Application of Pediococcus pentosaceus MPL5 in promoting the conversion of calcium bound in food into ionic calcium and improving osteoporosis

Abstract

The present invention discloses Pediococcus pentosaceus MPL5 and its application in promoting the conversion of bound calcium in food into ionic calcium and improving calcium bioavailability, belonging to the field of microbial technology. Pediococcus pentosaceus MPL5 provided by the present invention can effectively promote the conversion of bound calcium into ionic calcium (>60%) in both culture broth or composition fermentation broth, thereby improving the bioavailability of calcium; it can also improve and condition osteoporosis, specifically manifested in: significantly increasing the bone density, trabecular bone number, trabecular bone connectivity and trabecular bone connection density of osteoporosis model mice, and significantly reducing the trabecular bone separation degree and structure model index, thereby reducing the occurrence of osteoporosis. Pediococcus pentosaceus MPL5 of the present invention has a very wide range of application scenarios in the preparation of functional foods and health foods that promote the conversion of ionic calcium and assist in alleviating 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:

[0007] The first aspect of the present invention provides a Pediococcus pentosaceus, wherein the Pediococcus pentosaceus is Pediococcus pentosaceus ( Pediococcus pentosaceus ) MPL5 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on June 30, 2023, with the deposit number GDMCC No. 63606, and was disclosed in a Chinese patent application with a publication date of December 22, 2023 and a publication number of CN117264852A.

[0008] Pediococcus pentosaceus is a facultative anaerobe, belonging to the genus Pediococcus, which is a kind of lactic acid bacterium. It can use a variety of sugars as carbon sources to produce a large amount of organic acids, has no proteolytic activity, and is sensitive to hop preservatives. As a probiotic, Pediococcus pentosaceus is mainly distributed in fresh wort and fermented plant materials, such as pickled vegetables and silage. Pediococcus pentosaceus is an edible strain, and it has been proven to have probiotic functions such as improving the synthesis and metabolism of nutrients, inhibiting the growth of pathogenic bacteria, reducing serum cholesterol, and enhancing human immunity. However, no strain of Pediococcus pentosaceus that can promote the conversion of bound calcium in food into ionic calcium has been found so far.

[0009] In the present invention, the Guangdong Engineering Technology Research Center for Microecological Agents of South China Agricultural University accidentally discovered a new strain of Pediococcus pentosaceus in the stored 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 ionic calcium, and the conversion rate > 60%.

[0010] The second aspect of the present invention provides a microecological agent, which contains the above-mentioned Pediococcus pentosaceus MPL5.

[0011] Further, the microecological agent can include various preparations of the above-mentioned Pediococcus pentosaceus MPL5, including but not limited to bacterial suspensions, fermentation broths, etc.

[0012] The third aspect of the present invention provides a fermented product for improving osteoporosis, which is obtained by inoculating the above-mentioned Pediococcus pentosaceus MPL5 in a composition and fermenting it;

[0013] Among them, the composition includes the following components by weight: 30 - 40 parts of hazelnut kernels, 20 - 30 parts of oats, 10 - 15 parts of yellow millet, 10 - 20 parts of fructooligosaccharide, 10 - 30 parts of isomaltooligosaccharide, 5 - 10 parts of stachyose, and 500 - 700 parts of water.

[0014] Hazelnut kernels are highly nutritious and are known as the "king of nuts". They are rich in various minerals such as calcium, phosphorus, and iron. Among them, the calcium content is as high as 815 mg / 100 g, which is very beneficial for protecting bone health, strengthening the body, resisting fatigue, and delaying aging. Moreover, they also contain manganese that can make tissues such as human bones, skin, tendons, and ligaments firm, as well as various vitamins with important physiological functions, such as vitamin B1, B2, B6, and E. Among them, the vitamin E content is as high as 33.9 mg / 100 g, which has a strong antioxidant effect.

[0015] Oats are crops of the genus Avena in the family Poaceae and contain various nutrients such as rich protein, lipids, dietary fiber, carbohydrates, minerals, antioxidants (such as vitamin E, phenolic acids, flavonoids, sterols, and alkaloids, etc.). Among them, the iron content 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 diseases such as calcium loss and osteoporosis in the elderly, maintain the balance of calcium elements in the human body, but also help prevent premature aging, chronic diseases, cancer, cardiovascular diseases, and strokes.

