Application of pediococcus pentosaceus MPL5 in promoting conversion of combined calcium in food into ionized calcium and improving osteoporosis

Abstract

The invention discloses pediococcus pentosaceus MPL5 and application thereof in promoting conversion of combined calcium in food into ionized calcium and improving calcium bioaccessibility, and belongs to the technical field of microorganisms. The pediococcus pentosaceus MPL5 provided by the invention can effectively promote combined calcium to be converted into ionized calcium (greater than 60%) in a culture solution or a composition fermentation solution, so that the bioavailability of calcium is improved; in addition, osteoporosis can be improved and conditioned, specifically, the bone mineral density, the number of bone trabecula, the connectivity of the bone trabecula and the connection density of the bone trabecula of an osteoporosis model mouse are remarkably improved, the separation degree of the bone trabecula and the index of a structural model are remarkably reduced, and therefore osteoporosis is reduced. The pediococcus pentosaceus MPL5 disclosed by the invention has a very wide application range in preparation of functional foods and health foods for promoting ionized calcium conversion and assisting in relieving osteoporosis, and is healthy and safe.

Description

Technical Field

[0001] The present invention relates to the field of microecological technology, and in particular to the application of Pediococcus pentosaceus MPL5 (including its fermentation product) in promoting the conversion of bound calcium in food into ionized calcium and improving osteoporosis. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease characterized by low bone mass, destruction of bone microstructure, increased bone brittleness, and easy fractures. It is common in the elderly. With age, the metabolic activities of osteocytes and osteoblasts, gonadal metabolic activities, food digestion, and nutrient absorption are constantly declining, all of which can lead to the occurrence of osteoporosis. In addition, many people now have long-term bad living and eating habits, such as staying up late, being picky about food, and excessive drinking / coffee / carbonated drinks, which lead to a large loss of bone mass. Therefore, this disease also occurs in young people and even minors.

[0003] As an important substance that constitutes bones and teeth, calcium participates in many complex life activities such as muscle contraction, synthesis and release of neurotransmitters, synthesis and secretion of hormones, blood coagulation, etc. Adequate calcium intake is conducive to improving osteoporosis. For people with osteoporosis, even if the food in their daily diet is rich in calcium, if this calcium is not well converted and cannot meet the body's calcium needs, the symptoms of osteoporosis cannot be improved.

[0004] Most of the calcium in food exists in the form of bound calcium. If it is to be absorbed by the human body, it needs to be converted into ionized calcium so that it can be quickly absorbed by the intestine. How to effectively promote the conversion of bound calcium in food into ionized calcium has become a technical problem that needs to be solved in this field. Through a large number of experiments, we found that probiotics that can promote the conversion of bound calcium in food into ionized calcium greatly improve the absorption and utilization of calcium by the intestine. After being consumed by the human body, it is beneficial to improve osteoporosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Pediococcus pentosaceus MPL5, which can promote the conversion of bound calcium in food into ionic calcium, is beneficial to improving osteoporosis, has a significant effect, and is healthy and safe.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: The first aspect of the present invention provides a Pediococcus pentosaceus, wherein the Pediococcus pentosaceus is Pediococcus pentosaceus ( Pediococcus pentosaceus ) MPL5, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 30, 2023, with the deposit number GDMCC No: 63606, and disclosed in the Chinese invention patent application with the publication date of December 22, 2023 and the publication number CN117264852A.

[0007] Pediococcus pentosaceus is a facultative anaerobic bacterium belonging to the genus Pediococcus. It is a type of lactic acid bacteria that can produce a large amount of organic acid using a variety of sugars as carbon sources. It has no proteolytic effect and is sensitive to hop preservatives. As a probiotic, Pediococcus pentosaceus is mainly distributed in fresh wort and fermented plant materials, such as pickles and silage. Pediococcus pentosaceus is an edible fungus that has been proven to have beneficial functions such as improving nutrient synthesis and metabolism, inhibiting the growth of pathogens, lowering serum cholesterol, and improving human immunity. However, no strain of Pediococcus pentosaceus has been found that can promote the conversion of bound calcium in food into ionic calcium.

