A strain of Lactobacillus gasseri that reduces the risk of osteoporosis caused by low estrogen levels and its application.
By regulating the gut microbiota and increasing estrogen levels through Lactobacillus gasseri CCFM1255, the problem of osteoporosis caused by low estrogen levels has been solved, achieving safe and effective prevention and treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2022-09-16
- Publication Date
- 2026-05-26
Smart Images
Figure CN115584331B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of Lactobacillus gasseri that reduces the risk of osteoporosis caused by low estrogen levels and its applications, belonging to the field of microbial technology. Background Technology
[0002] Osteoporosis is a disease affecting bone integrity and strength, primarily affecting women 10-15 years postmenopause. Approximately 50% of women over 50 will experience an osteoporosis-related fracture in the remainder of their lives. The incidence of fractures increases exponentially with age. With an aging population, osteoporosis in menopausal women has become an emerging global public health concern. Estrogen deficiency is associated with increased bone turnover, resulting in bone resorption exceeding bone formation and thus bone loss. Estrogen plays a positive role in normalizing the bone turnover index and preventing bone loss in women. Estrogen or estrogen receptor modulator supplementation is considered one of the most effective treatments for osteoporosis in menopausal women.
[0003] In recent years, numerous studies have confirmed that probiotics can improve human health by regulating the gut microbiota. Similarly, probiotics have a positive impact on bone metabolism and bone density by regulating the gut microbiota structure. Probiotics mainly exert their potential beneficial effects on bone metabolism by increasing the production of short-chain fatty acids in the gut, increasing mineral solubility, reducing intestinal inflammation, and increasing bone density; or by increasing mineral bioavailability. With the deepening of research on probiotics, this invention has discovered that in cases of insufficient ovarian sex hormone secretion, probiotics can increase the production of estrogen in fat, thereby increasing the level of estrogen in the body. Therefore, moderately increasing the level of circulating estrogen through lactic acid bacteria may be a new approach to treat and prevent osteoporosis in menopausal women. Summary of the Invention
[0004] The first objective of this invention is to provide Lactobacillus gasseri CCFM1255, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 21, 2022, with accession number GDMCC No: 62305.
[0005] The Lactobacillus gasseri CCFM1255 has the following characteristics:
[0006] (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria;
[0007] (2) Colony characteristics: grayish-white, round, glossy, with slightly undulating and rough edges;
[0008] (3) Growth characteristics: Under constant temperature anaerobic conditions at 37℃, the growth reaches the end-log phase after about 12 hours of culture in MRS medium;
[0009] (4) It has a strong tolerance to simulated gastrointestinal fluid.
[0010] The present invention also provides a composition containing the Lactobacillus gasseri CCFM1255.
[0011] In one embodiment, the number of Lactobacillus gasseri CCFM1255 is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g.
[0012] In one embodiment, the number of Lactobacillus gasseri CCFM1255 is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.
[0013] In one embodiment, the composition includes, but is not limited to, microbial preparations, functional foods, health products, or pharmaceuticals.
[0014] In one embodiment, the composition contains a live strain, a dry strain, a strain metabolite, or an inactivated strain of Lactobacillus gasseri CCFM1255.
[0015] The present invention also provides the use of the Lactobacillus gasseri CCFM1255 in the preparation of a medicament for preventing osteoporosis, reducing the risk of osteoporosis and / or alleviating osteoporosis; wherein the osteoporosis is caused by abnormal estrogen levels; the abnormal estrogen levels include, but are not limited to, abnormal estrogen levels during menopause or postmenopause.
[0016] In one implementation, the application includes, but is not limited to, at least one of the following functions:
[0017] (1) Increase serum estradiol levels in mammals with abnormal estrogen levels;
[0018] (2) Increase blood calcium levels in mammals with abnormal estrogen levels;
[0019] (3) Reduce serum procollagen type I N-terminal propeptide (PINP) levels in mammals with abnormal estrogen levels.
[0020] (4) Reduce osteoprotegerin (OPG) levels in mammals with abnormal estrogen levels;
[0021] (5) Reduces serum IL-10, TNF-α and IL-6 levels in mammals with abnormal estrogen levels;
[0022] (6) Increase the expression levels of CYP19 and estrogen receptor ESR1 in the adipose tissue of mammals with abnormal estrogen levels.
