CN116814503A - Post-production meta-product for promoting calcium supplement and bone protection as well as preparation method and application thereof - Google Patents
Post-production meta-product for promoting calcium supplement and bone protection as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN116814503A CN116814503A CN202310934166.XA CN202310934166A CN116814503A CN 116814503 A CN116814503 A CN 116814503A CN 202310934166 A CN202310934166 A CN 202310934166A CN 116814503 A CN116814503 A CN 116814503A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- hours
- lactobacillus reuteri
- temperature
- calcium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000001737 promoting effect Effects 0.000 title claims abstract description 7
- 239000000047 product Substances 0.000 title abstract description 19
- 229940069978 calcium supplement Drugs 0.000 title abstract description 5
- 238000004519 manufacturing process Methods 0.000 title description 7
- 241000186604 Lactobacillus reuteri Species 0.000 claims abstract description 53
- 229940001882 lactobacillus reuteri Drugs 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims description 93
- 230000004151 fermentation Effects 0.000 claims description 82
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 38
- 229960005069 calcium Drugs 0.000 claims description 38
- 239000011575 calcium Substances 0.000 claims description 38
- 229910052791 calcium Inorganic materials 0.000 claims description 38
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 32
- 238000003756 stirring Methods 0.000 claims description 31
- 229910052757 nitrogen Inorganic materials 0.000 claims description 16
- 235000013305 food Nutrition 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
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- 238000009423 ventilation Methods 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 12
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- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 claims description 10
- 239000001527 calcium lactate Substances 0.000 claims description 10
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 4
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The invention belongs to the technical field of microbial preparations, and particularly relates to a metaplasia product for promoting calcium supplement and bone protection, and a preparation method and application thereof. The invention screens and obtains a lactobacillus reuteri (Lactobacillus reuteri) Nice-06 which is preserved in China center for type culture Collection, the preservation date is 2023 and 06 month 29 days, and the biological preservation number is CCTCC NO: m20231123. Based on the strain, a metaplasia product is successfully developed, which can promote skeletal development, and simultaneously improve the quality of metaplasia products by optimizing process parameters, and is more suitable for industrial production, so that the metaplasia product has good practical application value.
Description
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to a metaplasia product for promoting calcium supplement and bone protection, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Calcium is one of the necessary nutrient elements for human body, and the content of calcium in normal human body is 1200-1400 g, which is about 1.5% -2.0% of the weight of human body, and has important effect for maintaining various life activities such as human body circulation, respiration, nerve, endocrine, digestion, blood, muscle, bone, urinary tract, immunity, etc. The normal physiological state of all cells in the human body depends on the existence of calcium, and the balance of calcium metabolism maintains the overall balance of life health. However, the ability of the human body to absorb calcium gradually decreases for various reasons, such as age, life style, etc., thereby causing calcium-related diseases such as osteoporosis.
Dietary intake or calcium supplementation may achieve more calcium, but due to the limited dietary habits and foods rich in calcium, people are not always able to ingest more and sufficient dietary calcium. Therefore, calcium supplementation is effective in patients with inadequate dietary calcium intake. At present, the calcium supplementing products on the market mainly comprise inorganic calcium such as calcium carbonate, calcium lactate and the like and organic calcium such as protein calcium, bone collagen calcium and the like.
However, the bioavailability of inorganic calcium is low, side effects are easy to occur, and the bioavailability of organic calcium is high, but the cost is high. Moreover, blind and excessive calcium supplementation may result in excessive blood calcium levels in the body, increasing the burden on the kidneys, while also increasing calcium excretion, leading to urinary calculi and increased risk of cardiovascular disease. Therefore, research and development of a calcium supplementing product with high bioavailability and good absorption effect has important significance.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a metaplasia product for promoting calcium supplement and bone protection, and a preparation method and application thereof. Experiments prove that the metaproduct can effectively promote the absorption of the zebra fish to calcium and promote the growth and development of zebra fish bones. Based on the above results, the present invention has been completed.
In order to achieve the technical purpose, the invention relates to the following technical scheme:
in one aspect of the invention, a lactobacillus reuteri (Lactobacillus reuteri) Nice-06 is provided and is preserved in China center for type culture Collection (China, the preservation date is 2023 and 06 months 29 days, and the biological preservation number is CCTCC NO: m20231123.