[0008] In the present invention, the Guangdong Provincial Microecological Preparation Engineering Technology Research Center of South China Agricultural University accidentally discovered a new strain of Pediococcus pentosaceus in the storage of ripe mango pulp, and named it Pediococcus pentosaceus ( Pediococcus pentosaceus )MPL5. Through experiments, it was found that this strain has the function of promoting the conversion of bound calcium in food into ionized calcium, and the conversion rate is greater than 60%.

[0009] The second aspect of the present invention provides a microecological preparation, wherein the bacterial preparation contains the above-mentioned Pediococcus pentosaceus MPL5.

[0010] Furthermore, the microecological preparation may include various preparations of the above-mentioned Pediococcus pentosaceus MPL5, including but not limited to bacterial suspensions, fermentation broths, and the like.

[0011] The third aspect of the present invention provides a fermented product for improving osteoporosis, wherein the fermented product is obtained by inoculating the Pediococcus pentosaceus MPL5 into a composition and fermenting the mixture; The composition comprises the following components in parts by weight: 30-40 parts of hazelnut kernels, 20-30 parts of oats, 10-15 parts of yellow millet, 10-20 parts of oligofructose, 10-30 parts of isomaltooligosaccharide, 5-10 parts of stachyose, and 500-700 parts of water.

[0012] Hazelnut kernels have high nutritional value and are known as the "king of nuts". They are rich in various minerals such as calcium, phosphorus and iron. The calcium content is as high as 815 mg / 100g, which is very beneficial for protecting bone health, strengthening physical fitness, resisting fatigue and delaying aging; it also contains manganese that can make human bones, skin, tendons, ligaments and other tissues strong, and various vitamins with important physiological functions, such as vitamin B1, B2, B6 and E. The content of vitamin E is as high as 33.9 mg / 100g, which has a strong antioxidant effect.

[0013] Oats are a crop of the genus Avena in the Poaceae family. They are rich in various nutrients such as protein, lipids, dietary fiber, carbohydrates, minerals, antioxidants (such as vitamin E, phenolic acid, flavonoids, sterols and alkaloids), etc. The iron content of oats is 8 times that of rice, the calcium content is 5 times that of rice, and the selenium content is 35 times that of rice. Long-term consumption can not only effectively prevent calcium loss, osteoporosis and other diseases in the elderly, maintain the balance of calcium in the human body, but also help prevent premature aging, chronic diseases, cancer, cardiovascular disease and stroke.

[0014] Yellow millet is rich in trace elements such as calcium, iron, zinc, magnesium, and phosphorus, which play an indispensable role in human growth and immune regulation. In addition, the fatty acid composition of yellow millet is reasonable, and the content of unsaturated fatty acids such as linolenic acid and linoleic acid is more than 85%, especially linoleic acid, which is 65.05%. These fatty acids exist in the form of glycerides, which have a protective effect on the skin, microvessels, and central nervous system, and can prevent and treat arteriosclerosis and cirrhosis of the liver. They are good solvents for fat-soluble vitamins and fat-soluble pigments (carotene), which is conducive to the absorption of these nutrients by the human body.

[0015] Hazelnut kernels, oats and yellow millet are all rich in calcium, and these calcium elements mainly exist in the form of bound calcium. In the present invention, Pediococcus pentosaceus MPL5 is used as a fermentation strain, and a composition including hazelnut kernels, oats and yellow millet is fermented, and the characteristic of Pediococcus pentosaceus MPL5 that can promote the conversion of bound calcium into ionized calcium is utilized to convert the bound calcium rich in hazelnut kernels, oats and yellow millet into ionized calcium, so that the fermented product contains rich ionized calcium that is easily absorbed and utilized by the human body, which is beneficial to improving osteoporosis. In addition, hazelnut kernels, oats and yellow millet are also rich in protein, lipid, dietary fiber, carbohydrates, other minerals, trace elements and other nutrients, and are more easily absorbed by the human body after fermentation, and can better meet the needs of human growth and development.

