Saliva-associated lactobacillus for promoting calcium absorption and bone growth and application thereof

Abstract

The present invention provides a saliva-associated lactobacillus and its application for promoting calcium absorption and bone growth. The preservation number of saliva-associated lactobacillus LS60 is CGMCC No.30305. The present invention also provides the culture supernatant or dead bacteria of the saliva-associated lactobacillus. The saliva-associated lactobacillus LS60 strain is beneficial for intestinal cells to absorb calcium ions, promotes osteoblast differentiation, and increases the blood calcium and bone calcium content of mice. Most importantly, the probiotic can increase the level of IGF-1 and promote the growth of the femur of mice; in addition, the LS60 strain can also produce high GABA; in summary, the saliva-associated lactobacillus LS60 of the present invention has the potential to promote calcium absorption and bone growth.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and their applications, and particularly relates to a saliva-associated lactobacillus for promoting calcium absorption and bone growth and an application thereof. Background Art

[0002] The continuous self-renewal of bone tissue is of great physiological significance for the calcium content in the human blood and for maintaining bone strength. It is characterized by a dynamic balance between bone resorption (performed by osteoclasts) and bone formation (performed by osteoblasts). The bone formation process is the result of osteoblast differentiation under the combined action of a series of hormones (such as sex hormones and parathyroid hormone), growth factors (such as bone morphogenetic protein, insulin-like growth factor 1 (IGF-1), and vitamins C and D), and minerals such as calcium and magnesium, which eventually become mature osteocytes that participate in the formation of new bone. Among them, IGF-1 is a growth factor that affects bone growth. IGF-1 promotes bone growth mainly by acting on osteoblasts (bone formation), osteoclasts (bone resorption), and osteocytes. In other words, IGF-1 can enhance the differentiation function of osteoblasts.

[0003] Dynamic imbalances in bone tissue can lead to height limitations in adolescents and osteoporosis in adults. Children's height is an aspect of childhood and adolescent development that many parents are concerned about. Research has shown that 70% of height is determined by genetics, while the remaining 30% is primarily influenced by sleep, exercise, nutrition, and psychology. Beyond the 70% genetic factor, in order to keep children's height above average, it's crucial to ensure they have adequate sleep, balanced nutrition (especially calcium, essential for bone development), a healthy mindset, and appropriate exercise.

[0004] GABA generally refers to gamma-aminobutyric acid, also known as GABA. It is the primary inhibitory neurotransmitter in the human body, while dopamine, epinephrine, and norepinephrine are the primary excitatory neurotransmitters. Through the regulation of these two neurotransmitters, the body's neural responses are generally coordinated and stable. Therefore, GABA can often relieve tension and anxiety, maintain a stable mood, and thus promote sleep.

[0005] Probiotics are active microorganisms that colonize the human body and benefit the host. They promote nutrient absorption and maintain intestinal health by modulating host mucosal and systemic immune function or regulating the balance of intestinal flora, resulting in single microorganisms or a defined mixture of microorganisms with beneficial health effects. Through their long evolutionary journey alongside humans, probiotics have become inextricably linked to the body's absorption and metabolism. Modern research indicates that probiotics can synthesize many substances beneficial to the human body, including short-chain fatty acids (SCFAs), GABA, vitamins D and K, which are closely related to calcium absorption, and other trace elements that aid calcium absorption. Given their high safety and readily available availability, supplementing with appropriate probiotics is currently a preferred method for many consumers to maintain their health. Therefore, using beneficial microorganisms to moderately increase calcium absorption and supplement GABA, thereby regulating bone development, also holds significant development potential, particularly for adolescents who cannot undergo extensive medication and intervention. At present, such probiotics and related products are still very scarce in the domestic market, while many products of the same type have been launched in the foreign market. Therefore, the development of probiotics that can regulate bone development has great market prospects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention provides a probiotic Lactobacillus salivae that promotes calcium absorption and bone growth, and its use. The Lactobacillus salivae LS60 strain facilitates calcium ion absorption by intestinal cells, promotes osteoblast differentiation, and increases blood and bone calcium levels in mice. Most importantly, the probiotic can increase IGF-1 levels and promote femoral lengthening in mice. Furthermore, the LS60 strain can also produce high levels of GABA. Overall, the Lactobacillus salivae LS60 probiotic has the potential to promote calcium absorption and bone growth.