[0023] In one implementation, the mammal includes, but is not limited to, humans.
[0024] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier.
[0025] In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of the following: fillers, wetting agents, disintegrants, binders, or lubricants.
[0026] In one embodiment, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, croscarmellose sodium, croscarmellose, or low-substituted hydroxypropyl cellulose; the binder is one or more of starch paste, syrup, maltose, refined molasses, or liquid glucose; and the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talc, or silica.
[0027] This invention also claims protection for the use of the Lactobacillus gasseri CCFM1255 in the preparation of fermented foods.
[0028] In one embodiment, the application includes, but is not limited to, using Lactobacillus gasseri CCFM1255 as a fermentation microorganism to ferment food raw materials.
[0029] In one embodiment, the fermented food includes dairy products, soy products, and fruit and vegetable products; the dairy products include milk, sour cream, and cheese; and the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.
[0030] This invention also claims protection for fermented products prepared from the Lactobacillus gasseri CCFM1255; the fermented products include fermented foods, fermented milk, fermented fruit and vegetable beverages, etc.
[0031] Beneficial Effects: *Lactobacillus gasseri* CCFM1255 can increase serum estradiol levels in ovariectomized rats; it also increases serum calcium levels and decreases serum procollagen type I N-terminal propeptide (PINP) and osteoprotegerin (OPG) levels, thus slowing bone turnover. *Lactobacillus gasseri* CCFM1255 can significantly reduce serum IL-10, TNF-α, and IL-6 levels in ovariectomized rats. *Lactobacillus gasseri* CCFM1255 can increase the expression of CYP19 and estrogen receptor ESR1 in the adipose tissue of ovariectomized rats. Therefore, *Lactobacillus gasseri* CCFM1255 has broad application prospects in preventing osteoporosis, reducing osteoporosis risk, and / or alleviating osteoporosis caused by low estrogen levels.
[0032] Preservation of biological materials
[0033] Lactobacillus gasseri (CCFM1255), classified as Lactobacillus gasseri, was deposited on March 21, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 62305. Attached Figure Description
[0034] Figure 1 This describes the colony morphology of Lactobacillus gasseri CCFM1255.
[0035] Figure 2 The effect of Lactobacillus gasseri CCFM1255 on serum estradiol in ovariectomized rats;
[0036] Figure 3 The effect of Lactobacillus gasseri CCFM1255 on serum calcium levels in ovariectomized rats;
[0037] Figure 4 The effect of Lactobacillus gasseri CCFM1255 on serum N-terminal propeptide (PINP) levels of type I procollagen in ovariectomized rats;
[0038] Figure 5 The level of osteoprotegerin (OPG) in ovariectomized rats was measured by Lactobacillus gasseri CCFM1255.
[0039] Figure 6 The effect of Lactobacillus gasseri CCFM1255 on the production of IL-10, TNF-α, and IL-6 in ovariectomized rats;
[0040] Figure 7 The effect of Lactobacillus gasseri CCFM1255 on the expression of CYP19 and estrogen receptor ESR1 in the adipose tissue of ovariectomized rats;
[0041] Compared with the ovariectomized model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation
[0042] As used in this invention, a "composition" is a substance consisting of two or more components combined in a certain proportion, possessing specific properties and / or uses. There are no particular limitations on the properties of the composition. Compositions used to prevent and / or alleviate the risk of osteoporosis caused by low estrogen levels may be selected from food compositions, pharmaceutical compositions, food additives, functional foods, beverages, pet food compositions, powders, tablets, ointments, or gels.
[0043] The term "microbial preparation" as used in this invention refers to a live bacterial preparation made by processing microorganisms through separation and purification, strain improvement, industrial production and propagation, or any of the above processes. It is usually prepared into an adsorbable microbial liquid using simple organic matter as a carrier, or into a solid preparation by adding other auxiliary components.
[0044] As used in this invention, "functional food" refers to food that provides health benefits (as a fortified food, oral supplement, or dietary supplement).
[0045] The term "drug carrier" as used in this invention refers to a substance that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, or deliver a drug to a target organ.
