ltd; the following examples relate to sodium alendronate available from the European pharmaceuticals Ltd of Shijiazhuang Shiyao group.
The media involved in the following examples are as follows:
MRS solid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05 g/L, Tween 801 mL/L, agar 20g/L, cysteine hydrochloride 0.5g/L, and pH 6.8.
MRS liquid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05 g/L, Tween 801 mL/L, cysteine hydrochloride 0.5g/L, and pH 6.8.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
The method for preparing the suspension of lactobacillus fermentum referred to in the following examples is as follows:
streaking lactobacillus fermentum on MRS solid culture medium, and culturing at 37 deg.C for 48h to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min to obtain lactobacillus fermentum thallus; the lactobacillus fermentum thallus is washed by normal saline and then is suspended in skim milk powder solution with the concentration of 130g/L until the concentration of the lactobacillus fermentum thallus is 1 multiplied by 10 9 CFU/mL to obtain bacterial suspension, and storing the bacterial suspension at-80 ℃ for later use.
Example 1: acquisition of Lactobacillus fermentum
The method comprises the following specific steps:
taking fresh excrement of healthy people from the Enshi area in Hubei as a sample, sucking 0.5mL of the sample, adding the sample into 5mL of MRS liquid culture medium, culturing at 37 ℃ for 18-24 h, and enriching to obtain an enriched sample; 0.5mL of the enriched sample was aspirated and added to 4.5mL of sterile physiological saline to obtain 10 -1 The dilution was then pipetted 0.5mL 10 -1 The dilution was taken in 4.5mL of physiological saline to give 10 -2 The dilution was run in this order to give 10 -3 ，10 -4 ，10 -5 ，10 -6 Diluting the solution; draw 100. mu.L of the gradient diluent and spread on MRS solid medium, 10 -4 ，10 -5 ，10 -6 Culturing each gradient 1 plate at 37 ℃ for 48h to obtain bacterial colonies; selecting a bacterial colony with the typical characteristics of lactobacillus fermentum on an MRS solid culture medium according to the shape, the size, the edge, the transparency and the like of the bacterial colony, selecting the bacterial colony by using an inoculating loop, streaking the bacterial colony on the MRS solid culture medium, and culturing for 48h at 37 ℃ to obtain a purified single bacterial colony; selecting purified single colonies, respectively inoculating the single colonies into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 18-24 h to obtain a bacterial liquid; after numbering each strain corresponding to each bacterial solution, identification and gram staining of the strain were performed according to the procedures described in textbook "microbiology" (Shen Pink., Chengdong Shu Co., Ltd.)And physiological and biochemical experiments and the like, selecting strains with the typical characteristics of lactobacillus fermentum, and obtaining three strains through the experiments, wherein the three strains are named as CCFM1126, FZJTZ20 and L10 respectively;
wherein the strain identification process is as follows:
extracting genomes of CCFM1126, FZJTZ20 and L10, amplifying and sequencing 16S rDNA of CCFM1126, FZJTZ20 and L10 (finished by Shanghai Bioengineering Co., Ltd.), and comparing 16S rDNA sequences of CCFM1126, FZJTZ20 and L10 (the 16S rDNA sequence of CCFM1126 is shown as SEQ ID NO. 1) obtained by sequencing analysis in GenBank, wherein the three strains are Lactobacillus fermentum and are respectively named as Lactobacillus fermentum (Lactobacillus fermentum) CCFM1126, Lactobacillus fermentum (Lactobacillus fermentum) FZJTZ20 and Lactobacillus (Lactobacillus) L10;
the Lactobacillus fermentum (Lactobacillus fermentum) CCFM1126 has the morphological characteristics that: gram-positive bacteria; microscopic examination is in the shape of a short rod and arranged in pairs, and the examination has no flagellum and no spore;
the colony characteristics of Lactobacillus fermentum (CCFM 1126) are: round, white, smooth, no pigment production (colony features can be seen in fig. 1 in particular);
the physiological and biochemical characteristics of Lactobacillus fermentum (CCFM 1126) are as follows: arabinose, fructose, glucose, maltose, lactose, melibiose, ribose and sucrose can be utilized; without using aesculin, salicin, raffinose, sorbitol, inositol, dulcitol, rhamnose, galactose; catalase, H 2 S production, urea decomposition, nitrate reduction, etc. were all negative.
