CN119817819B - Application of Pediococcus pentosaceus MPL5 in promoting the conversion of bound calcium in food into ionized calcium and improving osteoporosis - Google Patents
Application of Pediococcus pentosaceus MPL5 in promoting the conversion of bound calcium in food into ionized calcium and improving osteoporosis Download PDFInfo
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Abstract
本发明公开了戊糖片球菌MPL5及其促进食物中结合钙转化成离子钙、改善钙生物可及性的应用，属于微生物技术领域。本发明提供的戊糖片球菌MPL5在培养液或组合物发酵液中均能够有效促进结合钙转化成离子钙（＞60%），进而提高钙的生物利用度；还能够改善和调理骨质疏松，具体体现在：显著提高骨质疏松模型小鼠的骨密度、骨小梁数目、骨小梁连通性和骨小梁连接密度，显著降低骨小梁分离度和结构模型指数，从而减少骨质疏松的发生。本发明的戊糖片球菌MPL5在制备促进离子钙转化、辅助缓解骨质疏松的功能性食品、保健食品中，具有非常广泛的应用场景，健康安全。The present invention discloses Pediococcus pentosaceus MPL5 and its application in promoting the conversion of bound calcium in food into ionized calcium and improving calcium bioaccessibility, belonging to the field of microbial technology. The Pediococcus pentosaceus MPL5 provided by the present invention can effectively promote the conversion of bound calcium into ionized calcium (>60%) in the culture solution or the fermentation solution of the composition, thereby improving the bioavailability of calcium; it can also improve and regulate osteoporosis, which is specifically reflected in: significantly improving the bone density, trabecular number, trabecular connectivity and trabecular connection density of osteoporosis model mice, and significantly reducing the trabecular separation and structural model index, thereby reducing the occurrence of osteoporosis. The 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 ionized calcium conversion and assist in alleviating osteoporosis, and is healthy and safe.
Description
Technical Field
The invention relates to the technical field of microecology, in particular to application of Pediococcus pentosaceus MPL5 (including fermentation products thereof) in promoting food to combine with calcium to be converted into ionized calcium and improving osteoporosis.
Background
Osteoporosis is a systemic metabolic bone disease characterized by low bone mass, increased bone fragility due to destruction of bone microstructure, and susceptibility to fracture, and is common in the elderly. With age, bone and osteoblast metabolic activity, gonadal metabolic activity, food digestion, nutrient absorption, etc. are continuously declining, which can lead to the occurrence of osteoporosis. In addition, many people now have long-term bad life and eating habits such as stay up, picky, excessive drinking/coffee/carbonated beverages, etc., resulting in a large loss of bone mass, and thus, the disease also occurs in young and even minor populations.
As important substances constituting bones and teeth, calcium participates in numerous complex vital activities such as muscle contraction, synthesis and release of neurotransmitters, synthesis and secretion of hormones, blood coagulation, and the like, and is fully ingested with calcium, thereby being beneficial to improving osteoporosis. For the osteoporosis population, even though foods in daily diet contain abundant calcium, when the calcium is not well converted, the calcium requirement of human body cannot be met, and osteoporosis symptoms cannot be improved.
Most of the calcium in the food exists in a form of combined calcium, and if the calcium is required to be absorbed by human body, the calcium needs to be converted into ionized calcium so as to be quickly absorbed by intestinal tracts. How to effectively promote the conversion of the combined calcium in the food into ionized calcium is a technical problem to be solved in the field. A large number of experiments show that the probiotic bacteria capable of promoting the conversion of the combined calcium in the food into the ionized calcium can greatly improve the absorption and utilization of the intestinal tract to the calcium, and the probiotic bacteria are beneficial to improving the osteoporosis after being eaten by human bodies.
Disclosure of Invention
The invention aims to solve the technical problem of providing the Pediococcus pentosaceus MPL5, wherein the Pediococcus pentosaceus MPL5 can promote the conversion of calcium combined with food into ionized calcium, is beneficial to improving osteoporosis, has obvious effect, and is healthy and safe.
In order to solve the technical problems, the invention provides the following technical scheme:
In a first aspect, the present invention provides pediococcus pentosaceus, which is pediococcus pentosaceus (Pediococcus pentosaceus) MPL5, deposited at the cantonese province microorganism strain deposit management center (GDMCC) at month 6 of 2023, deposit number GDMCC No.63606, and disclosed in the chinese patent application publication No. CN117264852a at month 22 of 2023.