[0016] Yellow millet contains rich trace elements such as calcium, iron, zinc, magnesium, and phosphorus, which play an indispensable role in human growth and development 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 above 85%, especially the linoleic acid content 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. They are good solvents for fat-soluble vitamins and fat-soluble pigments (carotenoids), which is beneficial for the human body to absorb these nutrients.

[0017] Hazelnut kernels, oats, and yellow millet all contain rich calcium elements, and these calcium elements mainly exist in the form of bound calcium. In the present invention, Pediococcus pentosaceus MPL5 is used as the fermentation strain, and a composition containing hazelnut kernels, oats, and yellow millet is fermented. Utilizing the characteristic that Pediococcus pentosaceus MPL5 can promote the conversion of bound calcium into ionic calcium, the rich bound calcium in hazelnut kernels, oats, and yellow millet is converted into ionic calcium, so that the fermented product contains rich ionic 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 also contain rich protein, lipids, dietary fiber, carbohydrates, other minerals, trace elements, and other various nutrient elements, and are more easily absorbed by the human body after fermentation treatment, and can better meet the needs of human growth and development.

[0018] Fructooligosaccharide, isomaltooligosaccharide, and stachyose belong to functional oligosaccharides with relatively high stability and are difficult to be decomposed by the enzyme system of the human digestive tract. However, they can serve as the carbon source for probiotics and can promote the reproduction and metabolism of probiotics.

[0019] In the above composition, the weight portion of hazelnut kernels is 30 - 40 portions, for example, it can be 30 portions, 31 portions, 32 portions, 33 portions, 34 portions, 35 portions, 36 portions, 37 portions, 38 portions, 39 portions, 40 portions, etc. The weight portion of oats is 20 - 30 portions, for example, it can be 20 portions, 21 portions, 22 portions, 23 portions, 24 portions, 25 portions, 26 portions, 27 portions, 28 portions, 29 portions, 30 portions, etc. The weight portion of yellow millet is 10 - 15 portions, for example, it can be 10 portions, 11 portions, 12 portions, 13 portions, 14 portions, 15 portions, etc. The weight portion of fructooligosaccharide is 10 - 20 portions, for example, it can be 10 portions, 11 portions, 12 portions, 13 portions, 14 portions, 15 portions, 16 portions, 17 portions, 18 portions, 19 portions, 20 portions, etc. The weight portion of isomaltooligosaccharide is 10 - 30 portions, for example, it can be 10 portions, 11 portions, 12 portions, 13 portions, 14 portions, 15 portions, 16 portions, 17 portions, 18 portions, 19 portions, 20 portions, 21 portions, 22 portions, 23 portions, 24 portions, 25 portions, 26 portions, 27 portions, 28 portions, 29 portions, 30 portions, etc. The weight portion of stachyose is 5 - 10 portions, for example, it can be 5 portions, 6 portions, 7 portions, 8 portions, 9 portions, 10 portions, etc. The weight portion of water is 500 - 700 portions, for example, it can be 500 portions, 550 portions, 600 portions, 650 portions, 700 portions, etc.

[0020] Further, in a preferred embodiment, the composition comprises the following components by weight portion: 30 portions of hazelnut kernels, 30 portions of oats, 15 portions of yellow millet, 10 portions of fructooligosaccharide, 10 portions of isomaltooligosaccharide, 5 portions of stachyose, and 500 portions of water.

[0021] The fourth aspect of the present invention provides a preparation method of a ferment for improving osteoporosis, comprising the following steps:

[0022] S1. Wash and mix the formula amounts of hazelnut kernels, oats, and yellow millet, add water for cell wall breaking to obtain a slurry; then boil the cell wall broken liquid and perform cell wall breaking again while it is hot; after filtration, collect the filtrate to obtain a slurry;

[0023] S2. Mix the formula amounts of fructooligosaccharide, isomaltooligosaccharide, and stachyose, add them to the above-mentioned material liquid, dissolve and mix them evenly, and then sterilize to obtain a culture medium matrix;

[0024] S3. Inoculate Pediococcus pentosaceus MPL5 into the above-mentioned culture medium matrix, and after anaerobic culture, obtain a semi-finished ferment;

[0025] S4. Perform low-temperature high-pressure homogenization on the above-mentioned semi-finished ferment to obtain the ferment.

[0026] Further, in step S2, the sterilization condition is sterilization at 121 °C for 15 min.