Example 2: effect of Lactobacillus fermentum on uterine coefficient of ovariectomized rats
The method comprises the following specific steps:
30 SPF-grade SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups respectively comprise: a sham operation group, a model control group, a positive control group of a perigastric sodium alendronate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 bacterial suspension, a FZJTZ20 group of a lactobacillus gasseri FZJTZ20 bacterial suspension and a L10 group of a lactobacillus gasseri L10 bacterial suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and 12h alternating day and night, the used feed is the rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology limited, and the experiment is carried out after the ordinary diet is adapted for one week.
After the first week, rats in the model control group, the positive control group, the CCFM1126 group, the FZJTZ20 group and the L10 group are anesthetized by intraperitoneal injection of 10% (v/v) chloral hydrate according to the dose of 3.3mL/kg, the abdomen is disinfected conventionally, the abdominal cavity is opened by a median abdominal incision, both ovaries are cut off, and the incision is sutured in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dose of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into rats of each group for anti-infection according to the dosage of 20000U/100g for 3 days continuously after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage the alendronate sodium solution at the dose of 1 mg/(kg. d) for the rats in the positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in the skim milk powder solution with the concentration of 130 g/L), gavage the bacteria suspension at the dose of 1.5 mL/day for the rats in the CCFM1126 group, the FZJTZ20 group and the L10 group, and gavage the skim milk powder solution with the concentration of 130g/L for the sham operation group and the model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
Killing all rats after the gavage is finished, measuring the wet weight of the uterus, calculating the uterus index of the rats, and obtaining a detection result shown in figure 2; wherein uterine coefficient (%) ═ uterine mass/rat body weight.
As can be seen from FIG. 2, compared with the sham operation group, the uterine coefficient of the model control group rat is significantly reduced, and it can be seen that after the removal of both ovaries, the rat is lack of estrogen and the uterus is significantly atrophied; after the lactobacillus fermentum is perfused, the uterine coefficient of the rat is improved compared with that of the rat of the model control group; the ability of the Lactobacillus fermentum FZJTZ20 and L10 to increase the uterine modulus of ovariectomized rats is significantly inferior to that of the Lactobacillus fermentum CCFM 1126.
Example 3: effect of Lactobacillus fermentum on bone Density and cortical volume in ovariectomized rats
The method comprises the following specific steps:
30 SPF-grade SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups respectively comprise: a sham operation group, a model control group, a positive control group of a perigastric alene sodium phosphate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 suspension, a lactobacillus gasseri FZJTZ20 group of a lactobacillus gasseri FZJTZ20 suspension, and a lactobacillus gasseri L10 group of a lactobacillus gasseri L10 suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and 12h alternating day and night, the used feed is the rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology limited, and the experiment is carried out after the ordinary diet is adapted for one week.
After the first week, injecting 10% (v/v) chloral hydrate into abdominal cavity of rats of model control group, positive control group, CCFM1126 group, FZJTZ20 group and L10 group according to the dose of 3.3mL/kg for anesthesia, sterilizing the abdomen, opening abdominal cavity through abdominal center incision, cutting bilateral ovaries, and suturing the incision in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dose of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into each group of rats to resist infection according to 20000U/100g dosage for 3 days after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage alendronate sodium solution at the dose of 1 mg/(kg. d) for rats in a positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in skim milk powder solution with the concentration of 130 g/L), gavage the suspension of bacteria at the dose of 1.5 mL/day for rats in a CCFM1126 group, an FZJTZ20 group and an L10 group, and gavage skim milk powder solution with the concentration of 130g/L for rats in a sham operation group and a model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
All rats were sacrificed after the completion of gavage, and left femurs of all groups of rats were uniformly sawed from the midpoint of the major diameterThe distal end of the femur was placed on a micro CT as required, and the bone density (BMD, g/cm) of the metaphysis of the femur was measured about 4mm from the intercondylar notch 3 ) Cortex Lycii volume (ct.v, mm) 3 ) The detection results are shown in FIGS. 3-4.
As can be seen from FIGS. 3 to 4, compared with the sham-operated group, the bone density and the cortical volume of the rats in the model control group were significantly reduced from 2.33. + -. 0.06g/cm 3 And 23.92. + -. 3.56mm 3 The reduction is 2.10 +/-0.03 g/cm 3 And 16.15. + -. 2.21mm 3 (ii) a The bone density and the cortical volume of the rats in the CCFM1126 group are obviously increased compared with the rats in the model control group, and are respectively 2.33 +/-0.06 g/cm 3 And 23.92. + -. 3.56mm 3 The rising is 2.16 +/-0.03 g/cm 3 And 25.80. + -. 3.49mm 3 While bone density and cortical volume were not significantly different in the FZJTZ20 and L10 groups compared to the model control group.