Pediococcus pentosaceus is facultative anaerobe, belongs to Pediococcus, is one kind of lactic acid bacteria, can produce great amount of organic acid with several kinds of sugar as carbon source, has no proteolytic action and is sensitive to hop preservative. 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 fungus, and has been proved to have the functions of improving the synthesis and metabolism of nutrients, inhibiting the growth of pathogenic bacteria, reducing serum cholesterol, improving the immunity of human body and the like. However, there is no strain of Pediococcus pentosaceus that has been found to promote the conversion of bound calcium in food to ionized calcium.
In the invention, a new strain of Pediococcus pentosaceus is discovered by accident in the well-stored mango pulp by a microecological preparation engineering technology research center in Guangdong province of agricultural university in south China, and is named as Pediococcus pentosaceus (Pediococcus pentosaceus) MPL5. Experiments show that the strain has the function of promoting the conversion of bound calcium in food into ionized calcium, and the conversion rate is more than 60%.
The second aspect of the invention provides a microecological preparation, wherein the microbial inoculum comprises the Pediococcus pentosaceus MPL5.
Further, the microecological formulation may comprise various formulations of Pediococcus pentosaceus MPL5 as described above, including but not limited to bacterial suspensions, fermentation broths, and the like.
The invention provides a ferment for improving osteoporosis, which is obtained by inoculating the Pediococcus pentosaceus MPL5 into a composition and fermenting;
The composition comprises, by weight, 30-40 parts of hazelnuts, 20-30 parts of oat, 10-15 parts of millet, 10-20 parts of fructo-oligosaccharides, 10-30 parts of isomaltooligosaccharides, 5-10 parts of stachyose and 500-700 parts of water.
The hazelnut kernel has high nutritive value, is called as nut king, is rich in various mineral matters such as calcium, phosphorus, iron and the like, has the content of calcium as high as 815mg/100g, is very beneficial to protecting bone health, strengthening physique, resisting fatigue and delaying aging, contains manganese elements which can enable tissues such as bones, skin, tendons and ligaments of human bodies to be firm, and also contains various vitamins with important physiological functions such as vitamins B1, B2, B6 and E, wherein the content of vitamin E is as high as 33.9 mg/100g, and has stronger antioxidation effect.
Oat is a gramineae oat crop and contains rich proteins, lipids, dietary fibers, carbohydrates, minerals, antioxidants (such as vitamin E, phenolic acid, flavone, sterol, alkaloids and the like) and other various nutrients, wherein 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, so that the oat can effectively prevent the diseases such as calcium loss and osteoporosis of the elderly and maintain the balance of calcium elements of human bodies after long-term consumption, and is also beneficial to preventing premature aging, chronic diseases, cancers, cardiovascular diseases and strokes.
The millet contains rich trace elements such as calcium, ferrum, zinc, magnesium, phosphorus and the like, which play an indispensable role in human growth and development and immunoregulation, and the millet has reasonable fatty acid composition, and the content of unsaturated fatty acids such as linolenic acid and linoleic acid is more than 85 percent, especially the content of linoleic acid is 65.05 percent. The fatty acids exist in glyceride form, have protective effect on skin, microvessels and central nervous system, can prevent and treat arteriosclerosis and liver cirrhosis, are good solvents for fat-soluble vitamins and fat-soluble pigments (carotenes), and are beneficial to human body to absorb the nutrients.
The hazelnut kernel, oat and millet all contain rich calcium elements which mainly exist in a form of combined calcium. According to the invention, the Pediococcus pentosaceus MPL5 is used as a fermentation strain, the composition containing hazelnut kernel, oat and millet is subjected to fermentation treatment, and the characteristic that the Pediococcus pentosaceus MPL5 can promote the conversion of bound calcium into ionized calcium is utilized, so that the bound calcium rich in the hazelnut kernel, oat and millet is converted into ionized calcium, so that the fermented product contains abundant ionized calcium which is easy to be absorbed and utilized by human bodies, and the osteoporosis is improved. In addition, the hazelnut kernel, oat and millet also contain rich protein, lipid, dietary fiber, carbohydrate, other mineral substances, trace elements and other nutrient elements, and the hazelnut kernel, oat and millet are easier to be absorbed by human body after fermentation treatment, so that the requirements of growth and development of the human body can be better met.