[0016] Fructooligosaccharides, isomaltooligosaccharides and stachyose are functional oligosaccharides with high stability. They are difficult to be broken down by the enzymes in the human digestive tract, but they can serve as carbon sources for probiotics and promote their reproduction and metabolism.

[0017] In the above composition, the weight portion of hazelnut kernel is 30-40 parts, for example, 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, etc. The weight portion of oats is 20-30 parts, for example, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, etc. The weight portion of yellow millet is 10-15 parts, for example, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, etc. The weight portion of oligofructose is 10-20 parts, for example, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, etc. The weight portion of isomaltooligosaccharide is 10 to 30 parts, for example, it can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, etc. The weight portion of stachyose is 5 to 10 parts, for example, it can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc. The weight portion of water is 500 to 700 parts, for example, it can be 500 parts, 550 parts, 600 parts, 650 parts, 700 parts, etc.

[0018] Further, in a preferred embodiment, the composition comprises the following components in parts by weight: 30 parts of hazelnut kernels, 30 parts of oats, 15 parts of yellow millet, 10 parts of fructooligosaccharides, 10 parts of isomaltooligosaccharides, 5 parts of stachyose, and 500 parts of water.

[0019] A fourth aspect of the present invention provides a method for preparing a fermented product for improving osteoporosis, comprising the following steps: S1. The formula amount of hazelnut kernels, oats and yellow millet was washed and mixed, and water was added to break the wall to obtain a slurry; the broken wall liquid was then boiled and broken again while hot; after filtering, the filtrate was collected to obtain a slurry; S2. The formulated amount of fructooligosaccharides, isomaltooligosaccharides and stachyose is mixed and added to the liquid, dissolved and mixed and sterilized to obtain a culture matrix; S3. The Pediococcus pentosaceus MPL5 was inoculated into the culture matrix, and after anaerobic culture, a fermentation semi-finished product was obtained; S4. homogenizing the fermentation semi-finished product at low temperature and high pressure to obtain the fermentation product.

[0020] Furthermore, in step S2, the sterilization condition is sterilization at 121° C. for 15 minutes.

[0021] Furthermore, in step S3, the inoculation amount of the Pediococcus pentosaceus MPL5 is as follows: the addition amount of the bacterial solution is 5 to 20 parts by weight, preferably 10 parts by weight; the concentration of the bacterial solution is 3.0×10 10 CFU / mL. The conditions of the anaerobic culture are: the culture temperature is 37°C and the culture time is 16h.

[0022] In step S4 of the present invention, the purpose of using low temperature and high pressure homogenization technology is that at low temperature, due to sudden decompression and high-speed impact, the cells are ruptured, and the steric hindrance between the intracellular enzyme and the substrate is eliminated. This technology can not only promote the autolysis of strain cells and fully release intracellular nutrients, but also protect the biological activity of strain metabolites, so that the efficacy and bioavailability of the fermented product are greatly improved. After the human body consumes this fermented product, osteoporosis can be significantly improved.

[0023] Further, in step S4, when low temperature and high pressure homogenization is performed, the homogenization pressure is 150-200 MPa, for example, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa, etc.; the temperature is 43-48°C, for example, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, etc. The number of cycles can be 2-5 times.

[0024] The fifth aspect of the present invention discloses the use of the Pediococcus pentosaceus, the microecological preparation or the fermentation product in the preparation of a preparation for promoting the conversion of bound calcium in food into ionic calcium.

[0025] The sixth aspect of the present invention discloses the use of the Pediococcus pentosaceus, the microecological preparation or the fermentation product in the preparation of a preparation for improving osteoporosis.

[0026] Furthermore, the Pediococcus pentosaceus includes but is not limited to live bacteria type and sterilized type.

[0027] Furthermore, the preparations include but are not limited to functional foods, foods and health foods.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. The Pediococcus pentosaceus MPL5 provided by the present invention can promote the conversion of bound calcium in food into ionized calcium, with a conversion rate of >60%, greatly improving the absorption and utilization of calcium by the intestine. After being consumed by the human body, it is beneficial to improve osteoporosis.