[0027] Further, in step S3, the inoculation amount of Pediococcus pentosaceus MPL5 is as follows: the addition amount of the bacterial liquid is 5 to 20 parts by weight, preferably 10 parts by weight; the concentration of the bacterial liquid is 3.0×10 10 CFU / mL. The conditions for anaerobic culture are: the culture temperature is 37°C, and the culture time is 16 h.

[0028] In step S4 of the present invention, the purpose of using the low-temperature high-pressure homogenization technology is: at low temperature, due to sudden pressure reduction and high-speed impact, cell rupture occurs, and the steric hindrance between intracellular enzymes and substrates 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, greatly improving the efficacy and bioavailability of the fermented product. After human consumption of this fermented product, osteoporosis can be significantly improved.

[0029] Further, in step S4, when performing low-temperature high-pressure homogenization, the homogenization pressure is 150 to 200 MPa, for example, it can be 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa, etc. The number of cycles can be 2 to 5 times.

[0030] The fifth aspect of the present invention discloses the application of the Pediococcus pentosaceus, the microecological preparation or the fermented product in the preparation of a preparation for promoting the conversion of bound calcium in food into ionic calcium.

[0031] The sixth aspect of the present invention discloses the application of the Pediococcus pentosaceus, the microecological preparation or the fermented product in the preparation of a preparation for improving osteoporosis.

[0032] Further, the Pediococcus pentosaceus includes, but is not limited to, viable bacteria type and sterilized type.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. The Pediococcus pentosaceus MPL5 provided by the present invention can promote the conversion of bound calcium in food into ionic calcium, and the conversion rate > 60%, greatly improving the absorption and utilization of calcium by the intestine. After human consumption, it is beneficial to improve osteoporosis.

[0035] 2. The present invention uses Pediococcus pentosaceus MPL5 to ferment a composition including hazelnut kernels, oats and yellow millet. Utilizing the characteristic that Pediococcus pentosaceus MPL5 can promote the conversion of bound calcium into ionic calcium, the bound calcium rich in hazelnut kernels, oats and yellow millet is converted into ionic calcium, making the fermented product contain rich ionic calcium and having high bioavailability, which is beneficial to improving osteoporosis.

[0036] 3. The low-temperature high-pressure homogenization technology in the present invention can not only promote the autolysis of strain cells, fully release intracellular nutrients, but also protect the biological activities of strain metabolites, greatly improving the efficacy and bioavailability of fermentation products. After human consumption of this fermented product, osteoporosis can be significantly improved. Detailed implementation manners

[0037] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description 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.

[0039] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0040] Strain screening:

[0041] Peel the ripe mangoes in storage, take the pulp, and first isolate multiple acid-producing strains on the bromocresol purple MRS solid medium by methods such as dilution plate coating and streaking purification; secondly, further screen out a strain with high organic acid production efficiency through the litmus milk color reaction test and the determination of acid production performance. According to morphological characteristics, physiological and biochemical characteristics, and third-generation 16S amplicon sequencing analysis and identification, this strain is Pediococcus pentosaceus, which is named MPL5.

[0042] Gene sequence identification results:

[0043]

[0044] Test Example 1: Determination of the Calcium Binding Conversion Rate of Pediococcus pentosaceus MPL5

[0045] 1. Experimental strain: Pediococcus pentosaceus MPL5

[0046] 2. Sample preparation

[0047] (1) Preparation of culture medium

[0048] Weigh 104.48 g of MRS liquid medium powder, add 2000 mL of pure water, stir to dissolve, measure 100 mL, denoted as solution A, and the rest is denoted 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 media, denoted as culture medium A and culture medium C respectively.

[0049] (2) Activation of bacterial strain

[0050] Take out the freeze-dried preservation tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile physiological saline, aspirate all the bacterial liquid onto the MRS solid medium, spread evenly, and anaerobically culture at 37 °C for 24 hours. Pick 2 loops of colonies into 10 mL of culture medium A, and after anaerobic culture at 37 °C for 24 hours, adjust the bacterial liquid concentration to 1.0×10 9 CFU / mL to obtain the bacterial liquid.

[0051] (3) Culture

[0052] Aspirate 1 mL of the bacterial liquid into 100 mL of culture medium C, mix evenly, and anaerobically culture for 0 h, 4 h, 8 h, 12 h, 16 h, and 20 h respectively, denoted as group A, group B, group C, group D, group E, and group F in sequence. Each group is made in 3 parallels, and the culture temperature is 37 °C to obtain the culture solutions.