Fructo-oligosaccharide, isomaltooligosaccharide and stachyose belong to functional oligosaccharides, have high stability and are difficult to decompose by enzyme systems of human digestive tracts, but can be used as a carbon source of probiotics, and can promote the propagation and metabolism of the probiotics.
In the composition, the weight 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 of oat is 20 to 30 parts, and for example, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, and the like. The weight parts of millet are 10 to 15 parts, for example, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, etc. The fructo-oligosaccharide may be 10 to 20 parts by weight, for example, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, or the like. The weight of the isomaltooligosaccharide is 10 to 30 parts, and may be, for example, 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, or the like. The stachyose is 5 to 10 parts by weight, and for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, and the like. The water may be 500 to 700 parts by weight, for example, 500 parts, 550 parts, 600 parts, 650 parts, 700 parts, or the like.
Further, in a preferred embodiment, the composition comprises, by weight, 30 parts of hazelnut kernel, 30 parts of oat, 15 parts of millet, 10 parts of fructo-oligosaccharide, 10 parts of isomaltooligosaccharide, 5 parts of stachyose and 500 parts of water.
In a fourth aspect, the present invention provides a method for preparing a ferment for improving osteoporosis, comprising the steps of:
S1, cleaning hazelnut kernels, oat and millet according to a formula amount, uniformly mixing, adding water for wall breaking to obtain slurry, boiling the wall-broken liquid, breaking the wall again when the wall is hot, filtering, and collecting filtrate to obtain slurry;
s2, uniformly mixing fructo-oligosaccharide, isomaltooligosaccharide and stachyose according to the formula amount, adding the mixture into the feed liquid, dissolving and uniformly mixing, and sterilizing to obtain a culture medium;
s3, inoculating the Pediococcus pentosaceus MPL5 into the culture medium, and performing anaerobic culture to obtain a fermentation semi-finished product;
s4, homogenizing the fermented semi-finished product at a low temperature and a high pressure to obtain the fermented product.
Further, in step S2, the sterilization condition is sterilization at 121 ℃ for 15min.
In step S3, the inoculation amount of the Pediococcus pentosaceus MPL5 is 5-20 parts by weight, preferably 10 parts by weight, of bacterial liquid, and the concentration of the bacterial liquid is 3.0X10 10 CFU/mL. The anaerobic culture condition is that the culture temperature is 37 ℃ and the culture time is 16 hours.
In step S4 of the present invention, the purpose of using a low temperature high pressure homogenization technique is that at low temperatures, due to sudden decompression and high speed impact, the cell breaks and steric hindrance between the intracellular enzyme and the substrate is eliminated. The technology can promote autolysis of bacterial strain cells, fully release intracellular nutrients, and protect the bioactivity of bacterial strain metabolites, so that the efficacy and bioavailability of the fermented product are greatly improved. After the human body eats the ferment, the osteoporosis can be obviously improved.
Further, in step S4, when the low-temperature high-pressure homogenization is performed, the homogenization pressure may be 150 to 200mpa, for example, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200MPa, or the like. The cycle number may be 2-5.
In a fifth aspect, the invention discloses the use of said pediococcus pentosaceus, said microecological formulation or said ferment for the preparation of a formulation for promoting the conversion of calcium-bound into ionized calcium in a foodstuff.
In a sixth aspect, the invention discloses the use of Pediococcus pentosaceus, the microecological formulation or the fermentation product in the preparation of an osteoporosis improving formulation.
Further, the pediococcus pentosaceus includes, but is not limited to, live bacteria type and sterilized type.
Compared with the prior art, the invention has the beneficial effects that:
1. the Pediococcus pentosaceus MPL5 provided by the invention can promote the conversion of calcium combined in food into ionic calcium, the conversion rate is more than 60%, and the absorption and utilization of calcium by intestinal tracts are greatly improved. After being eaten by human bodies, the food is beneficial to improving osteoporosis.
2. The invention utilizes the Pediococcus pentosaceus MPL5 to ferment the composition comprising hazelnut kernel, oat and millet, and utilizes the characteristic that the Pediococcus pentosaceus MPL5 can promote the conversion of combined calcium into ionized calcium, so that the combined calcium rich in the hazelnut kernel, oat and millet is converted into ionized calcium, the ferment contains rich ionized calcium, and the bioavailability is high, thereby being beneficial to improving osteoporosis.