[0008] The first aspect of the present invention provides Lactobacillus salivarius LS60, which has a deposit number of CGMCC No. 30305. The present invention also provides the culture supernatant or dead cells of the Lactobacillus salivarius.

[0009] Another aspect of the present invention provides a use of the saliva in combination with lactobacillus or its culture supernatant or dead bacteria for preparing gamma-aminobutyric acid.

[0010] Another aspect of the present invention provides the use of the saliva combined with lactobacillus or its culture supernatant or dead bacteria in preparing a product for synthesizing gamma-aminobutyric acid, promoting calcium absorption, bone cell differentiation, bone tissue development and growth, and increasing blood calcium and bone calcium content in mice.

[0011] Another aspect of the present invention provides the use of the saliva-associated Lactobacillus or its culture supernatant or dead cells in preparing a product that improves calcium absorption, osteocyte differentiation, bone tissue development and growth, and increases blood calcium and bone calcium content in mice. The present invention also provides a bacterial agent containing the saliva-associated Lactobacillus or its culture supernatant or dead cells. Preferably, the bacterial agent is a solid preparation or a liquid preparation. Preferably, the solid bacterial agent is a powder prepared by freeze-drying.

[0012] Another aspect of the present invention provides a food comprising the saliva-combined lactobacillus or its culture supernatant or dead bacteria. Preferably, the food is a functional food.

[0013] The present invention also provides a method for preparing a product composition, which comprises: adding bacterial substances and / or metabolites of the saliva-associated lactobacillus into a product matrix to obtain the product composition.

[0014] Biological sample deposit information: Ligilactobacillus salivarius LS60 was deposited with the China General Microbiological Culture Collection Center on April 11, 2024, under the accession number CGMCC No. 30305. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, 100101. The strain was confirmed to be viable by the collection center on April 11, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The effects of saliva combined with Lactobacillus LS60 on calcium ion absorption in Caco-2 cells are shown. (A) Detection of relative calcium ion content in Caco-2 cells; (B) Expression of key genes related to calcium ion transport in Caco-2 cells. *p < 0.05, **p < 0.01.

[0017] Figure 2The effects of saliva combined with Lactobacillus LS60 on the osteogenic differentiation of MC3T3-E1 cells are shown. (A) Alizarin red staining of osteoblast-differentiated bone nodules; (B) Alkaline phosphatase activity in osteoblasts. *p < 0.05, **p < 0.01, ***p < 0.001.

[0018] Figure 3 Figure 2 shows the effects of saliva combined with Lactobacillus LS60 on mouse skeletal development. (A) Mouse blood calcium levels; (B) Mouse bone calcium content; (C) Mouse femur length; (D) Serum IGF-1 levels. *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0019] To make the technical solutions, objectives and advantages of the present invention more clear, the present invention is further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0020] Unless otherwise defined, all terms (including technical and scientific terms) used in the present invention have the same meaning as commonly understood by those skilled in the art. In the event of a conflict between the prior art and the present invention, the present invention shall prevail.

[0021] Unless otherwise specified, the various reagents, materials, etc. used in the following examples are all products that can be obtained from commercial channels; unless otherwise specified, the various tests and detection methods used in the following examples are all conventional tests and detection methods in the field, which can be obtained from textbooks, reference books or academic journals.