In a second aspect of the invention, a metaproduct is provided comprising at least inactivated lactobacillus reuteri and fermentation metabolites thereof.
In a third aspect of the present invention, there is provided a method for producing the metaproduct described above, the method comprising:
s1, strain activation: streaking the lactobacillus reuteri Lactobacillus reuteri Nice-06 into an activation medium to obtain a pure strain;
s2, preparing primary seed liquid: obtaining a single colony of the lactobacillus reuteri Nice-06 obtained in the step S1, placing the single colony in a primary culture solution at 28-30 ℃ for static culture for 12-14 hours to obtain primary seed solution;
s3, preparing secondary seed liquid: inoculating the primary seed liquid into a seed tank containing a secondary culture liquid according to the inoculation amount of 0.1-5% (v/v), wherein the inoculation amount is 0.5-5%, and the secondary seed liquid is obtained by stirring and culturing at the temperature of 30-40 ℃ and the pH of 7.0 under the tank pressure of 0.03-0.06MPa for 10-14 hours at 20-50 rpm (preferably 30 rpm);
s4, primary aerobic fermentation of lactobacillus reuteri: inoculating the secondary seed solution into a fermentation tank containing sterilized culture solution, wherein the inoculation amount is 0.5-5%, the temperature is 30 ℃, the speed is 80-120r/min, the culture is carried out for 5-6 hours, the ventilation amount is 0.5-1.0L/min, and the pH value is regulated to be weak acidity;
s5, lactobacillus reuteri secondary anaerobic fermentation: after 5-6 hours, the fermentation temperature is increased to 35 ℃, the stirring rotation speed is reduced to 40-60r/min, the tank pressure is kept at 0.05MPa, nitrogen is introduced to perform anaerobic fermentation, and the nitrogen ventilation amount is 0.3-0.5L/min; natural pH, fermenting for 4-5 hours;
s6, lactobacillus reuteri three-stage facultative fermentation: after 9-11 hours, the stirring rotation speed is increased to 80-120r/min, the temperature is increased to 37 ℃, nitrogen is introduced, the tank pressure is kept at 0.05MPa, the pH value is regulated to 6.5, and the fermentation is carried out for 2-3 hours;
s7, lactobacillus reuteri four-stage aerobic fermentation: after 11-14 hours, the stirring rotation speed is unchanged, the fermentation temperature is unchanged, the tank pressure is kept at 0.05MPa, oxygen is introduced, the ventilation amount is 0.4-0.8L/min, the pH value is kept at 6.5, and the fermentation is carried out for 2-4 hours;
s8, lactobacillus reuteri five-stage facultative fermentation: 13-18 hours later, the stirring speed is reduced to 40-70r/min, the temperature is increased to 40 ℃, and 0.05-0.2% calcium lactate of the volume of the fermentation liquor is added into a fermentation tank for fermentation for 1-2 hours;
s9, lactobacillus reuteri six-stage fermentation: after 14-20 hours, the stirring speed is unchanged, the temperature is increased to 45 ℃, glycerin accounting for 0.1 percent of the volume of the fermentation broth is added, the temperature is increased to 75-95 ℃, the inactivation is kept for 60 minutes, and then the temperature is reduced to 30 ℃;
in a fourth aspect of the invention, there is provided the use of the metazoan described above in the manufacture of a product for calcium supplementation and bone protection.
The product for supplementing calcium and protecting bones can be food or medicine.
The beneficial technical effects of one or more of the technical schemes are as follows:
the lactobacillus reuteri strain is obtained through screening, a metaplasia product is successfully developed based on the lactobacillus reuteri strain, and experiments prove that the method can effectively promote the absorption of the zebra fish to calcium and promote the growth and development of zebra fish bones. Meanwhile, the preparation process condition parameters of the metagen preparation are optimized, so that the lactobacillus reuteri is subjected to hierarchical fermentation.