[0046] As used in this invention, the term "strain" refers to a microorganism of a specific species that shares common characteristics. Unless otherwise indicated, the terms "strain" and "cell" are used interchangeably herein.
[0047] The term "plate" as used in this invention refers to plate culture medium, which is the most commonly used form of solid culture medium for obtaining pure cultures of microorganisms. It is a solid surface of culture medium formed in a sterile petri dish after the solidified culture medium has cooled and solidified. It is often simply referred to as a culture plate or plate.
[0048] The term "culture medium" as used in this invention refers to a substrate or nutrient solution used for the cultivation of microorganisms, which includes nutrients and water, sufficient energy and a suitable pH environment required for the growth of said microorganisms.
[0049] The term "culture" as used in this invention refers to the continuous cultivation of the microorganisms over a period of time until a desired goal is achieved.
[0050] As used in this invention, the term "prevention and / or reduction of osteoporosis risk" means prevention and reduction of the frequency and / or incidence and / or severity and / or shortening of the duration of osteoporosis; incidence refers to the number of times any osteoporosis occurs; frequency refers to the number of times the same osteoporosis occurs; this prevention covers reducing the frequency and / or severity of such osteoporosis in later life.
[0051] The term "bacterial suspension" as used in this invention refers to a suspension containing microbial cells.
[0052] Furthermore, in the context of this invention, the terms "comprising" or "including" do not exclude other possible elements. The compositions mentioned in this invention (including the various embodiments described herein) may comprise, or consist of, the following elements: the basic elements described herein, and any other or optional ingredients or components described herein.
[0053] Example 1: Screening of Lactobacillus gasseri CCFM1255
[0054] (I) Isolation and screening of lactobacilli
[0055] Take 1g of fresh feces from a healthy infant. After serial dilution, spread it on LBS medium supplemented with 1% nystatin and incubate at 37°C for 48 hours.
[0056] After culturing, colonies are picked up with an inoculation loop and streaked for purification based on their color, size, and edge shape.
[0057] The purified colonies were subjected to Gram staining and catalase analysis. Gram-positive bacilli and catalase-negative bacteria were retained.
[0058] (II) Molecular biological identification of lactobacilli
[0059] (1) Single-strain genome extraction
[0060] (A) Culture the lactobacilli screened in step (I) overnight;
[0061] (B) Take 1 mL of the bacterial suspension that has been cultured overnight into a 1.5 mL centrifuge tube, centrifuge at 10000 r / min for 2 min, discard the supernatant to obtain the bacterial cells;
[0062] (C) After rinsing the bacterial cells with 1 mL of sterile water, centrifuge at 10000 r / min for 2 min, discard the supernatant to obtain the bacterial cells;
[0063] (D) Add 200 μL of SDS lysis buffer and incubate in a water bath at 80°C for 30 min;
[0064] (E) Add 200 μL of phenol-chloroform solution to the bacterial cell lysis buffer. The composition and volume ratio of the phenol-chloroform solution is Tris saturated phenol:chloroform:isoamyl alcohol = 25:24:1. After mixing by inversion, centrifuge at 12000 rpm for 5-10 min and take 200 μL of the supernatant.
[0065] (F) Add 400 μL of ice-cold ethanol or ice-cold isopropanol to 200 μL of supernatant, let stand at -20℃ for 1 h, centrifuge at 12000 rpm for 5-10 min, and discard the supernatant.
[0066] (G) Add 500 μL of 70% (v / v) ice-cold ethanol to resuspend the precipitate, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in an oven at 60℃ or air dry naturally;
[0067] (H) 50 μL ddH2O was used to redissolve the precipitate for PCR.
[0068] (2) 16S rDNA PCR
[0069] (A) Bacterial 16S rDNA 50μL PCR reaction system:
[0070] 10×Taq buffer, 5 μL; dNTP, 5 μL; primer 27F, 0.5 μL; primer 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template,
[0071] 0.5 μL; ddH2O, 38 μL.
[0072] (B) PCR conditions
[0073] 95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-4 30×; 72℃5min; 12℃2min.
[0074] (C) Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging.
[0075] (D) The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity in GenBank using BLAST. The product was identified as Lactobacillus gasseri. The strain was stored at -80℃.