3. The low-temperature high-pressure homogenization technology of the invention not only can promote the autolysis of cells of the strain and fully release intracellular nutrients, but also can protect the biological activity of metabolites of the strain, so that the efficacy and bioavailability of the fermentation product are greatly improved. After the human body eats the ferment, the osteoporosis can be obviously improved.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental methods used in the following examples and comparative examples are conventional methods unless otherwise specified, and materials, reagents and the like used, unless otherwise specified, are commercially available.
Strain screening:
Peeling mango well stored, taking pulp, firstly separating a plurality of acidogenic strains on a bromocresol purple MRS solid culture medium by methods of dilution plate coating, streaking purification and the like, and secondly, further screening out a strain with high organic acid production efficiency by a litmus milk chromogenic test and acid production energy measurement. According to morphological characteristics, physiological and biochemical characteristics and the third generation 16S amplicon sequencing analysis and identification, the strain is Pediococcus pentosaceus and named as MPL5.
Gene sequence identification results:
aagcatgcgcgtgctatacatgcagtcgaacgaacttccgttaattgattatgacgtacttgtactgattgagattttaacacgaagtgagtggcgaacgggtgagtaacacgtgggtaacctgcccagaagtaggggataacacctggaaacagatgctaataccgtataacagagaaaaccgcatggttttcttttaaaagatggctctgctatcacttctggatggacccgcggcgtattagctagttggtgaggcaaaggctcaccaaggcagtgatacgtagccgacctgagagggtaatcggccacattgggactgctagatggtggggactcctacgggaggcagcagtagggaatcttccacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaagggtttcggctcgtaaagctctgttgttaaagaagaacgtgggtaagagtaactgtttacccagtgacggtatttaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggatttattgggcgtaaagcgagcgcaggcggtcttttaagtctaatgtgaaagccttcggctcaaccgaagaagtgcattggaaactgggagacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaggcggctgtctggtctggacatggatacaggtggcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaaacgatgattactaagtgttggagggtttccgcccttcagtgctgcagctaacgcattaagtaatccgcctggggagtacgaccgcaaggttgaaactcaaaagaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagctacgcgaagaaccttaccaggtcttgacatcttctgacagtctaagagattagaggttcccttcggggacagaatgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattactagttgccagcattaagttgggcactctagtgagactgccggtgacaaaccggaggaaggtggggacgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagtcgcgagaccgcgaggttaagctaatctcttaaaaccattctcagttcggactgtaggctgcaactcgcctacacgaagtcggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagtttgtaacacccaaagccggtggggtaaccttttaggagctagccgtctaagcgat.
Test example one determination of calcium-binding conversion of Pediococcus pentosaceus MPL5
1. Experimental Strain Pediococcus pentosaceus MPL5
2. Sample preparation
(1) Preparation of the culture Medium
The MRS liquid culture medium powder 104.48g is weighed, 2000mL of purified water is added, stirring and dissolving are carried out, 100mL is measured, the solution is marked as solution A, and the rest is marked as solution B. 5.0g of calcium carbonate and 5.0g of milk calcium are added into the solution B and stirred uniformly, thus obtaining the solution C. Sterilizing the solution A and the solution C for 15min at 121 ℃, and cooling to 37 ℃ to obtain culture mediums which are respectively marked as culture medium A and culture medium C.
(2) Strain activation
Taking out the freeze-drying preservation tube of Pediococcus pentosaceus MPL5, opening, dissolving bacterial powder with 0.6mL of sterile physiological saline, absorbing all bacterial liquid onto MRS solid culture medium, uniformly coating, carrying out anaerobic culture at 37 ℃ for 24 hours, picking 2-ring bacterial colony into 10mL of culture medium A, carrying out anaerobic culture at 37 ℃ for 24 hours, and regulating the bacterial liquid concentration to 1.0X10 9 CFU/mL to obtain bacterial liquid.
(3) Culturing
And (3) sucking 1mL of bacterial liquid into 100mL of culture medium C, uniformly mixing, and performing anaerobic culture for 0h, 4h, 8h, 12h, 16h and 20h respectively, wherein the groups are sequentially marked as group A, group B, group C, group D, group E and group F, 3 groups are parallel, and the culture temperature is 37 ℃ to obtain the culture liquid.