[0029] 2. The present invention utilizes Pediococcus pentosaceus MPL5 to ferment a composition including hazelnut kernels, oats and yellow millet. By utilizing the property of Pediococcus pentosaceus MPL5 that can promote the conversion of bound calcium into ionized calcium, bound calcium rich in hazelnut kernels, oats and yellow millet is converted into ionized calcium, so that the fermented product contains rich ionized calcium and has high bioavailability, which is beneficial to improving osteoporosis.

[0030] 3. The low temperature and high pressure homogenization technology in the present invention can not only promote the autolysis of strain cells and fully release the intracellular nutrients, but also protect the biological activity of strain metabolites, so that the efficacy and bioavailability of fermented products are greatly improved. After the human body consumes this fermented product, it can significantly improve osteoporosis. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0033] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0034] Strain screening: The ripe mangoes were peeled and the pulp was taken. First, multiple acid-producing strains were isolated by dilution plate coating and streak purification on MRS solid medium containing bromocresol purple; secondly, a strain with high efficiency in producing organic acid was further screened out through litmus milk color development test and determination of acid production performance. According to morphological characteristics, physiological and biochemical characteristics and third-generation 16S amplicon sequencing analysis, the strain was identified as Pediococcus pentosaceus and named MPL5.

[0035] Gene sequence identification results:

[0036] Test Example 1: Determination of the conversion rate of bound calcium of Pediococcus pentosaceus MPL5 1. Experimental strain: Pediococcus pentosaceus MPL5 2. Sample preparation (1) Culture medium preparation Weigh 104.48 g of MRS liquid culture medium powder, add 2000 mL of purified water, stir to dissolve, measure 100 mL, record as solution A, and the rest as solution B. Add 5.0 g of calcium carbonate and 5.0 g of milk calcium to solution B, stir evenly, and obtain solution C. Sterilize solution A and solution C for 15 min at a sterilization temperature of 121°C, and cool to 37°C to obtain the culture medium, which are recorded as culture medium A and culture medium C, respectively.

[0037] (2) Bacterial activation Take out the freeze-dried storage tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile saline, draw all the bacterial solution onto MRS solid medium, spread it evenly, and culture it anaerobically at 37°C for 24 hours. Pick 2 loops of colonies and add them to 10 mL of medium A. After anaerobic culture at 37°C for 24 hours, adjust the bacterial solution concentration to 1.0×10 9 CFU / mL, and the bacterial solution is obtained.

[0038] (3) Cultivation Pipette 1 mL of bacterial solution into 100 mL of culture medium C, mix well, and culture anaerobically for 0 h, 4 h, 8 h, 12 h, 16 h, and 20 h, respectively, and record them as group A, group B, group C, group D, group E, and group F, respectively. Make 3 parallels for each group, and the culture temperature is 37 °C to obtain the culture solution.

[0039] (4) Centrifugation The culture solution was centrifuged for 10 minutes at 4°C and 8000 r / min. At the end of the centrifugation, the precipitate and the supernatant were separated to obtain the supernatant, which was weighed, and the precipitate was washed with 2 times the volume of clean water to obtain the precipitate, which was weighed.

[0040] 3. Determination of combined calcium and ion calcium conversion rate (1) Preparation of calcium colorimetric reagent Prepare the calcium colorimetric reagent by mixing storage solution 1, storage solution 2, and methanol in a ratio of 20:9:14 before use. The preparation method of storage solution 1 is as follows: weigh 32.5 mg of o-cresolphthalein complexing agent, add 100 ml of water, add 7.5 ml of concentrated hydrochloric acid, and dilute to a 500 ml volumetric flask after fully dissolving; the preparation method of storage solution 2 is as follows: weigh 21 ml of diethylamine, add water to a 500 ml volumetric flask, and mix well.

[0041] (2) Working curve drawing Pipette 0.25, 0.50, 0.75, 1.00, and 1.50 mL of 3 mg / mL calcium standard solution into a 10 mL volumetric flask to obtain calcium standard solutions of different concentrations. Measure the spectrophotometry of calcium standard solutions of different concentrations at 570 nm, and draw a standard curve with calcium ion concentration and absorbance as the horizontal and vertical coordinates.