It can be seen that lactobacillus fermentum CCFM1126 can significantly increase bone density and cortical volume in ovariectomized rats.
Example 4: effect of Lactobacillus fermentum on the content of tartrate phosphatase (TRACP) in serum of ovariectomized rats
The method comprises the following specific steps:
30 SPF SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups comprise: a sham operation group, a model control group, a positive control group of a perigastric alene sodium phosphate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 suspension, a lactobacillus gasseri FZJTZ20 group of a lactobacillus gasseri FZJTZ20 suspension, and a lactobacillus gasseri L10 group of a lactobacillus gasseri L10 suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and the day and night alternation of 12h for free diet, the used feed is rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology Limited, and the experiment is carried out after the ordinary diet adapts for one week.
After the first week, injecting 10% (v/v) chloral hydrate into abdominal cavity of rats of model control group, positive control group, CCFM1126 group, FZJTZ20 group and L10 group according to the dose of 3.3mL/kg for anesthesia, sterilizing the abdomen, opening abdominal cavity through abdominal center incision, cutting bilateral ovaries, and suturing the incision in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dose of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into each group of rats to resist infection according to 20000U/100g dosage for 3 days after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage alendronate sodium solution at the dose of 1 mg/(kg. d) for rats in a positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in skim milk powder solution with the concentration of 130 g/L), gavage the suspension of bacteria at the dose of 1.5 mL/day for rats in a CCFM1126 group, an FZJTZ20 group and an L10 group, and gavage skim milk powder solution with the concentration of 130g/L for rats in a sham operation group and a model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
After the completion of the gavage, all rats were sacrificed, blood was taken and the concentration of tartrate TRACP in the serum of each group of rats was measured by ELISA kit, and the results of the measurement are shown in FIG. 5.
As shown in FIG. 5, the content of TRACP in rat serum was increased from 7.20. + -. 0.72ng/mL to 9.13. + -. 0.75ng/mL by ovariectomy, whereas the content of TRACP in rat serum was reduced to 4.11. + -. 1.37ng/mL by the treatment with Lactobacillus fermentum CCFM1126, at a level close to that of the sham group; the ability of the L.fermentum FZJTZ20 and L10 to modulate serum tartrate TRACP is significantly inferior to that of L.fermentum CCFM 1126.
As can be seen, Lactobacillus fermentum CCFM1126 can significantly reduce the content of tartrate TRACP in the serum of ovariectomized rats.
Example 5: effect of Lactobacillus fermentum on the content of type I collagen-crosslinked carboxy-terminal peptide (CTX-I) in serum of ovariectomized rats
The method comprises the following specific steps:
30 SPF-grade SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups respectively comprise: a sham operation group, a model control group, a positive control group of a perigastric alene sodium phosphate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 suspension, a lactobacillus gasseri FZJTZ20 group of a lactobacillus gasseri FZJTZ20 suspension, and a lactobacillus gasseri L10 group of a lactobacillus gasseri L10 suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and the day and night alternation of 12h for free diet, the used feed is rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology Limited, and the experiment is carried out after the ordinary diet adapts for one week.
After the first week, injecting 10% (v/v) chloral hydrate into abdominal cavity of rats of model control group, positive control group, CCFM1126 group, FZJTZ20 group and L10 group according to the dose of 3.3mL/kg for anesthesia, sterilizing the abdomen, opening abdominal cavity through abdominal center incision, cutting bilateral ovaries, and suturing the incision in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dose of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into rats of each group for anti-infection according to the dosage of 20000U/100g for 3 days continuously after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage alendronate sodium solution at the dose of 1 mg/(kg. d) for rats in a positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in skim milk powder solution with the concentration of 130 g/L), gavage the suspension of bacteria at the dose of 1.5 mL/day for rats in a CCFM1126 group, an FZJTZ20 group and an L10 group, and gavage skim milk powder solution with the concentration of 130g/L for rats in a sham operation group and a model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
After the completion of the gavage, all rats were sacrificed, blood was collected and the concentration of type I collagen-crosslinked carboxy-terminal peptide (CTX-I) in the serum of each group of rats was measured by ELISA kit, and the results of the measurement are shown in FIG. 6.