(4) Centrifuging
The culture broth was centrifuged for 10 minutes with a centrifuge at 4℃and 8000 r/min. And (3) separating the precipitate and the supernatant after centrifugation to obtain supernatant, weighing, washing the precipitate with 2 times of clear water to obtain precipitate, and weighing.
3. Determination of conversion of bound calcium and ionized calcium
(1) Preparation of calcium developer
The storage solution 1, the storage solution 2 and the methanol are prepared into a calcium color developing agent according to the proportion of 20:9:14, and the calcium color developing agent is prepared before use. The preparation method of the storage liquid 1 comprises the steps of weighing 32.5 mg of o-cresolphthalein complexing agent, adding 100. 100 ml of water, adding 7.5 of mL of concentrated hydrochloric acid, fully dissolving, and then fixing the volume into a 500 mL volumetric flask, and the preparation method of the storage liquid 2 comprises the steps of weighing 21 mL of diethylamine, adding water, fixing the volume into the 500 mL volumetric flask, and uniformly mixing.
(2) Working curve drawing
And respectively sucking 0.25, 0.50, 0.75, 1.00 and 1.50 mL of 3mg/mL of calcium standard solution into a 10 mL volumetric flask for constant volume to obtain calcium standard solutions with different concentrations. The spectroscopies of calcium standard solutions with different concentrations are measured at 570nm, and standard curves are drawn by taking the calcium ion concentration and the absorbance as the abscissa.
(3) The precipitate and supernatant were weighed, 0.1mg of the sample was added to a 5mL cuvette with plug, and the sample was replaced with purified water equivalent to the sample as a reagent blank. Accurately adding 4mL of calcium color developing agent, and uniformly mixing. After 5min, the sample was subjected to colorimetric reaction at 570nm in a 0.5cm cuvette. The calcium content of the supernatant and precipitate of each group was measured by standard curve method, and the conversion of bound calcium was calculated, and the result was averaged (two bits after decimal place were retained by rounding off) and shown in table 1.
4. Results and analysis
Both calcium carbonate and milk calcium are bound calcium obtained by binding calcium with other compounds, and are insoluble in water, and therefore exist in the precipitate after centrifugation. During fermentation, a portion of the bound calcium can be converted to ionized calcium, i.e., the calcium is in ionic form and readily soluble in water and thus, after centrifugation, is present in the supernatant. As shown in Table 1, the calcium content of the precipitate from group B to group F was continuously decreased, the calcium content of the supernatant was continuously increased, and the conversion rate was continuously increased, which indicates that the combined calcium was continuously converted into ionized calcium by the strain with the increase of fermentation time. The conversion rate of the group F is highest, the conversion rate difference between the group E and the group F is smallest, and the growth condition of the strain tends to be saturated after 16h of fermentation, the conversion condition of the combined calcium in the culture solution tends to be saturated, and the conversion rate of the combined calcium into ionic calcium is more than 60%.
Example 1
This example provides a fermented product for promoting the conversion of bound calcium in food to ionized calcium comprising the steps of:
s1, selecting raw materials, namely, selecting dried hazelnut kernels, oat and millet which are free of mildew and worm damage, and enabling the raw materials to be fructo-oligosaccharides, isomaltooligosaccharides and stachyose which are qualified in quality.
S2, cleaning and weighing, namely cleaning hazelnut kernels, oat and millet with clear water for 3 times, draining, weighing 30 parts of hazelnut kernels, 30 parts of oat and 15 parts of millet, and uniformly mixing to obtain a mixture A.
S3, breaking the wall, namely weighing 500 parts of purified water, uniformly mixing the purified water with the mixture A, breaking the wall for 1min by using a wall breaking machine at the rotating speed of 36000r/min, pouring the mixture into a boiling pot, boiling for 20min, supplementing lost water, breaking the wall for 0.5min by using the wall breaking machine when the mixture is hot, filtering the mixture by using a 60-mesh and 100-mesh filter screen in sequence, and collecting filtrate which is recorded as feed liquid B.
And S4, blending, namely weighing 10 parts of fructo-oligosaccharide, 10 parts of isomaltooligosaccharide and 5 parts of stachyose, uniformly mixing, adding the mixture into the feed liquid B, stirring and dissolving to obtain the feed liquid C.
And S5, sterilizing the feed liquid C under the sterilization condition of 121 ℃ and 15min, and cooling to 37 ℃ to obtain a composition, namely a composition D.