[0042] (3) Weigh the precipitate and supernatant, take 0.1 mg of the sample and add it to a 5 mL stoppered colorimetric tube. Replace the sample with an equal amount of purified water as the reagent blank. Accurately add 4 mL of calcium colorimetric reagent and mix well. After 5 minutes, use a 0.5 cm cuvette to compare the color at 570 nm. Use the standard curve method to determine the calcium content in the supernatant and precipitate of different groups, calculate the conversion rate of bound calcium, and calculate the average value (round off to two decimal places). The results are shown in Table 1.

[0043] 4. Results and Analysis

[0044] Calcium carbonate and milk calcium are both bound calcium formed by the combination of calcium and other compounds. They are insoluble in water, so they exist in the precipitate after centrifugation. During the fermentation process, part of the bound calcium can be converted into ionic calcium, that is, calcium exists in the form of ions and is easily soluble in water. Therefore, after centrifugation, it exists in the supernatant. As shown in Table 1, compared with group A, the calcium content of the precipitate of groups B to F continued to decrease, the calcium content of the supernatant continued to increase, and the conversion rate continued to increase, indicating that with the extension of fermentation time, the bound calcium was continuously converted into ionic calcium by the strain. Among them, the conversion rate of group F was the highest, and the difference in conversion rate between group E and group F was the smallest, indicating that after 16 hours of fermentation, the growth of the strain tended to be saturated, the conversion of bound calcium in the culture medium tended to be saturated, and the conversion rate of bound calcium into ionic calcium was >60%. Example 1

[0045] This embodiment provides a fermented product for promoting the conversion of bound calcium in food into ionic calcium, comprising the following steps: S1. Raw material selection: Choose dry, mildew-free, insect-free hazelnut kernels, oats, yellow millet, and qualified quality fructooligosaccharides, isomaltooligosaccharides, and stachyose.

[0046] S2. Cleaning and weighing: Clean the hazelnut kernels, oats and yellow millet with clean water for 3 times, drain and weigh 30 parts of hazelnut kernels, 30 parts of oats and 15 parts of yellow millet, and mix them evenly to obtain a mixture A.

[0047] S3, wall breaking: weigh 500 parts of purified water, mix it evenly with mixture A, break the wall with a wall breaking machine for 1 min at a speed of 36000r / min, pour it into a boiling pot, boil for 20 min, replenish the lost water, break the wall with a wall breaking machine for 0.5 min while it is hot, filter with 60-mesh and 100-mesh filter screens in turn, collect the filtrate, and record it as liquid B.

[0048] S4. Preparation: Weigh 10 parts of oligofructose, 10 parts of oligomaltose and 5 parts of stachyose, mix well, add to liquid B, stir and dissolve, and obtain liquid C.

[0049] S5. Sterilization: Sterilize liquid C at 121° C. for 15 min, and cool to 37° C. to obtain a composition, which is recorded as composition D.

[0050] S6. Activation of bacterial strains: Take out the freeze-dried storage tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile physiological saline, draw all the bacterial solution onto MRS solid medium, spread it evenly, and culture it anaerobically at 37°C for 24 hours. Pick 2 loops of colonies and add them to 10 mL of composition D. After anaerobic culture at 37°C for 24 hours, adjust the bacterial solution concentration to 3.0×10 10 CFU / mL, the bacterial solution was obtained and recorded as bacterial solution E.

[0051] S7, inoculation and fermentation: 5 parts by weight of bacterial solution E is added to composition D, mixed evenly, cultured anaerobically at 37° C. for 16 hours, and refrigerated for 8 hours to obtain a semi-finished product, which is recorded as semi-finished product F.

[0052] S8. Homogenization: The semi-finished product F is homogenized at low temperature and high pressure, with a homogenization pressure of 150 MPa, 3 cycles, and a temperature of 4°C. The final product is the fermentation product.