As can be seen from FIG. 6, the content of CTX-I in rat serum was significantly reduced by the treatment of Lactobacillus fermentum CCFM 1126; the ability of Lactobacillus fermentum FZJTZ20 and L10 to modulate collagen I cross-linking carboxyl-terminal peptides (CTX-I) in serum was significantly inferior to that of Lactobacillus fermentum CCFM 1126.
As can be seen, Lactobacillus fermentum CCFM1126 can significantly reduce the content of type I collagen-crosslinked carboxy-terminal peptide (CTX-I) in serum of ovariectomized rats.
Example 6: effect of Lactobacillus fermentum on the content of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in serum of ovariectomized rat
The method comprises the following specific steps:
30 SPF-grade SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups respectively comprise: a sham operation group, a model control group, a positive control group of a perigastric alene sodium phosphate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 suspension, a lactobacillus gasseri FZJTZ20 group of a lactobacillus gasseri FZJTZ20 suspension, and a lactobacillus gasseri L10 group of a lactobacillus gasseri L10 suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and 12h alternating day and night, the used feed is the rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology limited, and the experiment is carried out after the ordinary diet is adapted for one week.
After the first week, injecting 10% (v/v) chloral hydrate into abdominal cavity of rats of model control group, positive control group, CCFM1126 group, FZJTZ20 group and L10 group according to the dose of 3.3mL/kg for anesthesia, sterilizing the abdomen, opening abdominal cavity through abdominal center incision, cutting bilateral ovaries, and suturing the incision in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dosage of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into each group of rats to resist infection according to 20000U/100g dosage for 3 days after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage the alendronate sodium solution at the dose of 1 mg/(kg. d) for the rats in the positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in the skim milk powder solution with the concentration of 130 g/L), gavage the bacteria suspension at the dose of 1.5 mL/day for the rats in the CCFM1126 group, the FZJTZ20 group and the L10 group, and gavage the skim milk powder solution with the concentration of 130g/L for the sham operation group and the model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
After the gavage is finished, all rats are sacrificed, blood is taken and the concentration of tumor necrosis factors TNF-alpha and interleukin-1 beta (IL-1 beta) in the serum of each group of rats is measured by an ELISA kit (product of Nanjing Senega Biotechnology Co., Ltd.), and the detection result is shown in figures 7-8.
As can be seen from FIG. 7, the ovariectomy operation increased the TNF- α content in rat serum from 17.99. + -. 1.71pg/mL to 20.91. + -. 1.84pg/mL, while the TNF- α content in rat serum was reduced to 17.30. + -. 0.67pg/mL by the treatment of Lactobacillus fermentum CCFM1126, which is close to the sham operation group; the ability of the Lactobacillus fermentum FZJTZ20 and L10 to modulate TNF- α in serum is significantly inferior to that of Lactobacillus fermentum CCFM 1126.
As can be seen, the content of tumor necrosis factor TNF-alpha in the serum of the ovariectomized rat can be obviously reduced by the lactobacillus fermentum CCFM 1126.
As can be seen from FIG. 8, the content of interleukin-1 β (IL-1 β) in rat serum was increased from 23.80. + -. 3.18pg/mL to 31.46. + -. 2.87pg/mL by ovariectomy, whereas the treatment with Lactobacillus fermentum CCFM1126 had no significant effect on the content of interleukin-1 β in rat serum.
Example 7: lactobacillus fermentum for estradiol (E) in ovariectomized rat serum 2 ) Influence of the amount
The method comprises the following specific steps:
30 SPF-grade SD female rats weighing 250 +/-20 g are randomly divided into 6 groups, wherein each group comprises 5, and the 6 groups respectively comprise: a sham operation group, a model control group, a positive control group of a perigastric sodium alendronate solution, a CCFM1126 group of a lactobacillus gasseri CCFM1126 bacterial suspension, a FZJTZ20 group of a lactobacillus gasseri FZJTZ20 bacterial suspension and a L10 group of a lactobacillus gasseri L10 bacterial suspension.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and 12h alternating day and night, the used feed is the rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology limited, and the experiment is carried out after the ordinary diet is adapted for one week.