Since aerobic fermentation, anaerobic fermentation and facultative fermentation conditions have different effects on the growth and metabolism of lactobacillus reuteri. The combination of different fermentation modes utilizes the difference of metabolic modes of the lactobacillus reuteri in different environments, and different fermentation processes are adopted to promote the growth and metabolic activity of the lactobacillus reuteri, so that the production efficiency and the yield of the lactobacillus reuteri are improved. The oxygen can supply energy to lactobacillus reuteri, improve the production of intracellular enzymes, and metabolize organic substances such as saccharides into a series of metabolites such as organic acids and amino acids. In anaerobic fermentation, lactobacillus reuteri can produce a large amount of lactic acid under the anoxic condition by utilizing anaerobic metabolic acids such as lactic acid, acetic acid and the like generated in the organic matter metabolic process, so that the fermentation liquor presents a slightly acidic environment with low pH value. During facultative fermentation, lactobacillus reuteri switches metabolic pathways according to the oxygen content in the system. When oxygen is present, lactobacillus reuteri will use oxygen as the final electron acceptor, producing more ATP and lactic acid. When the oxygen content decreases, lactobacillus reuteri will produce ATP by lactic acid fermentation and will produce a certain amount of ethanol and small amounts of lactic and acetic acids. The organic combination of different fermentation modes can promote the metabolism of lactobacillus reuteri cell nutrient substances, increase the cell number, increase the lactobacillus reuteri and improve the quality and the number of cell metabolic products. Therefore, the technical scheme has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing the phenotype of fluorescence intensity of vertebra of a postnatal effect zebra fish according to the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In one embodiment of the invention, provided is Lactobacillus reuteri (Lactobacillus reuteri) Nice-06, which is preserved in China center for type culture Collection (address: university of Wuchang mountain and Wuhan, hubei province), with a preservation date of 2023, month and 29, and a biological preservation number of CCTCC NO: m20231123.
In yet another embodiment of the invention, a metaproduct is provided comprising at least inactivated lactobacillus reuteri and fermentation metabolites thereof.
Accordingly, in yet another embodiment of the present invention, there is provided a method for producing the above-described metaproduct, the method comprising:
s1, strain activation: streaking the lactobacillus reuteri Lactobacillus reuteri Nice-06 into an activation medium to obtain a pure strain;
wherein the composition of the activation medium is as follows: every 1000 mL of culture medium contains 10g of protein, 10g of calcium phosphate, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 15g of agar powder.
S2, preparing primary seed liquid: obtaining a single colony of the lactobacillus reuteri Nice-06 obtained in the step S1, placing the single colony in a primary culture solution at 28-30 ℃ for static culture for 12-14 hours to obtain primary seed solution;
the primary culture solution comprises the following components: each 1000 mL of culture medium contains 10g of protein, 10g of calcium lactate, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate.
S3, preparing secondary seed liquid: inoculating the primary seed solution into a seed tank containing a secondary culture solution according to an inoculation amount of 0.1-5% (preferably 0.5%, v/v), wherein the inoculation amount is 0.5-5% (preferably 2%), stirring and culturing at a temperature of 30-40 ℃ (preferably 37 ℃) and a pH of 7.0, and a tank pressure of 0.03-0.06MPa (preferably 0.05 MPa) for 10-14 hours (preferably 12 hours) to obtain the secondary seed solution;
the composition of the secondary culture solution is as follows: every 1000 mL of culture medium contains 5g of protein, 5g of calcium lactate, 5g of beef powder, 4g of yeast powder, 5g of fructo-oligosaccharide, 1mL of Tween 80 and has natural pH.
S4, primary aerobic fermentation of lactobacillus reuteri: inoculating the secondary seed solution into a fermentation tank containing sterilized culture solution, wherein the inoculation amount is 0.5-5% (preferably 2%), the temperature is 30 ℃, the speed is 80-120r/min (preferably 100 r/min), the culture is carried out for 5-6 hours, the ventilation is 0.5-1.0L/min, and the pH value is regulated to be weak acidity;
the composition of the culture solution is as follows: every 1000 ml of culture medium contains 3g of xylooligosaccharide, 2g of protein, 2g of peptone, 5g of isomaltooligosaccharide, 3g of water-soluble starch, 1ml of tween-80, 5g of glycine, 5g of tyrosine, 0.2% of polyether defoamer and natural pH value.