[0053] (4) Centrifugation

[0054] Centrifuge the culture solution with a centrifuge for 10 minutes under the conditions of 4 °C and 8000 r / min. At the end of centrifugation, separate the precipitate and the supernatant to obtain the supernatant, weigh it, wash the precipitate with 2 times the volume of clear water to obtain the precipitate, and weigh it.

[0055] 3. Determination of the calcium binding and ionic calcium conversion rates

[0056] (1) Preparation of calcium color reagent

[0057] Prepare a calcium chromogenic reagent by mixing storage solution 1, storage solution 2, and methanol in a ratio of 20:9:14, and prepare it immediately before use. Among them, the preparation method of storage solution 1: Weigh 32.5 mg of o-cresolphthalein complexone, add 100 ml of water, add 7.5 mL of concentrated hydrochloric acid, fully dissolve it, and make up the volume to 500 mL in a volumetric flask; the preparation method of storage solution 2: Measure 21 mL of diethylamine, add water to make up the volume to 500 mL in a volumetric flask, and mix well.

[0058] (2)Drawing of working curve

[0059] Respectively pipette 0.25, 0.50, 0.75, 1.00, 1.50 mL of 3 mg / mL calcium standard solution into a 10 mL volumetric flask and make up the volume to obtain calcium standard solutions with different concentrations. Measure the absorbance of calcium standard solutions with different concentrations at 570 nm, and draw a standard curve with calcium ion concentration and absorbance as the abscissa and ordinate.

[0060] (3)Weigh the precipitate and supernatant. Take 0.1 mg of the sample and add it to a 5 mL stoppered colorimetric tube. Use purified water equal to the sample volume to replace the sample as a reagent blank. Accurately add 4 mL of the calcium chromogenic reagent and mix well. After 5 minutes, use a 0.5 cm colorimetric cell to measure the absorbance at 570 nm. Use the standard curve method to measure the calcium content in the supernatant and precipitate of different groups, calculate the conversion rate of bound calcium, and find the average value (round off according to the rounding method, retain two decimal places after the decimal point), and the results are shown in Table 1.

[0061] 4. Results and analysis

[0062]

[0063] Calcium carbonate and milk calcium are both bound calcium formed by the combination of calcium and other compounds and are insoluble in water. Therefore, after centrifugation, they exist in the precipitate. During the fermentation process, a 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 can be seen from Table 1, compared with Group A, the calcium content in the precipitate of Groups B - F continuously decreases, the calcium content in the supernatant continuously increases, and the conversion rate continuously increases, indicating that with the prolongation of the fermentation time, the bound calcium is continuously converted into ionic calcium by the strain. Among them, the conversion rate of Group F is the highest, and the difference in the conversion rate between Group E and Group F is the smallest, indicating that after 16 hours of fermentation, the growth status of the strain tends to be saturated, the conversion of bound calcium in the culture solution tends to be saturated, and the conversion rate of bound calcium into ionic calcium is > 60%. Example 1

[0064] This example provides a fermented product that promotes the conversion of bound calcium in food into ionic calcium, including the following steps:

[0065] S1. Raw material selection: Select dried hazelnut kernels, oats, and yellow millet that are free from mildew and insect damage, as well as qualified fructooligosaccharide, isomaltooligosaccharide, and stachyose.

[0066] S2. Cleaning and weighing: Wash the hazelnut kernels, oats, and yellow millet with clean water 3 times. After draining, weigh 30 parts of hazelnut kernels, 30 parts of oats, and 15 parts of yellow millet, and mix them evenly to obtain mixture A.

[0067] S3. Cell wall breaking: Weigh 500 parts of purified water, mix it evenly with mixture A, break the cells with a cell wall breaker for 1 minute at a rotation speed of 36000 r / min, then pour it into a boiling pot, boil for 20 minutes, replenish the lost water, and break the cells with a cell wall breaker for 0.5 minute while it is still hot. Filter successively through 60-mesh and 100-mesh filter screens, and collect the filtrate, denoted as liquid B.

[0068] S4. Blending: Weigh 10 parts of fructooligosaccharide, 10 parts of isomaltooligosaccharide, and 5 parts of stachyose, mix them evenly, add them to liquid B, and stir to dissolve to obtain the product, denoted as liquid C.