[0076] (3) Whole genome sequencing
[0077] The extracted whole genome was sent to a professional sequencing company, where a second-generation sequencer was used to sequence the bacterial genome. The obtained sequence results were then searched and compared for similarity in GenBank using BLAST. The sequencing results identified it as a newly discovered strain of Lactobacillus gasseri, which was stored at -80℃ for future use.
[0078] Example 2: Lactobacillus gastroenteritis CCFM1255 exhibits good tolerance to simulated gastrointestinal fluid.
[0079] Frozen Lactobacillus gasseri CCFM1255 was inoculated into MRS medium and cultured anaerobically at 37°C for 14 hours. After being passaged in MRS medium 2-3 times, the culture was then carried out.
[0080] Collect bacterial cells by centrifuging 3 mL of Lactobacillus gastroenteritis CCFM1255 culture medium at 8000×g for 2 min. Mix the mixture with 3 mL of pH 3.0 artificial gastric juice (containing 3 g / L pepsin and pH 3.0 physiological saline) and anaerobic culture at 37℃. Take samples at 0 h and 2 h, respectively, and perform plate colony counting on MRS agar medium to determine the number of viable bacteria and calculate their survival rate.
[0081] Collect bacterial cells by centrifuging 3 mL of Lactobacillus gasseri CCFM1255 culture medium at 8000×g for 2 min, add 3 mL of pH 8.0 artificial intestinal fluid (containing 1 g / L trypsin, 0.3% bile salts, and physiological saline at pH 8.0) and mix. Incubate anaerobically at 37℃. Take samples at 0 h, 2 h, and 4 h, respectively, and perform plate colony counting on MRS agar medium. Determine the number of viable bacteria and calculate their survival rate.
[0082] Survival rate (%) was calculated as the ratio of the number of viable bacteria in the culture medium at the time of sampling to the number of viable bacteria at 0 h. The experimental results are shown in Table 1, indicating that *Lactobacillus gastroenteritis* has good tolerance to artificial simulated gastrointestinal fluid.
[0083] Table 1. Tolerance of Lactobacillus gastroenteritis CCFM1255 in artificial gastrointestinal fluid.
[0084]
[0085] Example 3: Lactobacillus gasseri CCFM1255 has no toxic side effects on SD rats.
[0086] Lactobacillus gasseri CCFM1255 was cultured anaerobically in MRS medium at 37°C until the logarithmic growth phase. The bacterial cells were collected and resuspended in a 30 g / L sucrose solution to prepare a solution with a concentration of 4.0 × 10⁻⁶. 9CFU / mL bacterial suspension. Twelve healthy female SD rats weighing approximately 280-330g were used. After acclimatizing to the environment for one week, they were divided into a CCFM1255 group and a control group. The CCFM1255 group was given 1mL of the bacterial suspension at this concentration by gavage once daily, while the control group was given the same volume of a 30g / L sucrose solution without Lactobacillus gasseri CCFM1255 by gavage. The rats were observed for one week, and mortality and weight were recorded.
[0087] The results of these experiments are listed in Table 2. These results indicate that a feeding concentration of 1×10⁻⁶ is suitable. 9 CFU / rat of Lactobacillus gasseri CCFM1255 did not have a significant effect on rats; there was no significant change in body weight, and no deaths occurred. The rats showed no obvious pathological symptoms.
[0088] Table 2. Changes in rat body weight and mortality.
[0089]
[0090] Example 4: Lactobacillus gasseri CCFM1255 increases serum estradiol levels in ovariectomized rats
[0091] Twenty-four healthy female SD rats weighing 280-330g were randomly divided into four groups after one week of acclimatization culture: sham-operated group, ovariectomized model group, Lactobacillus gasseri CCFM1255 intervention group (CCFM1255), and estradiol drug (estradiol). Except for the sham-operated group, where only a small amount of fat was removed from the sides of the ovaries, all other groups underwent bilateral ovarian removal. All rats rested for two weeks post-surgery. Then, starting from day 15 post-surgery, the sham-operated group and the ovariectomized model group were given 10% skim milk, and each rat was given 1×10⁻⁶ CCFM1255. 9 CFU-containing Lactobacillus gasseri CCFM1255 was administered to rats at a dose of 28 μg / kg / day. The experimental grouping method is shown in Table 3.