[0022] Example 1: Culture and identification of saliva-associated Lactobacillus LS60

[0023] The probiotic strain of the present invention was isolated from breast milk of healthy people and identified as Ligilactobacillus salivarius by 16S rRNA sequence analysis (sequence shown in SEQ ID NO: 1). It is preserved in the General Microbiology Center of China National Committee for Microbiological Culture Collection with the deposit number: CGMCC No. 30305. Before use, the frozen strain was activated and subcultured for two generations, and then inoculated into MRS liquid culture medium at a rate of 2-4%, cultured continuously for 16h-24h, and centrifuged (6000r / min, 10min, 4°C). The obtained supernatant is the fermentation supernatant of the probiotic. The bacterial sludge precipitate was washed twice with sterile PBS, and then the bacterial count was adjusted to 5×10 9 CFU / mL was used for subsequent animal experiments.

[0024] Example 2: Evaluation of the tolerance of saliva combined with Lactobacillus LS60 in artificial gastrointestinal acid and bile salt solutions

[0025] The activated saliva-associated Lactobacillus LS60 strain was inoculated into liquid MRS and cultured overnight. 10 mL of culture solution was centrifuged at 8000 rpm / min for 5 min to obtain the bacteria. The bacteria were washed twice with sterile PBS buffer solution, resuspended and the bacterial concentration was adjusted to 5 × 10 8 CFU / mL. Mix 1 mL of bacterial solution with 9 mL of artificial simulated gastric fluid (MRS medium containing 1% pepsin, pH 3.0) or artificial simulated intestinal fluid (MRS medium containing 0.3% ox bile salts, 1% trypsin, pH 8.0), respectively. Incubate at 37°C under anaerobic conditions. Then, sample at 0 h, 1 h, and 3 h for plate counts. Determine the number of viable bacteria and calculate the survival rate. Survival rate = viable bacterial count (logarithmic value) at sampling / viable bacterial count (logarithmic value) at 0 h, expressed as %.

[0026] The activated saliva-associated Lactobacillus LS60 strain was inoculated into liquid MRS and cultured overnight. 10 mL of culture solution was centrifuged at 8000 rpm / min for 5 min to obtain the bacteria. The bacteria were washed twice with sterile PBS buffer solution, resuspended and the bacterial concentration was adjusted to 5 × 10 8 CFU / mL. Take 1mL of bacterial solution and mix it with 9mL of liquid MRS culture medium containing different bile salt concentrations (0.1%, 0.2%, 0.3%). Use MRS culture medium without bile salts (pH=6.4) as a control. Repeat three times for each group. After static anaerobic culture at 37℃ for 3h, take out and count. Calculate the number of surviving bacteria and survival rate of Lactobacillus salivais LS60 strain at different bile salt concentrations. Survival rate = logarithm of viable bacteria at sampling / logarithm of viable bacteria at 0h, expressed as %.

[0027] Probiotics must be able to tolerate the acid-base and bile salt environment in the digestive tract of the body in order to colonize and survive in the intestinal tract, thereby playing a function that is beneficial to the host body. The survival rate test results of the saliva-combined Lactobacillus LS60 strain in artificial simulated gastric acid and intestinal fluid are shown in Table 1. The viable bacteria survival rate of the LS60 strain after being treated in artificial gastric fluid and intestinal fluid for 3 hours was 76.2% and 77.3%, respectively, indicating that the saliva-combined Lactobacillus LS60 of the present invention has excellent gastric acid and intestinal fluid resistance. The bile salt tolerance experiment results showed that the LS60 strain still retained a survival rate of 76.4% after being cultured in a 3% bile salt solution for 3 hours. As the concentration increased, the survival rate of the probiotics in a 0.5% bile salt solution slightly decreased (65.7%). Overall, the saliva-combined Lactobacillus LS60 strain of the present invention has a strong ability to withstand the harsh environment of the gastrointestinal tract and can survive and colonize smoothly in the intestinal tract.