S5, lactobacillus reuteri secondary anaerobic fermentation: after 5-6 hours, the fermentation temperature is increased to 35 ℃, the stirring rotation speed is reduced to 40-60r/min (preferably 50 r/min), the tank pressure is kept at 0.05MPa, nitrogen is introduced to perform anaerobic fermentation, and the nitrogen ventilation amount is 0.3-0.5L/min; natural pH, fermenting for 4-5 hours;
s6, lactobacillus reuteri three-stage facultative fermentation: after 9-11 hours, the stirring rotation speed is increased to 80-120r/min (preferably 100 r/min), the temperature is increased to 37 ℃, nitrogen is introduced, the tank pressure is kept at 0.05MPa, the pH value is regulated to 6.5, and the fermentation is carried out for 2-3 hours;
s7, lactobacillus reuteri four-stage aerobic fermentation: after 11-14 hours, the stirring rotation speed is unchanged, the fermentation temperature is unchanged, the tank pressure is kept at 0.05MPa, oxygen is introduced, the ventilation amount is 0.4-0.8L/min, the pH value is kept at 6.5, and the fermentation is carried out for 2-4 hours;
s8, lactobacillus reuteri five-stage facultative fermentation: 13-18 hours later, the stirring speed is reduced to 40-70r/min (preferably 60 r/min), the temperature is increased to 40 ℃, and 0.05-0.2 percent (preferably 0.1 percent) of calcium lactate by the volume of fermentation broth is added into a fermentation tank for fermentation for 1-2 hours;
s9, lactobacillus reuteri six-stage fermentation: after 14-20 hours, stirring speed is unchanged, temperature is increased to 45 ℃, glycerin accounting for 0.1% of the volume of fermentation broth is added, temperature is increased to 75-95 ℃, and the mixture is kept for 60 minutes for inactivation, and then the mixture is cooled to 30 ℃.
The preparation method further comprises a step of spray drying the fermentation broth prepared in the step S9. Wherein, the spray drying is carried out at the inlet air temperature of 150-180 ℃ and the outlet air temperature of 90-100 ℃ to obtain the metapowder product.
In yet another embodiment of the present invention, there is provided the use of the metazoan described above in the preparation of a product for calcium supplementation and bone protection.
The calcium supplementing and bone protecting product is characterized by promoting bone development, bone cell generation and cartilage injury repair.
The product for supplementing calcium and protecting bones can be food or medicine.
Furthermore, the term "food" as used in the present invention may be in any form that can be consumed, including general foods and special foods, including health foods and special medical use formulas; whereas a general food is a food suitable for all people, as opposed to a special food.
Further, the food may also include food for human consumption and food for animal consumption (such as pet food, feed additives, etc.).
The medicine of the present invention may also contain common carrier, excipient, diluent, etc. Further, the composition can be formulated into various dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions by a usual method.
The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.
In yet another embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
The subject of the pharmaceutical product may be a human or non-human animal including vertebrates such as fish, amphibians, reptiles, birds and mammals. The present invention is not particularly limited herein.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
A metaplasia for supplementing calcium and protecting bones and a preparation method thereof comprise the following steps:
marking lactobacillus reuteri Lactobacillus reuteri Nice-06 preserved in a low-temperature refrigerator at-80 ℃ to a culture medium flat plate under a sterile operation table, screening single bacterial colonies, repeatedly activating the single bacterial colonies for 3 times to obtain pure strain, inoculating the cultured single bacterial colonies to a 100mL liquid culture medium in a triangular flask, and standing and culturing for 14 hours at 28 ℃; inoculating the above cultured seed solution into a seed tank under aseptic condition, culturing at 37deg.C, pH7.0 and tank pressure of 0.05MPa for 12 hr under stirring at 30 rpm with inoculum size of 0.5%.