[0092] Table 3 Grouping of experimental animals
[0093]
[0094] At the end of the experiment, fresh rat feces were collected and frozen at -80℃. At the end of the experiment, rats were fasted but allowed free access to water for 12 hours. After anesthesia with an intraperitoneal injection of 0.1 mL / 10 g 1% sodium pentobarbital solution, blood was collected from the abdominal aorta, and the rats were euthanized by cervical dislocation. Blood samples were centrifuged at 3500 rpm for 15 minutes, and the supernatant was collected and frozen at -80℃ for blood parameter analysis. Abdominal fat was removed, rapidly frozen in liquid nitrogen, and then transferred to a -80℃ freezer for subsequent determination of relevant parameters. Serum estradiol was measured according to the kit instructions.
[0095] The effect of Lactobacillus gasseri CCFM1255 on serum estradiol levels in rats, as follows: Figure 2 Compared with ovariectomized rats, Lactobacillus gasseri CCFM1255 significantly increased estrogen levels in ovariectomized rats, increasing serum estradiol levels from 52.72±12.31 pg / mL to 96.67±36.96 pg / mL, an increase of 83.36%. This estrogen level is close to that of the sham-operated group (106.1±14.44 pg / mL). Although the effect is not as good as that of the estradiol drug group (152.8±23.59 pg / mL), it can avoid the adverse reactions caused by excessively high estrogen levels. Therefore, the use of Lactobacillus gasseri CCFM1255 can prevent and reduce a series of menopausal symptoms caused by excessively low estrogen levels, such as menopausal osteoporosis.
[0096] Example 5: Lactobacillus gasseri CCFM1255 promotes calcium absorption and increases serum calcium levels in rats.
[0097] The grouping and processing of experimental animals were the same as in Example 4. At the end of the experiment, rat serum was collected for serum calcium level analysis. Experimental methods were performed according to the kit instructions.
[0098] The results show ( Figure 3 The serum calcium level in ovariectomized rats (2.278±0.079 mmol / L) was lower than that in sham-operated rats (2.557±0.134 mmol / L), indicating that ovariectomy alters calcium absorption. Lactobacillus gasseri CCFM1255 (2.434±0.075 mmol / L) could reverse this change and promote calcium absorption, thus increasing serum calcium levels. Meanwhile, the estradiol group also showed a significant effect in promoting calcium absorption, raising calcium levels to 2.756±0.060 mmol / L.
[0099] Example 6: Lactobacillus gasseri CCFM1255 alleviates bone turnover abnormalities caused by low estrogen levels
[0100] The experimental animals were grouped and treated in the same way as in Example 4. Bone health and bone turnover related indicators were determined according to the kit instructions.
[0101] Type I collagen is the most abundant protein in bone, and serum N-terminal propeptide (PINP) concentration of type I procollagen reflects the total amount of new bone formation. Studies have shown that diseases with high bone turnover are associated with high serum PINP concentrations. Figure 4The results showed that after ovariectomy, the serum PINP level in rats (79.66±8.19 ng / ml) was significantly higher than that in the sham-operated group (66.84±3.51 ng / ml). The CCFM1255 and estradiol groups significantly reduced the PINP level to 69.49±4.021 ng / ml and 67.40±3.294 ng / ml, respectively, restoring it to normal levels.
[0102] Osteoporosis protectin (OPG) belongs to the tumor necrosis factor receptor superfamily and plays a crucial role in bone remodeling. It can competitively bind to RANKL to block RANK-RANKL signaling, thereby inhibiting osteoclast recruitment and activation, and ultimately inducing osteoclast apoptosis. Figure 5 The results showed that the serum OPG level in ovariectomized rats (3739±146.8 pg / ml) was significantly higher than that in the sham-operated group (3516±78.66 pg / ml). CCFM1255 intervention significantly reduced OPG levels to 3527±169.9 pg / ml, while the estradiol group failed to significantly reduce OPG levels (3594±129.7 pg / ml).
[0103] Example 7: Lactobacillus gasseri CCFM1255 alleviates inflammatory response caused by decreased estrogen levels.
[0104] The grouping and treatment of experimental animals were the same as in Example 4, and the detection of inflammatory factors was performed according to the kit method.