S6, activating strains, namely taking out a freeze-drying preservation tube of the Pediococcus pentosaceus MPL5, opening, dissolving bacterial powder by using 0.6mL of sterile physiological saline, sucking all bacterial liquid onto an MRS solid culture medium, uniformly coating, carrying out anaerobic culture for 24 hours at 37 ℃, picking 2-ring bacterial colonies into 10mL of composition D, carrying out anaerobic culture for 24 hours at 37 ℃, and regulating the concentration of the bacterial liquid to 3.0 multiplied by 10 10 CFU/mL to obtain bacterial liquid, and recording as bacterial liquid E.
S7, inoculating and fermenting, namely adding 5 parts by weight of bacterial liquid E into the composition D, uniformly mixing, carrying out anaerobic culture at 37 ℃ for 16 hours, and refrigerating for 8 hours to obtain a semi-finished product, and marking the semi-finished product as a semi-finished product F.
S8, homogenizing the semi-finished product F at a low temperature and a high pressure, wherein the homogenizing pressure is 150MPa, the cycle times are 3 times, and the temperature is 4 ℃, so that the obtained final product is the fermented product.
Examples 2 to 4 and comparative example 1
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 the bacterial liquid added is different. Specific amounts of the bacterial liquids added are shown in Table 2.
TABLE 2
Test example II measurement of ionized calcium content
1.5 G of the fermented product was washed with 5g of purified water, centrifuged, and the supernatant was collected, and 3 sets of replicates were made for each example and comparative example.
2. The calcium content of the supernatant was measured by flame atomic absorption spectrometry according to GB 5009.92 food safety national standard food, and the results are shown in Table 3, wherein the measurement was averaged (the correction was performed by rounding and the decimal place was retained).
3. Results and analysis
TABLE 3 Table 3
After fermentation of the Pediococcus pentosaceus MPL5, the combined calcium in the composition is gradually converted into ionized calcium, and the ionized calcium can be dissolved in water after washing with purified water. As is clear from Table 3, the amounts of ionic calcium in examples 1 to 4 were all increased as compared with comparative example 1, indicating that Pediococcus pentosaceus MPL5 promoted the conversion of bound calcium in the food into ionic calcium, wherein the fermented product of example 4 had the highest amount of ionic calcium, and the amounts of the bacterial liquids added in examples 2, 3 and 4 were all similar to the amounts of ionic calcium in the fermented products of example 2 and 3, indicating that the metabolic activities of the strains were near saturation. Therefore, the amount of the bacterial liquid added in example 2 was selected as the optimum amount, and the resultant calcium-binding fermented product was sufficiently converted into ionized calcium.
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 proportions of the fermentation raw materials are different. The specific proportions of the fermentation raw materials are shown in Table 4.
Comparative example 9
The preparation method of comparative example 9 is the same as that of example 2, except that the high-pressure homogenization technique is not adopted in comparative example 9, and the step S8 is that the semi-finished product F is heated to normal temperature (25 ℃) and homogenized by a colloid mill for 3 times, and the obtained final product is a fermented product.
Example 5
The embodiment provides a preparation method of a solid preparation, which comprises the following steps:
S1, taking out a freeze-drying preservation tube of the Pediococcus pentosaceus MPL5, opening, dissolving bacterial powder by using 0.6mL of sterile physiological saline, sucking all bacterial liquid onto an MRS solid culture medium, uniformly coating, carrying out anaerobic culture at 37 ℃ for 24 hours, picking 2-ring bacterial colonies into 10mL of MRS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 24 hours, and regulating the bacterial liquid concentration to 1.0X10 11 CFU/mL to obtain bacterial liquid, namely bacterial liquid A.
S2, centrifuging the bacterial liquid A, separating the precipitate and the supernatant, uniformly mixing the precipitate and the freeze-drying protective agent to obtain bacterial liquid, and marking the bacterial liquid as bacterial liquid B.
S3, performing vacuum freeze drying on the bacterial liquid B, crushing and sieving to obtain powder, wherein the obtained powder is live bacterial Pediococcus pentosaceus MPL5 bacterial powder.
S4, uniformly mixing 10 parts of Pediococcus pentosaceus MPL5 bacterial powder with 90 parts of fructo-oligosaccharide to obtain powder, wherein the obtained powder is a solid preparation A.
