CN114164112A - Microbial protection composition and preparation method and application thereof - Google Patents
Microbial protection composition and preparation method and application thereof Download PDFInfo
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- CN114164112A CN114164112A CN202111342704.3A CN202111342704A CN114164112A CN 114164112 A CN114164112 A CN 114164112A CN 202111342704 A CN202111342704 A CN 202111342704A CN 114164112 A CN114164112 A CN 114164112A
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The invention provides a microbial protection composition and a preparation method and application thereof, and relates to the fields of microbes and new medicines and pharmacy. The microbial protection composition includes a composition of a polysaccharide, an acidic amino acid, and a basic amino acid. The preparation method comprises the following steps: s1, dissolving polysaccharide, stirring or high shearing, and sterilizing; s2, dissolving acidic amino acid and basic amino acid in water, stirring and sterilizing; s3, mixing the polysaccharide solution and the amino acid solution to obtain the microbial protection composition. The microbial protection composition provided by the invention is simple in component and wide in application range, and has a good protection effect on various microorganisms. The preparation method of the microbial protection composition has the advantages of simple process, low cost and good industrial application prospect.
Description
Technical Field
The invention belongs to the field of microorganisms and new medicine pharmacy, and particularly relates to a microorganism protection composition and a preparation method and application thereof.
Background
The intestinal flora is the "forgotten organ" of the human body, which is closely related to human health, and the imbalance of the intestinal flora has been shown to be associated with many diseases. Restoration of intestinal flora imbalance by means of Fecal transplantation (FMT) has been applied in clinical practice. The fecal strain transplantation is to transplant functional flora in feces of healthy people into gastrointestinal tracts of patients to reconstruct new intestinal flora so as to realize treatment of intestinal tract and parenteral diseases. There are several clinical medical guidelines and consensus that recommend coprophilous transplantation for the treatment of recurrent or refractory Clostridium Difficile Infection (CDI).
The fecal bacteria transplantation mainly has two modes, one mode is invasive transplantation, for example, the fecal bacteria liquid is transplanted to the intestinal tract of a patient through a nose intestinal tube, an enteroscope and other modes; the other is oral capsule, namely, the fecal bacteria is delivered to the intestinal tract of a patient in a targeted way in the form of oral enteric capsule. The coprophilous fungi used for treatment at present mainly have two forms of freezing and freeze-drying, and the preservation of the activity of the coprophilous fungi in the freezing or freeze-drying production process is a technical basic premise and a key difficulty of coprophilous fungi transplantation. Therefore, the preparation of a freezing or freeze-drying protective agent is the key for ensuring the activity retention of the coprophila in the freezing or freeze-drying production process.
At present, glycerol is the most commonly used microbial cryoprotectant, but transplantation of fecal bacteria containing glycerol often causes symptoms such as dizziness, nausea and diarrhea, and is difficult to be used as a cryoprotectant due to its extremely strong hygroscopicity. Trehalose is also a commonly used lyoprotectant, however, experimental evidence has shown that trehalose is metabolized by some strains of Clostridium difficile in the human gut, thereby increasing the virulence of these Clostridium difficile (Dietary fungal organisms, Nature, j.collins et al, 2018). And the trehalose mainly focuses on protecting the biological components in the drying process, and the protection effect on low-temperature freezing is not good enough. Skim milk can also be used as a cryoprotectant, and is generally widely used for the lyophilization protection of microorganisms. However, skim milk is a mixture of animal sources and is limited to be used as a pharmaceutical adjuvant, and components in dairy products may cause allergic reactions in some people.
In addition, most of the microbial freezing or freeze-drying protective agents are only used for a single strain at present, and the protective agents can simultaneously protect complex flora. And is limited by the detection method, the protective effect of the protective agent is usually detected by a plate detection method or a membrane fluorescent staining method, and the method only shows that the protective agent can protect the proliferation capability and the membrane integrity of the microorganism. However, the various functions of the microorganism are often dependent on the metabolic activity of the cell, and the intracellular ATP concentration is used as a biological energy marker to better reflect the metabolic activity of the microorganism, but the marker is rarely used in the detection of freezing and freeze-drying protective agents.