Primary fermentation: inoculating the second-level seed liquid into a sterilized fermentation tank, wherein the inoculum size is 2%, the temperature is 30 ℃, the culture is 100r/min, the ventilation is 0.5-1.0L/min, and the pH value is regulated to 6.0 by sodium hydroxide;
secondary anaerobic fermentation: after 5 hours, the fermentation temperature is reduced to 35 ℃, the stirring rotation speed is reduced to 50r/min, the tank pressure is kept at 0.05MPa, the oxygen is stopped to be introduced, 99% nitrogen is introduced instead, the nitrogen introducing amount is 0.4L/min, the pH value is natural, and the fermentation is carried out for 5 hours;
and (3) three-stage facultative fermentation: after 10 hours, the stirring rotation speed is increased to 100r/min, the temperature is increased to 37 ℃, nitrogen is introduced, the tank pressure is kept at 0.05MPa, the pH value is regulated to 6.5 by sodium hydroxide, and the fermentation is carried out for 2 hours;
four-stage aerobic fermentation: after 12 hours, the stirring rotation speed is unchanged, the fermentation temperature is unchanged, the tank pressure is kept at 0.05MPa, oxygen is introduced, the ventilation amount is 0.6L/min, the pH value is kept at 6.5 through an acid and alkali automatic regulation system, and the fermentation is carried out for 3 hours;
five-stage facultative fermentation: after 15 hours, the stirring speed is reduced to 60r/min, the temperature is increased to 40 ℃, and calcium lactate accounting for 0.1 percent of the volume of the fermentation broth is added into a fermentation tank for fermentation for 2 hours;
six-stage fermentation: after 17 hours, the stirring speed is unchanged, the temperature is increased to 45 ℃, glycerin accounting for 0.1 percent of the volume of the fermentation broth is added, the temperature is increased to 80 ℃, the inactivation is carried out for 60 minutes, and then the temperature is reduced to 30 ℃;
spray drying: setting the air inlet temperature at 150-180 ℃ and the air outlet temperature at 90-100 ℃ and carrying out spray drying to obtain the metapowder.
Example 2
A metaplasia for supplementing calcium and protecting bones and a preparation method thereof comprise the following steps:
marking lactobacillus reuteri Lactobacillus reuteri Nice-06 preserved in a low-temperature refrigerator at-80 ℃ to a culture medium flat plate under a sterile operation table, screening single bacterial colonies, repeatedly activating the single bacterial colonies for 3 times to obtain pure strain, inoculating the cultured single bacterial colonies to a 100mL liquid culture medium in a triangular flask, and standing and culturing for 14 hours at 28 ℃; inoculating the above cultured seed solution into a seed tank under aseptic condition, culturing at 37deg.C, pH7.0 and tank pressure of 0.05MPa for 12 hr under stirring at 30 rpm with inoculum size of 0.5%.
Primary fermentation: inoculating the second-level seed liquid into a sterilized fermentation tank, wherein the inoculum size is 2%, the temperature is 30 ℃, the speed is 100r/min, the culture is carried out for 12 hours, the ventilation is 0.5-1.0L/min, and the pH value is regulated to 6.0 by sodium hydroxide;
secondary anaerobic fermentation: after 12 hours, the fermentation temperature is reduced to 35 ℃, the stirring rotation speed is reduced to 50r/min, the tank pressure is kept at 0.05MPa, the oxygen is stopped to be introduced, 99% nitrogen is introduced instead, the nitrogen introducing amount is 0.4L/min, the pH value is natural, and the fermentation is carried out for 4 hours;
four-stage facultative fermentation: after 16 hours, the stirring speed is reduced to 60r/min, the temperature is increased to 40 ℃, and calcium lactate accounting for 0.1 percent of the volume of the fermentation broth is added into a fermentation tank for fermentation for 2 hours;
five-stage fermentation: after 18 hours, the stirring speed is unchanged, the temperature is increased to 45 ℃, glycerin accounting for 0.1 percent of the volume of the fermentation broth is added, the temperature is increased to 80 ℃, the inactivation is carried out for 60 minutes, and then the temperature is reduced to 30 ℃;
spray drying: setting the air inlet temperature at 150-180 ℃ and the air outlet temperature at 90-100 ℃ and carrying out spray drying to obtain the metapowder.