[0069] S5. Sterilization: Sterilize liquid C under the conditions of 121 °C for 15 minutes, and cool it to 37 °C to obtain the composition, denoted as composition D.

[0070] S6. Strain activation: Take out the freeze-dried preservation tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile physiological saline, suck all the bacterial liquid onto the MRS solid medium, spread it evenly, and culture it anaerobically at 37 °C for 24 hours. Pick 2 loops of colonies into 10 mL of composition D, and after culturing anaerobically at 37 °C for 24 hours, adjust the bacterial liquid concentration to 3.0×10 10 CFU / mL to obtain the bacterial liquid, denoted as bacterial liquid E.

[0071] S7. Inoculation and fermentation: Take 5 parts by weight of bacterial liquid E, add it to composition D, mix it evenly, culture it anaerobically at 37 °C for 16 hours, and refrigerate it for 8 hours to obtain the semi-finished product, denoted as semi-finished product F.

[0072] S8. Homogenization: Homogenize semi-finished product F at low temperature and high pressure. The homogenization pressure is 150 MPa, the number of cycles is 3 times, and the temperature is 4 °C. The final product obtained is the fermented product.

[0073] Examples 2 to 4 and Comparative Example 1

[0074] The preparation methods of Examples 2 to 4 and Comparative Example 1 are the same as those of Example 1, except that: the addition amount of the bacterial liquid is different. The specific addition amounts of the bacterial liquid are shown in Table 2.

[0075] Table 2

[0076]

[0077] Test Example 2: Determination of ionic calcium content

[0078] 1. Take 5 g of the fermented product, wash and centrifuge it with 5 g of purified water, collect the supernatant, and perform 3 sets of parallel tests for each example and comparative example.

[0079] 2. According to the flame atomic absorption spectrometry method in the "National Food Safety Standard for the Determination of Calcium in Foods GB 5009.92", determine the calcium content of the supernatant, and calculate the average value (rounding according to the rounding-off method, retaining two decimal places), and the results are shown in Table 3.

[0080] 3. Results and analysis

[0081] Table 3

[0082]

[0083] After fermentation by Pediococcus pentosaceus MPL5, the bound calcium in the composition is gradually converted into ionic calcium. After washing with purified water, the ionic calcium can dissolve in water. As can be seen from Table 3, compared with Comparative Example 1, the ionic calcium content in Examples 1 to 4 has increased, indicating that Pediococcus pentosaceus MPL5 can promote the conversion of bound calcium in food into ionic calcium. Among them, the ionic calcium content of the fermented product in Example 4 is the highest, and it is close to the ionic calcium content of the fermented products in Examples 2 and 3, indicating that the addition amounts of the bacterial solutions in Examples 2, 3, and 4 can all cause the bound calcium in food to be fully converted into ionic calcium, and the metabolic activities of the strains are close to saturation. Therefore, the addition amount of the bacterial solution in Example 2 is selected as the optimal addition amount, and the bound calcium of the obtained fermented product can be fully converted into ionic calcium.

[0084] Comparative Examples 2 - 8

[0085] The preparation methods of Comparative Examples 2 - 8 are the same as those of Example 2, except that: the ratios of the fermentation raw materials are different. The specific ratios of the fermentation raw materials are shown in Table 4.

[0086] Comparative Example 9

[0087] The preparation method of Comparative Example 9 is the same as that of Example 2, except that: Comparative Example 9 does not adopt the high-pressure homogenization technology, and its step S8 is: heat the semi-finished product F to room temperature (25°C), homogenize it with a colloid mill, and the number of circulation times is 3 times. The obtained final product is the fermented product. Example 5

[0088] This example provides a preparation method for a solid preparation, including the following steps:

[0089] S1. Take out the freeze-dried preservation tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile normal saline, aspirate all the bacterial liquid onto the MRS solid medium, spread it evenly, and culture anaerobically at 37 °C for 24 hours. Pick 2 loops of colonies into 10 mL of MRS liquid medium, and after culturing anaerobically at 37 °C for 24 hours, adjust the bacterial liquid concentration to 1.0×10 11 CFU / mL to obtain the bacterial liquid, denoted as bacterial liquid A.