[0028] Table 1 Analysis of acid-base and bile salt tolerance of saliva-associated Lactobacillus LS60

[0029]

[0030] Example 3: Evaluation of the Adhesion Ability of Saliva Combined with Lactobacillus LS60 to Intestinal Cells

[0031] In this example, human colon cancer cell line Caco-2 cells (purchased from the National Laboratory Cell Resource Sharing Platform) were co-incubated with saliva-associated Lactobacillus LS60 strain to detect its adhesion to intestinal cells. Specifically, Caco-2 cells were cultured at a concentration of 1×10 5 The concentration of cells / well was plated into a 12-well plate with a cell slide, and each well contained 2 mL of DMEM complete medium. The overnight cultured LS60 strain was washed and centrifuged with sterile PBS buffer and the bacterial concentration was adjusted to 1x10 9 CFU / mL. After the cells have completely adhered, rinse the cells with sterile PBS, then add 1mL of DMEM medium and 1mL of probiotic suspension respectively. After incubation at 37℃ 5% CO2 for 2h, rinse the cells three times with PBS to remove non-adherent bacteria, then add 1mL of methanol to fix them. After 20 minutes, aspirate the liquid in the well plate and wash with PBS buffer. Remove the cell slide and perform Gram staining. Observe and photograph the number of bacteria adhered to the cells in 20 random fields of view under a microscope, and calculate the number of bacteria adhered around every 100 cells.

[0032] Generally, the residence time of probiotics in the intestine is 1-4 hours. After incubation with intestinal cells Caco-2 for 2 hours, the salivary Lactobacillus LS60 strain in the present invention has an adhesion number of 332.6±32.12 (unit: number of adherent bacteria / 100 cells), indicating that the saliva-associated Lactobacillus LS60 strain has the ability to colonize intestinal cells.

[0033] Example 4: Detection of GABA synthesis by saliva combined with Lactobacillus LS60

[0034] This example uses chemical chromatography to detect the amount of GABA synthesized in the fermentation supernatant of the LS60 strain. The probiotic supernatant, MRS culture medium, and GABA standard solutions (0, 250, 500, 1000, and 1500 mg / L) were spotted on chromatography paper and then developed in a developing solvent (n-butanol: glacial acetic acid: water = 5:3:2). After chromatography, drying, and color development, the spot corresponding to the standard was cut out and eluted with 5 mL of eluent (75% ethanol: 0.6% copper sulfate = 38:2). 200 μL of the mixed eluent was taken and measured for absorbance at 510 nm. Calculations show that the probiotics of the present invention can produce 754.81 mg / L of GABA (average of three measurements) without exogenous L-glutamate stimulation. Compared with existing strains, the saliva-associated Lactobacillus LS60 strain described in the present invention can effectively synthesize GABA and has the potential for development of related probiotic products.

[0035] Example 5: Effect of saliva combined with Lactobacillus LS60 on intestinal cell calcium absorption

[0036] Caco-2 cells grown in culture flasks were digested and evenly seeded into 96-well fluorescent cell culture plates. The plates were cultured in a 37°C incubator with 5% CO₂. After 24 hours, the plates were washed two to three times with HBS (calcium-free). Fresh culture medium containing varying concentrations of calcium solution and different volume ratios of LS60 fermentation supernatant (10% and 20%) was added. After a further 24 hours of incubation, the old culture medium was discarded and the plates were washed three times with HBS. 50 μL of HBS (calcium-free) containing 5 μM Fluo- / AM and 2.5 mM Pluronic F-127 was added to each well. The plates were incubated at 37°C for 30 minutes. Excess probe was washed away, and fluorescence intensity was measured using a fluorescence microplate reader with excitation at 488 nm and emission at 525 nm. The absorbance reflects the relative calcium content in the Caco-2 cells.