Example 6
The embodiment provides a preparation method of a solid preparation, which comprises the following steps:
S1, taking out a freeze-drying preservation tube of the Pediococcus pentosaceus MPL5, opening, dissolving bacterial powder by using 0.6mL of sterile physiological saline, sucking all bacterial liquid onto an MRS solid culture medium, uniformly coating, carrying out anaerobic culture at 37 ℃ for 24 hours, picking 2-ring bacterial colonies into 10mL of MRS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 24 hours, and regulating the bacterial liquid concentration to 1.0X10 11 CFU/mL to obtain bacterial liquid, namely bacterial liquid A.
S2, centrifuging the bacterial liquid A, separating the precipitate and the supernatant, uniformly mixing the precipitate and the freeze-drying protective agent to obtain bacterial liquid, and marking the bacterial liquid as bacterial liquid B.
S3, performing vacuum freeze drying on the bacterial liquid B, crushing and sieving to obtain powder, wherein the obtained powder is the Pediococcus pentosaceus MPL5 bacterial powder.
S4, heating the Pediococcus pentosaceus MPL5 powder at 60 ℃ for 45min, heating to 80 ℃ and keeping for 20min, and finally crushing and sieving to obtain powder, wherein the obtained powder is the inactivated Pediococcus pentosaceus MPL5 powder.
S5, uniformly mixing 10 parts of inactivated pediococcus pentosaceus MPL5 bacterial powder with 90 parts of fructo-oligosaccharides to obtain powder, wherein the obtained powder is a solid preparation B.
Test example III determination of the Ionic calcium content
1. 5G of the fermented product was washed with 5g of purified water, centrifuged, and the supernatant was collected, and 3 groups of each example were performed in parallel.
2. The calcium content of the supernatant was measured by flame atomic absorption spectrometry according to GB 5009.92 food safety national standard food, and the results are shown in Table 5.
Referring to Table 5, the results show that the fermented product prepared with different raw material compositions all have different ionic calcium contents, wherein the ionic calcium content in the fermented product of example 2 is the highest. This demonstrates that the raw material formulation has an effect on the amount of ionized calcium in the final fermented product, and that the raw material of the specific formulation in example 2 has the highest amount of ionized calcium in the fermented product obtained by fermentation of Pediococcus pentosaceus MPL 5.
Test example IV bone shortness mouse model test
1. Solution preparation
(1) 0.5% Sodium carboxymethyl cellulose (0.5% CMC-Na) solution, precisely weighing 5g sodium carboxymethyl cellulose, dissolving in deionized water, ultrasonic treating, stirring to dissolve uniformly, constant volume to 1000 mL, and preserving at 4deg.C.
(2) D-galactose solution (D-gal solution) 1.5g D-galactose is precisely weighed and dissolved in physiological saline, and stirred to be fully and uniformly dissolved, and the volume is fixed to 100mL and stored at 4 ℃ for standby.
(3) And accurately weighing 14mg of alendronate sodium powder, carrying out ultrasonic treatment and stirring to uniformly distribute the powder in 0.5% CMC-Na solution, and keeping the volume to 10mL and 4 ℃ for later use.
2. ICR mice, 3 months old, male, body weight 28+ -2 g, clean grade.
3. Grouping and molding
Mice were randomly divided into 14 groups of 6 mice each, designated as groups A through N. Group A was intraperitoneally injected with an equal volume of physiological saline and the remaining 13 groups of mice were intraperitoneally injected with D-gal solution at a dose of 150mg/kg, 3 times per week for 12 weeks. The specific modes of administration are shown in Table 6.
4. Administration of drugs
The administration dose of the group C mice is 14mg/kg, the administration dose is 1 time per week by gastric lavage, the administration dose of the fermented product of the group D mice to the group L mice is 260mg/kg, the group A mice and the group B mice are filled with the same amount of 0.5% CMC-Na solution, and the gastric lavage is carried out every day for 12 weeks. The specific modes of administration are shown in Table 6.
5. Mouse bone specimen collection and treatment
Taking the right femur of the mouse, removing redundant tissues, taking the upward 100 layers of growth plates as the ROI area, scanning the distal femur with the thickness of 12 mu m, and carrying out Micro-CT detection. The measurement indexes include bone density (BMD), bone trabecular separation degree (Tb.Sp), bone trabecular number (Tb.N), structure Model Index (SMI), bone trabecular connectivity (Conn) and bone trabecular connection density (Conn.Dn), and the results are shown in Table 7.