Chinese patent CN202110087737.1 discloses a bifidobacterium lactis freeze-drying protective agent, which comprises the following raw materials: 6-8% of trehalose, 6-8% of maltodextrin, 1-4% of sucrose, 0.01-0.05% of vitamin C, 1-5% of glycerol, 0.1-1% of L-arginine and the balance of water. The freeze-drying protective agent has complex components, has freeze-drying protection effect only aiming at the bifidobacterium lactis, and greatly reduces the protection effect on complex flora.
Chinese patent CN202010561524.3 discloses a freeze-dried powder composite protective agent for bifidobacteria, which comprises 10-30% of skimmed milk powder, 0.1-5.0% of amino acid and salt thereof, 3-25% of disaccharide or/and polysaccharide, 1-5% of micromolecular polyhydric alcohol and the balance of phosphate buffer solution by weight percent, and the pH value of the composite protective agent is 6.5-8.5. The compound protective agent has complex components, contains animal-derived skim milk powder, is relatively limited in application, and simultaneously, the components in the dairy product can cause anaphylactic reaction of partial crowds.
Disclosure of Invention
The invention provides a microbial protection composition, a preparation method and application thereof for solving the problems. The composition has simple components and wide application range, has a protective effect on various microorganisms, and has good protective effect in the freezing and freeze-drying processes of the microorganisms.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the present invention provides a microbial protection composition comprising a combination of a polysaccharide, an acidic amino acid and a basic amino acid.
Preferably, the microbial protection composition further comprises a medicinal filler, wherein the medicinal filler is one or more of glucose, fructose, lactose, trehalose, sucrose, sorbitol, mannitol, xylitol, silicon dioxide, zinc oxide, calcium phosphate and calcium carbonate.
Preferably, the content of the polysaccharide is 1-60% by mass; more preferably, from 8% to 40%; further preferably, from 8% to 30%; still more preferably, it is 10%.
Preferably, the polysaccharide is at least one of stachyose, arabinogalactan, polydextrose, xylan, alginic acid, alginate, microcrystalline cellulose, inulin, fructooligosaccharide, maltodextrin and starch.
The polysaccharide can provide basic filling effect, and can reduce mechanical damage of pure water crystallization to microbial cells in a low-temperature process. Meanwhile, the polysaccharide has good hydrophilicity and a polyhydroxy structure, and can replace water molecules to support bacterial cell structures in the drying process.
Preferably, the total content of the acidic amino acid and the basic amino acid is 0.1 to 10 percent by mass; more preferably, from 1.05 to 5%; further preferably 2.1%.
Preferably, the acidic amino acid is one or more of aspartic acid, cysteine, glutamic acid, glycine, glutamine and the salt forms corresponding to the five amino acids.
Preferably, the basic amino acid is histidine, arginine, lysine, or one of the salt forms corresponding to the three amino acids.
Preferably, the combination of the above acidic amino acid and basic amino acid gives a pH of the microbial protection composition of 7 to 10, more preferably 7.1 to 9.8, further preferably 7.3 to 9.5, still further preferably 8.7.
The amino acid has both acidic and basic groups and has a certain buffering effect, and the combination of the acidic amino acid and the basic amino acid can form a buffering system, so that compared with a phosphate buffering system commonly used in the field and a buffering system consisting of the double amino acids, the buffering system cannot form huge pH fluctuation due to solute precipitation in the concentration process. In addition, amino acids such as glutamic acid, histidine, arginine, glycine and the like have a certain stabilizing effect on protein and are often used as freeze-drying stabilizers for protein active substances, but part of amino acid aqueous solution has strong pH value and is not beneficial to the growth of microorganisms, but the adverse factors can be effectively avoided after a buffer system consisting of acid-base amino acids is adopted.