[0037] like Figure 1 As shown in Figure A, the relative content of calcium ions in Caco-2 cells increased significantly after co-incubation with the supernatant of the high-content LS60 strain, and the promoting effect was stronger in the presence of exogenous calcium; the low-content LS60 probiotics had almost no effect on the calcium ion content in Caco-2 cells in the absence of exogenous calcium. When stimulated by the addition of calcium ions, the low-content probiotics could also significantly increase the intracellular calcium ions, indicating that the saliva-combined Lactobacillus LS60 strain of the present invention can promote the absorption of calcium ions by intestinal cells.

[0038] Referring to the above experimental method, the Caco-2 cells grown in the culture flask were digested and evenly inoculated into 6-well cell culture plates, and cultured in an incubator at 37°C containing 5% CO2. After 24 hours, they were washed 2-3 times with HBS solution (calcium-free), and fresh culture medium containing 2% calcium solution and different volume ratios of LS60 strain fermentation supernatant (10% and 20%) was added. After continuing to culture in the incubator for 24 hours, the old culture medium was discarded, and the cells were washed 3 times with HBS. Then the cells were collected, RNA was extracted, and qRT-PCR experiments were performed to detect the expression of key genes related to calcium ion transport. The detected genes included DMT1, TRPV6, and Calbindin-D9K. The results are as follows Figure 1 B shows that after intestinal cells were treated with LS60 probiotics (low and high concentrations), the gene expression of the ion channel TRPV6 and transporter Calbindin-D9k responsible for transmembrane calcium ion transport was significantly upregulated. This result just explains the phenomenon of increased calcium ion content in Caco-2 cells.

[0039] Example 6: Effect of saliva combined with Lactobacillus LS60 on osteoblast differentiation in mice

[0040] Mouse embryonic osteoblast MC3T3-E1 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) were plated into 6 cm cell culture dishes and cultured in DMEM medium in a 37°C incubator with 5% CO2. Based on the experimental treatments, the cells were divided into the following groups: ① negative control (DMEM); ② low-concentration LS60 probiotic fermentation supernatant group (DMEM + 10% (v / v) probiotics); and ③ low-concentration LS60 probiotic fermentation supernatant group (DMEM + 20% (v / v) probiotics). At the beginning of the experiment, the cells were treated with a set dose of probiotics. Fresh culture medium (with or without probiotics) was replaced every 3 days. The culture was terminated after 14 days, the culture medium was discarded, the cells were rinsed 3 times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then rinsed 3 times with PBS. Then, alizarin red dye was added for 5 minutes. After rinsing with PBS, the alizarin red dye in the cells was dissolved with a solvent (acetic acid: glycerol = 5:1). The cell debris was removed by centrifugation at 3000g for 5 minutes, and an appropriate volume of supernatant was taken to detect the absorbance at 405nm using a microplate reader.

[0041] When osteoblasts undergo osteoblast differentiation and mature, calcium ions in the cells will accumulate and form osteoblastic nodules. The chemical dye Alizarin Red can identify these osteoblastic nodules. The greater the absorbance value, the more bone differentiation. Figure 2 The results shown in A show that the saliva combined with the Lactobacillus LS60 probiotic fermentation supernatant of the present invention can promote the osteoblast differentiation and maturation.

[0042] During osteoblast differentiation, intracellular alkaline phosphatase (ALP) activity increases significantly. In this example, referring to the experimental method for detecting bone nodule formation described above, the culture supernatant of the LS60 strain was incubated with the osteoblast cell line MC3T3-E1 for 3 days. The cells were then lysed with a protein lysis buffer (10% SDS) and reacted with a substrate (1.0 mg / ml p-nitrophenyl phosphate, 0.2 M Tris buffer, and 5 mM magnesium chloride) for 30 minutes, and the absorbance was measured at 405 nm. Figure 2 Results B showed that the alkaline phosphatase activity in MC3T3-E1 cells was significantly increased after 3 days of treatment with the LS60 probiotics of the present invention.

[0043] The above results indicate that the cells treated with LS60 probiotics have entered the osteoblast differentiation process, indicating that the saliva-combined Lactobacillus LS60 probiotics of the present invention have the ability to promote osteoblast differentiation and maturation.