6. Results and analysis
The results in table 7 show that the BMD, tb.n, conn, conn.dn, etc. index of the mice in group B are significantly reduced, and the tb.sp, SMI, etc. index is significantly increased, compared with the mice in group a, indicating that the modeling of the senile osteoporosis mice was successful and the bone loss was serious.
Compared with the group B, the indexes of BMD, tb.N, conn, conn.Dn and the like of the group C mice are obviously increased, and the indexes of Tb.Sp, SMI and the like are obviously reduced, so that the positive medicament (alendronate sodium) has obvious treatment effect on senile osteoporosis.
Compared with the group B, the indexes of BMD, tb.N, conn, conn.Dn and the like of the mice in the group D-group K are increased to different degrees, and the indexes of Tb.Sp, SMI and the like are reduced to different degrees, so that the improvement effect of the fermented products prepared by the compositions with different formulas on osteoporosis is different. Wherein, the indexes of BMD, tb.N, conn, conn.Dn and the like of the J group mice are highest, the indexes of Tb.Sp, SMI and the like are lowest, which indicates that the osteoporosis improvement effect of the mice is best, and the lost bone is well rebuilt and repaired.
Compared with the group B, the indexes of BMD, tb.N, conn, conn.Dn and the like of the mice in the group M-N are increased to different degrees, the indexes of Tb.Sp, SMI and the like are reduced to different degrees, which indicates that the live bacterial type and the inactivation type of the Pediococcus pentosaceus MPL5 bacterial powder have an improving effect on osteoporosis and different improving effects.
Compared with the group J, the indexes of BMD, tb.N, conn, conn.Dn and the like of the mice in the group L are obviously reduced, the indexes of Tb.Sp, SMI and the like are obviously increased, which proves that the osteoporosis improvement effect of the mice in the group L is worse than that of the mice in the group J, and the effect of the fermented product can be obviously influenced by the change of the homogenizing condition.
Therefore, in combination with the experimental results of tables 5 and 7, the fermented product of group J, i.e., example 2, was selected as the optimal formulation, and the obtained fermented product had the highest ionic calcium content and the best osteoporosis improving effect.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Those skilled in the art will appreciate that various substitutions, changes, omissions, additions and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and that the scope of the invention is therefore not limited to the embodiments disclosed.
Claims (2)
1. A Pediococcus pentosaceus fermentation product is characterized in that Pediococcus pentosaceus MPL5 is used as Pediococcus pentosaceus, the preservation number of the Pediococcus pentosaceus MPL5 is GDMCCNo:63606, the fermentation product is obtained by inoculating Pediococcus pentosaceus MPL5 into a composition and fermenting the composition, wherein the composition is prepared from, by weight, 30 parts of hazelnut kernel, 30 parts of oat, 15 parts of millet, 10 parts of fructo-oligosaccharide, 10 parts of isomaltooligosaccharide, 5 parts of stachyose and 500 parts of water;
The preparation method of the ferment comprises the following steps:
s1, cleaning hazelnut kernels, oat and millet according to a formula amount, uniformly mixing, adding water for wall breaking to obtain slurry, boiling the slurry, breaking the wall again when the slurry is hot, filtering, and collecting filtrate to obtain feed liquid;
s2, uniformly mixing fructo-oligosaccharide, isomaltooligosaccharide and stachyose according to the formula amount, adding the mixture into the feed liquid, dissolving and uniformly mixing, and sterilizing to obtain a culture medium;
s3, inoculating the Pediococcus pentosaceus MPL5 into the culture medium, and performing anaerobic culture to obtain a fermentation semi-finished product;
S4, homogenizing the fermented semi-finished product at low temperature and high pressure to obtain the fermented product;
in the step S3, 10 parts by weight of bacterial liquid is adopted when the Pediococcus pentosaceus MPL5 is inoculated, and the concentration of the bacterial liquid is 3.0X10 10 CFU/mL;
In step S4, when low-temperature high-pressure homogenization is performed, the homogenization pressure is 150MPa, the temperature is 4 ℃, and the number of cycles is 3.
2. Use of pediococcus pentosaceus ferment according to claim 1 for the preparation of a formulation for increasing bone density, characterized in that the pediococcus pentosaceus MPL5 is of the viable bacterial type.
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