The polysaccharide and the amino acid can generate a synergistic effect, the amino acid can partially enter the cells to protect important intracellular organelles, the polysaccharide can form an environment support outside the cells to reduce extracellular crystallization damage and water loss damage of membranes, and pores which cannot be filled by part of the polysaccharide form a stabilizing effect by the amino acid. Polysaccharides and amino acids can form more complete protection for the cellular structure of microorganisms at different positions through different mechanisms. While the amino acid is rich in-NH3And the-COOH can effectively neutralize surplus-OH which is not combined with the microbial cell structure by polysaccharide, thereby greatly reducing the hygroscopicity of the microbial freeze-dried finished product and being beneficial to the long-term storage of freeze-dried microbes.
The invention also provides a cell protection preparation, which comprises the microbial protection composition.
The invention also provides an application of the microbial protection composition in microbial treatment.
Preferably, the microbial treatment is microbial preservation, microbial storage or microbial transport.
Preferably, the microorganisms are bacteria and fungi.
The invention also provides a preparation method of the microbial protection composition, which comprises the following steps:
s1, dissolving the polysaccharide in water, stirring or high shearing, and sterilizing for later use;
s2, dissolving the acidic amino acid and the basic amino acid in water, stirring, and sterilizing for later use to obtain a composition of the acidic amino acid and the basic amino acid;
s3, mixing the polysaccharide solution obtained in the step S1 and the composition of the acidic amino acid and the basic amino acid obtained in the step S2 to obtain the microbial protection composition.
Preferably, the sterilization in the step S1 is performed by 110-122 ℃ for 15-30 minutes.
Preferably, the sterilization in the step S2 is performed by sterilizing at 110-122 ℃ for 15-30 minutes or performing filtration sterilization by using a filter membrane with a diameter less than or equal to 0.45 μm.
The invention also provides a method for detecting the microbial protection effect of the microbial protection composition, which is used for detecting the metabolic activity of microbial cells.
Preferably, the above-mentioned detection of the metabolic activity of the microbial cells is carried out by detecting the ATP content in the microbial cells.
The components and the proportion in the microbial protection composition are screened and optimized through a large number of tests, and compared with the prior art, the microbial protection composition has the following beneficial effects:
1. the microbial protection composition provided by the invention does not contain animal-derived skim milk commonly used in the prior art, avoids the problem of intolerance of part of people, and widens the applicability of the composition. The components are simple in composition, and a better protection effect is achieved ingeniously by screening specific components and proportions.
2. The microbial protection composition provided by the invention is different from a protectant composition which only protects one process of freezing or freeze-drying in the prior art, and can play an effective protection role on microbes in the processes of low-temperature freezing and freeze-drying.
3. The microbial protection composition provided by the invention has a protection effect on only a single strain or a single strain, and is different from the prior art, and the protection effect is embodied in the protection of the whole flora.
4. The method for detecting the microbial protection effect of the microbial protection composition provided by the invention is different from the prior art which only detects the proliferation capacity and the membrane integrity of microorganisms and mainly detects the metabolic activity of microbial cells, and the detection method provided by the invention is more accurate because various functions of the microorganisms depend on the metabolic activity of the cells.
Drawings
FIG. 1 shows the preservation rates of the proliferation potency of microorganisms in examples 1 to 32 and comparative examples 1 to 10, which were measured by the plate method after freezing treatment.
FIG. 2 preservation of microbial membrane integrity for each of the sets of flow cytometry methods after freezing treatment of examples 1-32 and comparative examples 1-10.
FIG. 3 shows that the preservation rates of the metabolic energies of the microorganisms in examples 1 to 32 and comparative examples 1 to 10 are quantitatively obtained for each ATP group after freezing treatment.
FIG. 4 shows the preservation rates of the proliferation capacities of the microorganisms of examples 1 to 32 and comparative examples 1 to 10, which were measured by plate method after the lyophilization treatment.
FIG. 5 shows that flow cytometry results in preservation of microbial membrane integrity for each of examples 1-32 and comparative examples 1-10 after lyophilization.