[0053] Examples 2 to 4 and Comparative Example 1

[0054] The preparation methods of Examples 2 to 4 and Comparative Example 1 are the same as those of Example 1, except that the amount of bacterial solution added is different. The specific amount of bacterial solution added is shown in Table 2.

[0055]

[0056] Test Example 2: Determination of ionized calcium content 1. Take 5 g of the fermented product, wash it with 5 g of purified water, centrifuge it, and collect the supernatant. Make 3 parallel groups for each example and comparative example.

[0057] 2. According to the flame atomic absorption spectrometry of "GB 5009.92 National Food Safety Standard Determination of Calcium in Food", the calcium content of the supernatant was determined and the average value was calculated (rounded off to two decimal places). The results are shown in Table 3.

[0058] 3. Results and analysis

[0059] After fermentation by Pediococcus pentosaceus MPL5, the bound calcium in the composition is gradually converted into ionized calcium. After washing with purified water, the ionized calcium can be dissolved in water. As shown in Table 3, compared with Comparative Example 1, the ionized calcium content of Examples 1 to 4 is increased, indicating that Pediococcus pentosaceus MPL5 can promote the conversion of bound calcium in food into ionized calcium, among which the ionized calcium content of the fermented product of Example 4 is the highest, and is close to the ionized calcium content of the fermented products of Examples 2 and 3, indicating that the bacterial liquid addition amounts of Examples 2, 3, and 4 can fully convert the bound calcium in food into ionized calcium, and the metabolic activity of the strain is close to saturation. Therefore, the bacterial liquid addition amount of Example 2 is selected as the optimal addition amount, and the bound calcium of the resulting fermented product can be fully converted into ionized calcium.

[0060] Comparative Examples 2 to 8 The preparation methods of Comparative Examples 2 to 8 are the same as those of Example 2, except that the ratio of the fermentation raw materials is different. The specific ratio of the fermentation raw materials is shown in Table 4.

[0061]

[0062] Comparative Example 9 The preparation method of Comparative Example 9 is the same as that of Example 2, except that: Comparative Example 9 does not use high-pressure homogenization technology, and its step S8 is: heating the semi-finished product F to room temperature (25°C), homogenizing it with a colloid mill, and repeating it for 3 times. The final product obtained is the fermented product.

[0063] Example 5 This embodiment provides a method for preparing a solid preparation, comprising the following steps: S1. Take out the freeze-dried storage tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile saline, draw all the bacterial solution onto MRS solid medium, spread it evenly, culture it anaerobically at 37°C for 24 hours, pick 2 loops of colonies and add them to 10 mL of MRS liquid medium, culture it anaerobically at 37°C for 24 hours, adjust the bacterial solution concentration to 1.0×10 11 CFU / mL, and the bacterial solution was obtained, which was recorded as bacterial solution A.

[0064] S2. Centrifuge the bacterial solution A to separate the precipitate and the supernatant, and mix the precipitate and the freeze-drying protective agent evenly to obtain the bacterial solution, which is recorded as bacterial solution B.

[0065] S3. The bacterial solution B is freeze-dried in vacuum, and then crushed and sieved to obtain powder. The obtained powder is a live bacterial type of Pediococcus pentosaceus MPL5 bacterial powder.

[0066] S4. Evenly mix 10 parts of Pediococcus pentosaceus MPL5 powder and 90 parts of oligofructose to obtain powder, which is solid preparation A.

[0067] Example 6 This embodiment provides a method for preparing a solid preparation, comprising the following steps: S1. Take out the freeze-dried storage tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile saline, draw all the bacterial solution onto MRS solid medium, spread it evenly, culture it anaerobically at 37°C for 24 hours, pick 2 loops of colonies and add them to 10 mL of MRS liquid medium, culture it anaerobically at 37°C for 24 hours, adjust the bacterial solution concentration to 1.0×10 11 CFU / mL, and the bacterial solution was obtained, which was recorded as bacterial solution A.

[0068] S2. Centrifuge the bacterial solution A to separate the precipitate and the supernatant, and mix the precipitate and the freeze-drying protective agent evenly to obtain the bacterial solution, which is recorded as bacterial solution B.