[0105] Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-6 may play a key role in osteoclast formation. Figure 6The results showed that TNF-α and IL-6 levels significantly increased after ovariectomy, reaching 278.9±21.32 pg / ml and 172.0±9.759 pg / ml, respectively, while the TNF-α and IL-6 levels in the sham surgery group were 248.9±5.141 pg / ml and 154.4±10.77 pg / ml, respectively. CCFM1255 intervention significantly reduced the levels of these inflammatory factors (TNF-α: 251.1±6.809 pg / ml; IL-6: 153.5±5.21 pg / ml). The estradiol group only alleviated the abnormally high IL-6 level (155.9±2.166 pg / ml), but did not significantly alleviate the abnormally high TNF-α level after ovariectomy (estradiol group: 266.6±6.809 pg / ml). In addition, CCFM1255 intervention significantly reduced IL-10 levels (CCFM1255 group: 43.73±3.626 pg / ml; oophorectomy model group: 48.6±3.323 pg / ml). Although there was no significant difference in IL-10 levels in the oophorectomy model group (oophorectomy model group: 48.6±3.323 pg / ml) compared with the sham surgery group (46.09±1.443 pg / ml) and the estradiol drug group (46.01±3.494 pg / ml), it is not difficult to see that oophorectomy increased IL-10 levels.
[0106] Example 8: Lactobacillus gasseri CCFM1255 increases CYP19 levels and estrogen receptor ESR1 expression in adipose tissue.
[0107] The grouping and treatment of experimental animals were the same as in Example 4. ESR1 and CYP19 mRNA assays in adipose tissue: Approximately 20 mg of abdominal adipose tissue was added to 500 μL of Trizol, homogenized in an ice bath, and RNA was extracted from the adipose tissue using standard methods. cDNA synthesis was performed according to the reverse transcription kit instructions. The sample was mixed with the fluorescent dye SYBR Green super mix (Qiagen, Germany). The PCR system consisted of 5 μL mix, 1 μL cDNA, and 1 μL of forward and reverse primers, with ddH2O added to a total volume of 10 μL. The results were analyzed using a CFX96 real-time quantitative gene amplification instrument. TM The detection was performed on a Real-Time System (Bio-Rad, USA), with three parallel wells for each sample and GAPDH as an internal reference. The results were analyzed using 2... -ΔΔCt The analysis was performed using the method described in Table 4; the primer sequences used are shown in Table 4.
[0108] Table 4 qPCR primer sequences
[0109]
[0110]
[0111] The results show ( Figure 7 In the sham-operated group, the expression level of ESR1 receptor in the adipose tissue of mice was 2.262±1.154, significantly higher than that in the ovariectomized model group (0.7575±0.6859). Compared with the ovariectomized model group, CCFM1255 significantly increased the expression of ESR1 receptor in the adipose tissue of ovariectomized mice (ESR1 expression level: 2.214±0.8242). The mRNA level of CYP19 in adipose tissue also showed that CCFM1255 significantly increased CYP19 expression, increasing it to 4.354±2.966 compared to the ovariectomized model group (0.8626±0.5674). CYP19 encodes the enzyme cytochrome P450c19, a key site in estrogen biosynthesis, catalyzing the conversion of androgens to estrogens. Therefore, CCFM1255 may promote estradiol production and activate ESR1 by increasing CYP19 expression.
[0112] Example 9: Using Lactobacillus gasseri CCFM1255 of the present invention to produce fermented food containing this bacterium.
[0113] Fresh vegetables are selected, washed, and juiced. The juice is then subjected to high-temperature instantaneous sterilization at 140°C for 2 seconds, followed by immediate cooling to 37°C. The Lactobacillus gasseri CCFM1255 inoculum prepared according to this invention is then inoculated to achieve a concentration of 10. 8 Fruit and vegetable beverages containing live Lactobacillus gasseri CCFM1255 of the present invention were obtained by refrigerating at 4°C with a concentration of CFU / mL or higher.
[0114] This invention enables the production and preparation of other fermented foods using Lactobacillus gasseri CCFM1255, including solid foods, liquid foods, and semi-solid foods. The fermented foods include dairy products, soy products, and fruit and vegetable products. The dairy products include milk, sour cream, and cheese; the fruit and vegetable products include products made from cucumbers, carrots, beets, celery, and cabbage.