After the first week, rats in the model control group, the positive control group, the CCFM1126 group, the FZJTZ20 group and the L10 group are anesthetized by intraperitoneal injection of 10% (v/v) chloral hydrate according to the dose of 3.3mL/kg, the abdomen is disinfected conventionally, the abdominal cavity is opened by a median abdominal incision, both ovaries are cut off, and the incision is sutured in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dosage of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into each group of rats to resist infection according to 20000U/100g dosage for 3 days after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage alendronate sodium solution at the dose of 1 mg/(kg. d) for rats in a positive control group (the alendronate sodium solution is obtained by dissolving alendronate sodium in skim milk powder solution with the concentration of 130 g/L), gavage the suspension of bacteria at the dose of 1.5 mL/day for rats in a CCFM1126 group, an FZJTZ20 group and an L10 group, and gavage skim milk powder solution with the concentration of 130g/L for rats in a sham operation group and a model control group at the dose of 1.5 mL/day; gavage for 4 weeks.
After completion of gavage, all rats were sacrificed, and blood was collected and measured for estradiol concentration in serum of each group of rats by using an ELISA kit (product of Wuhan Eleret Biotech Co., Ltd.), and the measurement results are shown in FIG. 9.
As can be seen from FIG. 9, the content of estradiol in the serum of the rats in the sham operation group is 117.90 + -4.92 pg/mL, the content of estradiol in the serum of the rats in the model control group is significantly reduced to 70.40 + -7.55 pg/mL, the content of estradiol in the serum of the rats in the CCFM1126 group is significantly increased to 88.38 + -6.46 pg/mL, and the content of estradiol in the serum of the rats in the FZJTZ20 group and the L10 group is not significantly different from that of the rats in the sham operation group.
Therefore, the content of the estradiol in the serum of the ovariectomized rat can be obviously improved by the lactobacillus fermentum CCFM 1126.
Example 8: application of lactobacillus fermentum
The lactobacillus fermentum CCFM1126 can be used for preparing bacterial powder, and the bacterial powder is prepared by the following specific steps:
lactobacillus fermentum CCFM1126 streaked on MRS solid medium, cultured at 37 deg.C for 48h, obtaining a single bacterial colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min to obtain bacterial mud; washing the bacterial mud with normal saline for 3 times, and resuspending the bacterial mud with protective agent to a concentration of 1 × 10 10 CFU/mL to obtain bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus fermentum CCFM1126 bacterial powder;
the protective agent is a skim milk powder solution with the concentration of 130 g/L.
Example 9: application of lactobacillus fermentum
The lactobacillus fermentum CCFM1126 can be used for preparing milk drinks, and the specific preparation process of the milk drinks is as follows:
the lactobacillus fermentum CCFM1126 is streaked on an MRS solid culture medium and cultured for 48h at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min to obtain bacterial mud; washing the bacterial mud with normal saline for 3 times, and resuspending the bacterial mud with protective agent to a concentration of 1 × 10 10 CFU/mL to obtain a bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus fermentum CCFM1126 bacterial powder;
wherein the protective agent is skim milk powder solution with the concentration of 130 g/L.
Sterilizing skimmed milk at 95 deg.C for 20min, and cooling to 4 deg.C to obtain raw material; adding lactobacillus fermentum CCFM1126 bacteria powder to the raw material to make the concentration not less than 1 × 10 6 CFU/mL to obtain milk beverage (the milk beverage needs to be stored at 4 ℃ for refrigeration).
Example 10: application of lactobacillus fermentum
The lactobacillus fermentum CCFM1126 can be used for preparing fruit and vegetable beverages, and the fruit and vegetable beverages are prepared by the following specific preparation process:
lactobacillus fermentum CCFM1126 streaking onCulturing for 48h on MRS solid culture medium at 37 ℃ to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min to obtain bacterial mud; washing the bacterial mud with normal saline for 3 times, and resuspending the bacterial mud with protective agent to a concentration of 1 × 10 10 CFU/mL to obtain bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus fermentum CCFM1126 bacteria powder;
wherein the protective agent is skim milk powder solution with the concentration of 130 g/L.
Cleaning fresh fruits and vegetables, and squeezing to obtain fruit and vegetable juice; thermally sterilizing the fruit and vegetable juice at 140 deg.C for 2 s to obtain sterilized fruit and vegetable juice; cooling the sterilized fruit and vegetable juice to about 37 deg.C, adding lactobacillus fermentum CCFM1126 bacteria powder to the sterilized fruit and vegetable juice to reach concentration of not less than 1 × 10 6 CFU/mL to obtain fruit and vegetable beverage (the fruit and vegetable beverage needs to be refrigerated at 4 ℃).