Example 3
A metaplasia for supplementing calcium and protecting bones and a preparation method thereof comprise the following steps:
marking lactobacillus reuteri Lactobacillus reuteri Nice-06 preserved in a low-temperature refrigerator at-80 ℃ to a culture medium flat plate under a sterile operation table, screening single bacterial colonies, repeatedly activating the single bacterial colonies for 3 times to obtain pure strain, inoculating the cultured single bacterial colonies to a 100mL liquid culture medium in a triangular flask, and standing and culturing for 14 hours at 28 ℃; inoculating the above cultured seed solution into a seed tank under aseptic condition, culturing at 37deg.C, pH7.0 and tank pressure of 0.05MPa for 12 hr under stirring at 30 rpm with inoculum size of 0.5%.
Primary fermentation: inoculating the second-level seed liquid into a sterilized fermentation tank, wherein the inoculum size is 2%, the temperature is 30 ℃, the speed is 100r/min, the culture is carried out for 12 hours, the ventilation is 0.5-1.0L/min, and the pH value is regulated to 6.0 by sodium hydroxide;
secondary fermentation: after 12 hours, the fermentation temperature is reduced to 35 ℃, the stirring rotation speed is reduced to 50r/min, the tank pressure is kept at 0.05MPa, the oxygen is stopped to be introduced, 99% nitrogen is introduced instead, the nitrogen introducing amount is 0.4L/min, the pH value is natural, and the fermentation is carried out for 6 hours;
and (3) tertiary fermentation: after 18 hours, the stirring speed is unchanged, the temperature is increased to 45 ℃, glycerin accounting for 0.1 percent of the volume of the fermentation broth is added, the temperature is increased to 80 ℃, the inactivation is carried out for 60 minutes, and then the temperature is reduced to 30 ℃;
spray drying: setting the air inlet temperature at 150-180 ℃ and the air outlet temperature at 90-100 ℃ and carrying out spray drying to obtain the metapowder.
Effect verification
1. Zebra fish experiment
Normal development 3dpf (day post fertilization) wild AB-series zebra fish are taken as experimental animals, normal zebra fish are selected under a stereoscopic microscope, the normal zebra fish are randomly divided into a normal control group and a metaplasia group, 10 tails of each group are arranged, and 3 compound holes are arranged. Adding zebra fish culture water into a normal control group, adding sample solution with a final concentration of 1.0% into metazoan groups, placing the zebra fish groups into a constant temperature incubator with a temperature of 28 ℃ for culture, changing water every day, and observing and detecting the bone calcium level of the zebra fish by using calcein after 96 hours of treatment. Bones mainly comprise calcified structures, and calcein is a fluorescent dye which can be combined with calcium ions in vivo and can be used for detecting the bone calcium level of zebra fish.
The calcium supplementation efficacy of the samples was evaluated by measuring the number of vertebrae in the zebra fish spine and the calcium level in the zebra fish body. As shown in FIG. 1, it can be seen that the metaproduct obtained in example 1 using a combination of aerobic fermentation, anaerobic fermentation and facultative fermentation can cause a significant increase in bone calcium.
2. Crowd experiment
40 inclusion groups aged 30-60 years, 20 subjects orally administrated 600mg of metazoan (product prepared in example 1) on an empty stomach every morning, 20 subjects took the same dose of placebo for 8 weeks, and bone density increase was measured after the end of administration. The original eating habit is not changed during drinking, and the food is normally eaten.
After the end of the administration period, 100% of the subjects indicated that knee pain was present before administration, and symptoms disappeared or reduced after administration.
Table 1 crowd test statistics table
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.
Claims (10)
1. Lactobacillus reuteri (Lactobacillus reuteri) Nice-06, which is preserved in China center for type culture Collection, with a preservation date of 2023 and a biological preservation number of CCTCC NO: m20231123.