[0044] Example 7: Effects of saliva combined with Lactobacillus LS60 on bone growth and development in mice

[0045] Thirty C57BL / 6 male mice of about 6 weeks old were purchased from Weitonglihua. After one week of adaptive feeding, they were randomly divided into three groups, each with 10 mice. There was one normal control group and two experimental treatment groups: a low-dose LS60 probiotic group and a high-dose probiotic group. The mice in the treatment group received live probiotic suspension orally every day, and the high-dose group received 1×10 9 CFU / mouse / day, the oral dose of the low-dose group was 1×10 8 The control group received 100 CFU / mouse / day, while the normal group received the same volume of saline. The entire gavage experiment lasted for 6 weeks. At the end of the experiment, the mice were fasted for 12 hours, and blood was collected from the orbital vein. The supernatant was centrifuged at 4000 rpm and separated for serum samples. The mice were then sacrificed, and the femurs of the hind limbs were isolated. The soft tissue and cartilage were shaved, and the femur length was recorded. The bone tissue was stored in a freezer at -80°C. The mouse bone calcium, blood calcium, and IGF-1 levels were measured strictly according to the kit instructions.

[0046] according to Figure 3 A. After gavage with saliva combined with Lactobacillus LS60 probiotics, the blood calcium content of mice increased, especially the high dose of LS60 probiotics treatment had a significant effect. Similar results were also found in the bone calcium content test ( Figure 3 B), indicating that high-dose LS60 probiotic treatment can promote calcium absorption and bone tissue development in mice. Correspondingly, femoral length measurement also showed that the femur of mice tended to lengthen after gavage with LS60 strain ( Figure 3 C), the effect of high-dose LS60 probiotics after oral administration was better than that of low-dose treatment.

[0047] In addition, insulin-like growth factor 1 (IGF-1) is a polypeptide hormone that promotes growth and development. It is beneficial to promote bone growth and development in the body, thereby enabling normal growth of the body. When its level is reduced or abnormal, it is likely to lead to insufficient secretion of growth hormone, which will lead to growth stagnation. In adolescents and children, it is reflected in symptoms such as developmental abnormalities and short stature. Through the detection of serum samples, it was found that the level of IGF-1 increased after saliva was treated with Lactobacillus LS60 probiotics ( Figure 3 D), this result is consistent with the above phenotype.

[0048] In summary, these experimental results show that the saliva-combined Lactobacillus LS60 probiotic strain of the present invention can improve calcium absorption, produce high GABA, promote bone tissue development and growth, and has the potential to be developed into a food related to promoting bone development.

[0049] Sequence Listing:

[0050] Lactobacillus salivarius LS60 16S rRNA sequence:

Claims

1. Saliva-associated Lactobacillus (CGMCC No. 30305) Ligilactobacillus salivarius ).

2. Use of the saliva-associated lactobacillus according to claim 1 in the preparation of gamma-aminobutyric acid.

3. Use of the saliva-associated lactobacillus according to claim 1 in preparing a product that promotes calcium absorption.

4. Use of the saliva-lactobacillus according to claim 1 in preparing a product for promoting osteoblast differentiation, bone tissue development and growth, and increasing blood calcium and bone calcium content in mice. A bacterial agent comprising the Lactobacillus salivarius according to claim 1 .

6. The bacterial agent according to claim 5, wherein The bacterial agent is a solid preparation or a liquid preparation.

7. A food comprising the salivarius-associated Lactobacillus according to claim 1.

8. The food according to claim 7, wherein The food is a functional food, a special medical food or a health food.

9. The food according to claim 8, comprising 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g of the saliva-associated lactobacillus of claim 1.

10. A method for preparing a product composition, characterized in that: The method comprises: adding the bacterial substance of the saliva-associated Lactobacillus according to claim 1 to a product matrix to obtain the product composition.

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