[0090] S2. Centrifuge the bacterial liquid A to separate the precipitate and the supernatant, and mix the precipitate evenly with the lyoprotectant to obtain the bacterial liquid, denoted as bacterial liquid B.

[0091] S3. Perform vacuum freeze-drying on the bacterial liquid B, and then pulverize and sieve it to obtain the powder. The obtained powder is the viable Pediococcus pentosaceus MPL5 bacterial powder.

[0092] S4. Mix 10 parts of the Pediococcus pentosaceus MPL5 bacterial powder evenly with 90 parts of fructooligosaccharide to obtain the powder. The obtained powder is the solid preparation A.

[0093] Example 6

[0094] This example provides a preparation method of a solid preparation, including the following steps:

[0095] S1. Take out the freeze-dried preservation tube of Pediococcus pentosaceus MPL5, open it, dissolve the bacterial powder with 0.6 mL of sterile normal saline, aspirate all the bacterial liquid onto the MRS solid medium, spread it evenly, and culture anaerobically at 37 °C for 24 hours. Pick 2 loops of colonies into 10 mL of MRS liquid medium, and after culturing anaerobically at 37 °C for 24 hours, adjust the bacterial liquid concentration to 1.0×10 11 CFU / mL to obtain the bacterial liquid, denoted as bacterial liquid A.

[0096] S2. Centrifuge the bacterial liquid A to separate the precipitate and the supernatant, and mix the precipitate evenly with the lyoprotectant to obtain the bacterial liquid, denoted as bacterial liquid B.

[0097] S3. Perform vacuum freeze-drying on the bacterial liquid B, and then pulverize and sieve it to obtain the powder. The obtained powder is the Pediococcus pentosaceus MPL5 bacterial powder.

[0098] S4. Heat the Pediococcus pentosaceus MPL5 bacterial powder at 60 °C for 45 min, then raise the temperature to 80 °C and keep it for 20 min, and finally pulverize and sieve it to obtain the powder. The obtained powder is the inactivated Pediococcus pentosaceus MPL5 bacterial powder.

[0099] S5. Mix 10 parts of the inactivated Pediococcus pentosaceus MPL5 bacterial powder evenly with 90 parts of fructooligosaccharide to obtain the powder. The obtained powder is the solid preparation B.

[0100] Test Example 3: Determination of ionic calcium content

[0101] 1. Take 5 g of the fermentation product, wash it with 5 g of purified water, centrifuge it, and collect the supernatant. Three parallel groups are made for each example.

[0102] 2. According to the flame atomic absorption spectrometry method of "GB 5009.92 National Food Safety Standard - Determination of Calcium in Foods", determine the calcium content of the supernatant, and calculate the average value (rounding off according to the rounding method, retaining two decimal places). The results are shown in Table 5.

[0103]

[0104] Please refer to Table 5. The results show that there are differences in the ionic calcium content of the fermentation products prepared with different raw material formulations. Among them, the ionic calcium content in the fermentation product of Example 2 is the highest. This indicates that the raw material formulation has an impact on the ionic calcium content in the final fermentation product. The fermentation product obtained by fermenting the raw materials with a specific formulation in Example 2 by Pediococcus pentosaceus MPL5 has the highest ionic calcium content.

[0105] Test Example 4: Osteoporosis Mouse Model Experiment

[0106] 1. Solution Preparation

[0107] (1) 0.5% sodium carboxymethylcellulose (0.5% CMC-Na) solution: Weigh 5 g of sodium carboxymethylcellulose accurately, dissolve it in deionized water, sonicate and stir to make it dissolve evenly, make up the volume to 1000 mL, and store it at 4°C for later use.

[0108] (2) D-galactose solution (D-gal solution): Weigh 1.5 g of D-galactose accurately, dissolve it in physiological saline, stir to make it dissolve evenly, make up the volume to 100 mL, and store it at 4°C for later use.

[0109] (3) Alendronate sodium solution: Weigh 14 mg of alendronate sodium powder accurately, sonicate and stir to make it evenly distributed in the 0.5% CMC-Na solution, make up the volume to 10 mL, and store it at 4°C for later use.

[0110] 2. Experimental animals: ICR mice, 3 months old, male, body weight 28 ± 2 g, clean grade.