Example 11: application of lactobacillus fermentum
The lactobacillus fermentum CCFM1126 can be used for preparing capsule products, and the specific preparation process of the capsule products is as follows
The lactobacillus fermentum CCFM1126 is streaked on an MRS solid culture medium and cultured for 48h at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min to obtain bacterial sludge; washing the bacterial sludge with normal saline for 3 times, and suspending with protectant to 1 × 10 10 CFU/mL to obtain a bacterial suspension; adding the bacterial suspension into a sodium alginate solution with the concentration of 30g/L to reach the concentration of 2 x 10 9 After CFU/mL, fully stirring to uniformly disperse cells of the lactobacillus fermentum CCFM1126 in the sodium alginate solution to obtain a mixed solution; squeezing the mixed solution into 20g/L calcium chloride solution to formColloidal particles; standing and solidifying the formed colloidal particles for 30min, and filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; and filling the powder into a medicinal capsule to obtain a capsule product.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> lactobacillus fermentum with effect of relieving osteoporosis and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 953
<212> DNA
<213> Lactobacillus fermentum
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tacctggtct gcaactgacg ctgagactcg aaagcatggg tagcgaacag gattagatac 780
cctggtagtc catgccgtaa acgatgagtg ctaggtgttg gagggtttcc gcccttcagt 840
gccggagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa 900
aggaattgac gggggcccgc acaagcggtg gagctgtggt ttaattcgaa gct 953
Claims (9)
1. A strain of lactobacillus fermentum (Lactobacillus fermentatum) The lactobacillus fermentum is preserved in Guangdong provincial microorganism culture collection center with the preservation number of GDMCC No.61020 and the preservation date of 2020, 05 and 06 days.
2. Use of lactobacillus fermentum according to claim 1 for the preparation of a medicament for the prevention and/or treatment of osteoporosis due to menopause.
3. A product comprising the Lactobacillus fermentum of claim 1, wherein the product is a pharmaceutical product.
4. The product according to claim 3, wherein the viable count of the Lactobacillus fermentum according to claim 1 is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
5. A product according to claim 4, wherein the product comprises Lactobacillus fermentum according to claim 1, a pharmaceutical carrier and/or a pharmaceutical excipient.
6. A product according to claim 5, wherein the drug carrier is a microcapsule, microsphere, nanoparticle and/or liposome.
7. A product according to claim 6, wherein the pharmaceutical excipient is an excipient and/or an additive.
8. A product according to claim 7, wherein the excipients are colorants, absorbents, diluents, flocculants, and deflocculants; the additive is microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
9. A product as claimed in any one of claims 5 to 8, wherein the pharmaceutical product is in the form of a powder, granules, capsules, tablets, pills or oral liquid.
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| WO2020080736A1 (en) * | 2018-10-19 | 2020-04-23 | Clinical Nutrition Intl (M) Sdn Bhd | Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain |
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| KR101252639B1 (en) * | 2010-06-23 | 2013-04-09 | 한국 한의학 연구원 | Composition for Prevention or Treatment of Osteoporosis Comprising Ssanghwatang and Fermentation Product Thereof with Lactic Acid Bacteria |
| WO2018030838A1 (en) * | 2016-08-12 | 2018-02-15 | 한국식품연구원 | Composition comprising lactobacillus intestinalis for prevention or treatment of climacteric symptom |
| TWI604052B (en) * | 2017-02-20 | 2017-11-01 | 景岳生物科技股份有限公司 | Lactobacillus plantarum strain gmnl-662 and composition having the same for promoting bone regrowth |
| KR102120479B1 (en) * | 2018-10-30 | 2020-06-09 | 주식회사 종근당바이오 | Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics |
| KR20200049999A (en) * | 2018-10-30 | 2020-05-11 | 주식회사 종근당바이오 | Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics |
| GB201905386D0 (en) * | 2019-04-16 | 2019-05-29 | Probi Ab | Probiotic compositions and uses thereof |
| CN111066991A (en) * | 2019-12-31 | 2020-04-28 | 扬州江大食生一生食品有限公司 | Preparation method of composite prebiotics solid beverage for improving gastrointestinal tract function |
| CN111718903A (en) * | 2020-06-24 | 2020-09-29 | 江南大学 | Method for screening medicine capable of preventing and/or treating osteoporosis |
| CN113151070B (en) * | 2021-04-02 | 2022-09-06 | 江南大学 | Lactobacillus fermentum capable of improving relative abundance of Guttiferae in intestinal tract |
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