[0115] The fermented food can alleviate abnormal bone turnover caused by low estrogen levels in ovariectomized rats, promote calcium absorption, alleviate inflammatory responses caused by decreased estrogen, and increase the expression of CYP19 and estrogen receptor ESR1 in adipose tissue.
[0116] Example 10: Lactobacillus gasseri CCFM1255 used in drug preparation
[0117] A drug is prepared by mixing Lactobacillus gasseri CCFM1255 with a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier includes, but is not limited to, one or more of the following: fillers, wetting agents, disintegrants, binders or lubricants;
[0118] The filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, croscarmellose sodium, croscarmellose, or low-substituted hydroxypropyl cellulose; the binder is one or more of starch paste, syrup, maltose, refined molasses, or liquid glucose; and the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talc, or silica.
[0119] Optionally, the specific preparation process of tablets containing Lactobacillus gasseri CCFM1255 is as follows:
[0120] Single colonies of *Lactobacillus gasseri* CCFM1255 obtained in Example 1 were inoculated into MRS liquid medium and cultured at 37°C for 24 h to obtain an activation solution. The activation solution was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a primary seed culture. The primary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a secondary seed culture. The secondary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a bacterial culture. The bacterial culture was centrifuged at 6000g for 15 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer at pH 7.4, and then centrifuged again at 6000g for 10 min to obtain bacterial cells. The *Lactobacillus gasseri* CCFM1255 bacterial cells were resuspended in a protective solution containing 130 g / L skim milk, 20 g / L trehalose, and 20 g / L sucrose to a cell concentration of 1 × 10⁻⁶ cells. 10 CFU / mL was used to obtain Lactobacillus gasseri CCFM1255 bacterial suspension; the Lactobacillus gasseri CCFM1255 bacterial suspension was freeze-dried to obtain Lactobacillus gasseri CCFM1255 bacterial powder; the freeze-dried bacterial powder accounted for 10% of the total amount, followed by the addition of 2% stearic acid as a lubricant, 3% CMC-Na, 15.5% galactooligosaccharides, 7.8% xylooligosaccharides, 7.8% inulin, lactitol, erythritol, xylitol, and other excipients such as starch, and then compressed into tablets to obtain tablets.
[0121] Gavage administration of 1g of the above tablets to ovariectomized rats daily for four consecutive weeks can effectively alleviate abnormal bone turnover caused by low estrogen levels, promote calcium absorption, alleviate inflammatory responses caused by decreased estrogen, and increase the expression of CYP19 and estrogen receptor ESR1 in adipose tissue.
[0122] Example 11: Lactobacillus gasseri CCFM1255 used to prepare bacterial powder
[0123] Lactobacillus gasseri CCFM1255 can be used to prepare bacterial powder. The specific preparation process for the bacterial powder is as follows:
[0124] Single colonies of *Lactobacillus gasseri* CCFM1255 obtained in Example 1 were inoculated into MRS liquid medium and cultured at 37°C for 24 h to obtain an activation solution. The activation solution was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a primary seed culture. The primary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a secondary seed culture. The secondary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C for 24 h to obtain a bacterial culture. The bacterial culture was centrifuged at 6000g for 15 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer at pH 7.4, and then centrifuged again at 6000g for 10 min to obtain bacterial cells. The *Lactobacillus gasseri* CCFM1255 bacterial cells were resuspended in a protective solution containing 130 g / L skim milk, 20 g / L trehalose, and 20 g / L sucrose to a cell concentration of 1 × 10⁻⁶ cells. 10 The bacterial culture of Lactobacillus gasseri CCFM1255 was obtained by lyophilizing the bacterial culture at CFU / mL. The bacterial culture of Lactobacillus gasseri CCFM1255 was then freeze-dried to obtain bacterial powder.
[0125] Take a sample containing a total live bacteria count of 1×10⁻⁶. 9 CFU powder administered orally to ovariectomized rats daily for four consecutive weeks effectively alleviated abnormal bone turnover caused by low estrogen levels, promoted calcium absorption, alleviated inflammatory responses caused by decreased estrogen, and increased CYP19 levels and estrogen receptor ESR1 expression in adipose tissue.