[0111] 3. Grouping and Modeling

[0112] Randomly divide the mice into 14 groups, with 6 mice in each group, denoted as Group A to Group N. Group A is intraperitoneally injected with an equal volume of physiological saline, and the remaining 13 groups of mice are intraperitoneally injected with D-gal solution at a dosage of 150 mg / kg, 3 times a week for 12 weeks. The specific administration method is shown in Table 6.

[0113] 4. Administration

[0114] The dosage of the drug administered to the mice in group C was 14 mg / kg, and the drug was administered by gavage once a week; the mice in groups D - L were gavaged with the fermented product of the example, and the dosage of the drug administered was 260 mg / kg. The mice in groups A and B were gavaged with an equal amount of 0.5% CMC - Na solution every day for 12 weeks. The specific drug administration method is shown in Table 6.

[0115]

[0116] 5. Collection and processing of mouse bone specimens

[0117] Take the right femur of the mouse, remove the excess tissue, and take 100 layers upward from the growth plate as the ROI area. Scan the distal femur with a layer thickness of 12 μm and perform Micro - CT detection. The detection indexes include: bone mineral density (BMD), trabecular bone separation (Tb.Sp), trabecular bone number (Tb.N), structure model index (SMI), trabecular bone connectivity (Conn), and trabecular bone connection density (Conn.Dn). The results are shown in Table 7.

[0118] 6. Results and analysis

[0119]

[0120] The results in Table 7 show that compared with group A, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of the mice in group B were significantly decreased, and the indexes such as Tb.Sp and SMI were significantly increased, indicating that the model of senile osteoporosis in mice was successfully established and the bone loss was serious.

[0121] Compared with group B, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of the mice in group C were significantly increased, and the indexes such as Tb.Sp and SMI were significantly decreased, indicating that the positive drug (alendronate sodium) has an obvious therapeutic effect on senile osteoporosis.

[0122] Compared with group B, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of the mice in groups D - K all increased to varying degrees, and the indexes such as Tb.Sp and SMI all decreased to varying degrees, indicating that the fermented products prepared with the compositions of different formulas have different improvement effects on osteoporosis. Among them, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of the mice in group J were the highest, and the indexes such as Tb.Sp and SMI were the lowest, indicating that the improvement effect of osteoporosis in mice was the best and the lost bone was well rebuilt and repaired.

[0123] Compared with Group B, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of mice in Groups M to N increased to varying degrees, while the indexes such as Tb.Sp and SMI decreased to varying degrees, indicating that both the viable and inactivated forms of Pediococcus pentosaceus MPL5 bacterial powder have an improvement effect on osteoporosis, and the improvement effects are different.

[0124] Compared with Group J, the indexes such as BMD, Tb.N, Conn, and Conn.Dn of mice in Group L decreased significantly, while the indexes such as Tb.Sp and SMI increased significantly, indicating that the improvement effect of osteoporosis in Group L mice is worse than that in Group J, and the change of homogenization conditions will have a significant impact on the effect of fermented products.

[0125] Therefore, combining the experimental results in Table 5 and Table 7, the fermented product of Group J, that is, Example 2, was selected as the best formulation. The obtained fermented product has the highest ionic calcium content and the best effect on improving osteoporosis.

[0126] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Those skilled in the art of this technology can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes, deletions, additions, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A fermentation product of Pediococcus pentosaceus, characterized in that: The Pediococcus pentosaceus is Pediococcus pentosaceus MPL5, and its deposit number is GDMCC No: 63606; the fermented product is obtained by inoculating Pediococcus pentosaceus MPL5 into a composition and fermenting it; wherein the composition is made of the following components by weight: 30 parts of hazelnut kernels, 30 parts of oats, 15 parts of yellow millet, 10 parts of oligofructose, 10 parts of isomaltooligosaccharide, 5 parts of stachyose, and 500 parts of water; 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. The fermentation semi-finished product is homogenized at low temperature and high pressure to obtain the fermentation product; In step S3, the bacterial solution used for inoculating Pediococcus pentosaceus MPL5 is 10 parts by weight, and the concentration of the bacterial solution is 3.0×10 10 CFU / mL; In step S4, when low-temperature and high-pressure homogenization is performed, the homogenization pressure is 150 MPa, the temperature is 4° C., and the number of cycles is 3 times.

2. Use of the fermented product of Pediococcus pentosaceus according to claim 1 in preparing a preparation for increasing bone density, characterized in that: The Pediococcus pentosaceus MPL5 is a viable bacteria type.

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