2. A metagen comprising at least the inactivated lactobacillus reuteri of claim 1 and its fermentation metabolites.
3. A method of producing a metafile according to claim 2, wherein the method of producing comprises:
s1, strain activation: streaking the lactobacillus reuteri Lactobacillus reuteriNice-06 into an activation medium to obtain a pure strain;
s2, preparing primary seed liquid: obtaining a single colony of the lactobacillus reuteri Nice-06 obtained in the step S1, placing the single colony in a primary culture solution at 28-30 ℃ for static culture for 12-14 hours to obtain primary seed solution;
s3, preparing secondary seed liquid: inoculating the primary seed solution into a seed tank containing a secondary culture solution according to an inoculation amount of 0.1-5% (preferably 0.5%, v/v), wherein the inoculation amount is 0.5-5% (preferably 2%), stirring and culturing at a temperature of 30-40 ℃ (preferably 37 ℃) and a pH of 7.0, and a tank pressure of 0.03-0.06MPa (preferably 0.05 MPa) for 10-14 hours (preferably 12 hours) to obtain the secondary seed solution;
s4, primary aerobic fermentation of lactobacillus reuteri: inoculating the secondary seed solution into a fermentation tank containing sterilized culture solution, wherein the inoculation amount is 0.5-5% (preferably 2%), the temperature is 30 ℃, the speed is 80-120r/min (preferably 100 r/min), the culture is carried out for 5-6 hours, the ventilation is 0.5-1.0L/min, and the pH value is regulated to be weak acidity;
s5, lactobacillus reuteri secondary anaerobic fermentation: after 5-6 hours, the fermentation temperature is increased to 35 ℃, the stirring rotation speed is reduced to 40-60r/min (preferably 50 r/min), the tank pressure is kept at 0.05MPa, nitrogen is introduced to perform anaerobic fermentation, and the nitrogen ventilation amount is 0.3-0.5L/min; natural pH, fermenting for 4-5 hours;
s6, lactobacillus reuteri three-stage facultative fermentation: after 9-11 hours, the stirring rotation speed is increased to 80-120r/min (preferably 100 r/min), the temperature is increased to 37 ℃, nitrogen is introduced, the tank pressure is kept at 0.05MPa, the pH value is regulated to 6.5, and the fermentation is carried out for 2-3 hours;
s7, lactobacillus reuteri four-stage aerobic fermentation: after 11-14 hours, the stirring rotation speed is unchanged, the fermentation temperature is unchanged, the tank pressure is kept at 0.05MPa, oxygen is introduced, the ventilation amount is 0.4-0.8L/min, the pH value is kept at 6.5, and the fermentation is carried out for 2-4 hours;
s8, lactobacillus reuteri five-stage facultative fermentation: 13-18 hours later, the stirring speed is reduced to 40-70r/min (preferably 60 r/min), the temperature is increased to 40 ℃, and 0.05-0.2 percent (preferably 0.1 percent) of calcium lactate by the volume of fermentation broth is added into a fermentation tank for fermentation for 1-2 hours;
s9, lactobacillus reuteri six-stage fermentation: after 14-20 hours, stirring speed is unchanged, temperature is increased to 45 ℃, glycerin accounting for 0.1% of the volume of fermentation broth is added, temperature is increased to 75-95 ℃, and the mixture is kept for 60 minutes for inactivation, and then the mixture is cooled to 30 ℃.
4. The process according to claim 3, wherein in step S1,
the composition of the activation medium is as follows: every 1000 mL of culture medium contains 10g of protein, 10g of calcium phosphate, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 15g of agar powder.
5. The process according to claim 3, wherein in step S2,
the primary culture solution comprises the following components: each 1000 mL of culture medium contains 10g of protein, 10g of calcium lactate, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate.
6. The process according to claim 3, wherein in step S3,
the composition of the secondary culture solution is as follows: every 1000 mL of culture medium contains 5g of protein, 5g of calcium lactate, 5g of beef powder, 4g of yeast powder, 5g of fructo-oligosaccharide, 1mL of Tween 80 and has natural pH.
7. A method of preparation as claimed in claim 3, wherein the composition of the broth used in the multistage fermentation process is as follows: every 1000 ml of culture medium contains 3g of xylooligosaccharide, 2g of protein, 2g of peptone, 5g of isomaltooligosaccharide, 3g of water-soluble starch, 1ml of tween-80, 5g of glycine, 5g of tyrosine, 0.2% of polyether defoamer and natural pH value.
8. The method of any one of claims 3 to 7, further comprising the step of spray-drying the fermentation broth obtained in step S9.
9. Use of lactobacillus reuteri according to claim 1, the metazoan according to claim 2 for the preparation of a calcium-supplementing bone-care product.
10. The use according to claim 9, wherein the product is a food or pharmaceutical product;
the calcium supplementing and bone protecting product is characterized by promoting bone development, bone cell generation and cartilage injury repair.
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