[0126] Comparative Example 1
[0127] The specific implementation method is the same as in Implementation 4, except that Lactobacillus gasseri CCFM1255 is replaced with Lactobacillus gasseri JSWX33-L2 (described in Zhou Xingya's master's thesis "Screening, Genomic Comparison and Safety Evaluation of Lactobacillus gasseri and Lactobacillus paragne"). The serum estradiol level of rats was measured. The results showed that the serum estradiol level of rats in the Lactobacillus gasseri JSWX33-L2 group was 43.21±26.37μmol / L, which was significantly different from that in the ovariectomized model group (52.72±12.31μmol / L).
[0128] Comparative Example 2
[0129] The specific implementation method is the same as in Example 5, except that Lactobacillus gasseri CCFM1255 is replaced with Lactobacillus gasseri JSWX33-L2 (described in Zhou Xingya's master's thesis "Screening, Genomic Comparison and Safety Evaluation of Lactobacillus gasseri and Lactobacillus paragneiss"). The calcium level in the serum of rats was measured. The results showed that the calcium level in the serum of rats in the Lactobacillus gasseri JSWX33-L2 group was 2.350±0.1115mmol / L. Compared with the ovariectomized model group (2.278±0.07918mmol / L), Lactobacillus gasseri JSWX33-L2 increased the calcium level in the serum of ovariectomized rats by 3.16%, which was not statistically significant.
[0130] Comparative Example 3
[0131] The specific implementation method is the same as in Example 4, except that Lactobacillus gasseri CCFM1255 is replaced with Lactobacillus gasseri ATCC19992 (described in Mayur Bagad, DOI:10.1016 / j.apjtb.2017.09.005). The serum estradiol level of rats was measured. The results showed that the serum estradiol level of rats in the Lactobacillus gasseri ATCC 19992 group was 56.83±13.21 μmol / L. Compared with the ovariectomized model group (52.72±12.31 μmol / L), Lactobacillus gasseri ATCC 19992 increased the serum estradiol level of ovariectomized rats by 7.80%, which was not statistically significant.
[0132] Comparative Example 4
[0133] The specific implementation method is the same as in Example 8, except that Lactobacillus gasseri CCFM1255 is replaced with Lactobacillus gasseri ATCC19992 (described in MayurBagad, DOI:10.1016 / j.apjtb.2017.09.005). The ESR1 mRNA level in the abdominal fat of rats was measured. The results showed that the ESR1 mRNA level in the abdominal fat of rats in the Lactobacillus gasseri ATCC 19992 group was 1.23±0.24, which was much lower than the expression level of Lactobacillus gasseri CCFM1255 in the abdominal fat tissue.
[0134] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. Lactobacillus gasseri ( Lactobacillus gasseri CCFM1255 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 21, 2022, with accession number GDMCC No: 62305.
2. A composition containing Lactobacillus gasseri CCFM1255 as described in claim 1.
3. The composition according to claim 2, characterized in that, The number of Lactobacillus gasseri CCFM1255 is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g.
4. The composition according to claim 2 or 3, characterized in that, The composition includes microbial preparations or pharmaceuticals.
5. The composition according to claim 2 or 3, characterized in that, The composition contains a live or dried strain of Lactobacillus gasseri CCFM1255.
6. The use of Lactobacillus gasseri CCFM1255 according to claim 1 in the preparation of a medicament for relieving osteoporosis; wherein the osteoporosis is caused by abnormal estrogen levels; the use includes, but is not limited to, at least one of the following effects: (1) Increase serum estradiol levels in mammals with abnormal estrogen levels; (2) Increase blood calcium levels in mammals with abnormal estrogen levels; (3) Reduce the level of type I procollagen N-terminal propeptide in the serum of mammals with abnormal estrogen levels; (4) Reduce osteoprotegerin levels in mammals with abnormal estrogen levels; (5) Reduce the levels of IL-10, TNF-α and IL-6 in the serum of mammals with abnormal estrogen levels; (6) Increase the expression levels of CYP19 and estrogen receptor ESR1 in adipose tissue of mammals with abnormal estrogen levels.
7. The application according to claim 6, characterized in that, The drug also contains a pharmaceutically acceptable carrier.