[0069] S3. The bacterial solution B is freeze-dried in vacuum, and then crushed and sieved to obtain powder, which is the bacterial powder of Pediococcus pentosaceus MPL5.

[0070] S4. Heat the powder of Pediococcus pentosaceus MPL5 at 60° C. for 45 min, then raise the temperature to 80° C. and maintain for 20 min, and finally crush and sieve to obtain powder. The obtained powder is inactivated powder of Pediococcus pentosaceus MPL5.

[0071] S5. Evenly mix 10 parts of inactivated Pediococcus pentosaceus MPL5 powder and 90 parts of oligofructose to obtain a powder, which is a solid preparation B.

[0072] Test Example 3: Determination of ionized calcium content 1. Take 5 g of the fermented product, wash it with 5 g of purified water, centrifuge it, and collect the supernatant. Make 3 parallel groups for each example.

[0073] 2. According to the flame atomic absorption spectrometry of "GB 5009.92 National Food Safety Standard Determination of Calcium in Food", the calcium content of the supernatant was determined and the average value was calculated (rounded off to two decimal places). The results are shown in Table 5.

[0074]

[0075] Please refer to Table 5. The results show that the fermented products prepared with different raw material formulas have different ion calcium contents, among which the ion calcium content in the fermented product of Example 2 is the highest. This shows that the raw material formula has an effect on the ion calcium content in the final fermented product. The fermented product obtained by fermenting the raw materials of the specific formula in Example 2 with Pediococcus pentosaceus MPL5 has the highest ion calcium content.

[0076] Test Example 4: Osteoporosis Mouse Model Test 1. Solution preparation (1) 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) solution: Accurately weigh 5 g of sodium carboxymethyl cellulose and dissolve it in deionized water. Ultrasonicate and stir to make it dissolve evenly. Make up to 1000 mL and store at 4°C until used.

[0077] (2) D-galactose solution (D-gal solution): Accurately weigh 1.5 g of D-galactose and dissolve it in physiological saline. Stir to make it fully dissolved and evenly dissolved. Make up to 100 mL and store at 4°C until used.

[0078] (3) Alendronate sodium solution: Accurately weigh 14 mg of alendronate sodium powder, ultrasonically mix and stir to evenly distribute it in 0.5% CMC-Na solution, make up to 10 mL, and store at 4°C until use.

[0079] 2. Experimental animals: ICR mice, 3 months old, male, weighing 28±2g, clean grade.

[0080] 3. Grouping and modeling The mice were randomly divided into 14 groups, 6 mice in each group, and recorded as groups A to N. Group A was intraperitoneally injected with an equal volume of normal saline, and the other 13 groups of mice were intraperitoneally injected with D-gal solution at a dose of 150 mg / kg, 3 times a week, for 12 weeks. The specific administration method is shown in Table 6.

[0081] 4. Drug administration The mice in group C were given a dose of 14 mg / kg, once a week by gavage; the mice in groups D to L were given a dose of 260 mg / kg by gavage of the fermented product of the example, and the mice in groups A and B were given an equal amount of 0.5% CMC-Na solution by gavage every day for 12 weeks. The specific dosing methods are shown in Table 6.

[0082]

[0083] 5. Collection and processing of mouse bone specimens The right femur of the mouse was removed, and the excess tissue was removed. The ROI area was 100 layers above the growth plate, and the distal femur was scanned with a layer thickness of 12 μm and tested by Micro-CT. The test indicators include: bone density (BMD), trabecular separation (Tb.Sp), trabecular number (Tb.N), structural model index (SMI), trabecular connectivity (Conn) and trabecular connection density (Conn.Dn). The results are shown in Table 7.

[0084] 6. Results and Analysis

[0085] The results in Table 7 show that compared with group A, the BMD, Tb.N, Conn, Conn.Dn and other indicators of group B mice were significantly reduced, and the Tb.Sp, SMI and other indicators were significantly increased, indicating that the osteoporosis model in elderly mice was successfully established and the bone loss was serious.

[0086] Compared with group B, the BMD, Tb.N, Conn, Conn.Dn and other indicators of mice in group C were significantly increased, and the Tb.Sp, SMI and other indicators were significantly decreased, indicating that the positive drug (alendronate sodium) has a significant therapeutic effect on senile osteoporosis.

[0087] Compared with group B, the BMD, Tb.N, Conn, Conn.Dn and other indicators of mice in groups D to K increased to varying degrees, and the Tb.Sp, SMI and other indicators decreased to varying degrees, indicating that fermented products prepared with different compositions have different effects on improving osteoporosis. Among them, the BMD, Tb.N, Conn, Conn.Dn and other indicators of mice in group J were the highest, and the Tb.Sp, SMI and other indicators were the lowest, indicating that the osteoporosis of mice was improved the best, and the lost bone was well rebuilt and repaired.

[0088] Compared with group B, the BMD, Tb.N, Conn, Conn.Dn and other indicators of mice in groups M to N increased to varying degrees, and the Tb.Sp, SMI and other indicators decreased to varying degrees, indicating that both the live and inactivated types of Pediococcus pentosaceus MPL5 powder have an improving effect on osteoporosis, and the improvement effects are different.

[0089] Compared with group J, the BMD, Tb.N, Conn, Conn.Dn and other indicators of mice in group L were significantly decreased, and the Tb.Sp, SMI and other indicators were significantly increased, indicating that the osteoporosis improvement effect of mice in group L was worse than that in group J, and changes in homogenization conditions will have a significant impact on the effect of fermented products.

[0090] Therefore, combined with the experimental results in Tables 5 and 7, the fermented product of Group J, namely Example 2, was selected as the best formulation. The obtained fermented product has the highest ion calcium content and the best effect in improving osteoporosis.

[0091] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Those skilled in the art can understand that various substitutions, changes, deletions, additions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims, and therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. Use of Pediococcus pentosaceus or its fermented product in the preparation of a preparation for promoting the conversion of bound calcium in food into ionized calcium and / or improving osteoporosis, characterized in that: The Pediococcus pentosaceus is Pediococcus pentosaceus MPL5, and its deposit number is GDMCC No: 63606.

2. The use according to claim 1, characterized in that: The Pediococcus pentosaceus MPL5 is a live bacteria type or an inactivated type.

3. The use according to claim 1, characterized in that: The preparations include food, functional food and health food.

4. The use according to claim 1, characterized in that: The fermented product is obtained by inoculating Pediococcus pentosaceus MPL5 into the composition and fermenting it; The composition comprises the following components in parts by weight: 30-40 parts of hazelnut kernels, 20-30 parts of oats, 10-15 parts of yellow millet, 10-20 parts of oligofructose, 10-30 parts of isomaltooligosaccharide, 5-10 parts of stachyose, and 500-700 parts of water.

5. The use according to claim 4, characterized in that: The composition comprises the following components in parts by weight: 30 parts of hazelnut kernels, 30 parts of oats, 15 parts of yellow millet, 10 parts of fructooligosaccharides, 10 parts of isomaltooligosaccharides, 5 parts of stachyose, and 500 parts of water.

6. The use according to claim 4 or 5, characterized in that: The method for preparing the fermented product comprises the following steps: S1. The formula amount of hazelnut kernels, oats and yellow millet was washed and mixed, and water was added to break the wall to obtain a slurry; the slurry was then boiled and broken again while hot; after filtering, the filtrate was collected to obtain a liquid feed; S2. The formulated amount of fructooligosaccharides, isomaltooligosaccharides and stachyose is mixed and added to the liquid, dissolved and mixed and sterilized to obtain a culture matrix; S3. The Pediococcus pentosaceus MPL5 was inoculated into the culture matrix, and after anaerobic culture, a fermentation semi-finished product was obtained; S4. homogenizing the fermentation semi-finished product at low temperature and high pressure to obtain the fermentation product.

7. The use according to claim 6, characterized in that: In step S4, when low-temperature and high-pressure homogenization is performed, the homogenization pressure is 150-200 MPa, the temperature is 43-48° C., and the number of cycles is 2-5 times.

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