FIG. 6 shows that after the freeze-drying treatment, the preservation rate of the metabolic energy of the microorganisms in examples 1 to 32 and comparative examples 1 to 10 is obtained quantitatively for each ATP group.
FIG. 7 is a graph showing the average retention rate of each index.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. 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 experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. All the raw materials are commercially available, and all the raw materials meet the hygienic standard and quality standard related to food safety, and the sources of the raw materials are not particularly limited.
Basic embodiment
The present invention provides a microbial protection composition comprising a combination of a polysaccharide, an acidic amino acid and a basic amino acid.
Preferably, the microbial protection composition further comprises a pharmaceutical filler, wherein the pharmaceutical filler is at least one of glucose, fructose, lactose, trehalose, sucrose, sorbitol, mannitol, xylitol, silicon dioxide, zinc oxide, calcium phosphate and calcium carbonate.
Preferably, the content of the polysaccharide is 1-60% by mass; more preferably, from 8% to 40%; further preferably, from 8% to 30%; still more preferably, it is 10%.
Preferably, the polysaccharide is at least one of stachyose, arabinogalactan, polydextrose, xylan, alginic acid, alginate, microcrystalline cellulose, inulin, fructooligosaccharide, maltodextrin and starch.
Preferably, the total content of the acidic amino acid and the basic amino acid is 0.1 to 10 percent by mass; more preferably, from 1.05 to 5%; further preferably 2.1%.
Preferably, the acidic amino acid is one or more of aspartic acid, cysteine, glutamic acid, glycine, glutamine and the salt forms corresponding to the five amino acids.
Preferably, the basic amino acid is histidine, arginine, lysine, or one of the salt forms corresponding to the three amino acids.
Preferably, the combination of the above acidic amino acid and basic amino acid gives a pH of the microbial protection composition of 7 to 10, more preferably 7.1 to 9.8, further preferably 7.3 to 9.5, still further preferably 8.7.
The invention also provides a cell protective agent, which comprises the microbial protection composition.
The invention also provides an application of the microbial protection composition in microbial treatment.
Preferably, the microbial treatment is microbial preservation, microbial storage or microbial transport.
Preferably, the microorganisms are bacteria and fungi.
The invention also provides a preparation method of the microbial protection composition, which comprises the following steps:
s1, dissolving the polysaccharide in water, stirring or high shearing, and sterilizing for later use;
s2, dissolving the acidic amino acid and the basic amino acid in water, stirring, and sterilizing for later use to obtain a composition of the acidic amino acid and the basic amino acid;
s3, mixing the polysaccharide solution obtained in the step S1 and the composition of the acidic amino acid and the basic amino acid obtained in the step S2 to obtain the microbial protection composition.
Preferably, the sterilization in the step S1 is performed by 110-122 ℃ for 15-30 minutes.
Preferably, the sterilization in the step S2 is performed by sterilizing at 110-122 ℃ for 15-30 minutes or performing filtration sterilization by using a filter membrane with a diameter less than or equal to 0.45 μm.
Examples 1-32 microbial protective compositions and methods of making the same
The raw material composition (mass ratio) of each composition is shown in the following table:
the preparation method comprises the following steps:
s1, dissolving the polysaccharide in water, stirring for 30min, and sterilizing for 15min at 110 ℃ for later use;
s2, stirring the amino acid composition until the amino acid composition is completely dissolved, and filtering and sterilizing the mixture by using a filter membrane with the diameter equal to 0.45 micrometer for later use;
s3, mixing the polysaccharide solution obtained in the step S1 and the amino acid composition obtained in the step S2 to obtain the microbial protection composition.
Comparative examples 1 to 10 microbial protective compositions and methods of preparation thereof
The raw material composition of each composition is shown in the following table:
the preparation method comprises the following steps:
comparative examples 1 to 8 were prepared in the same manner as in the above examples.
Comparative example 9 the preparation method was as follows:
s1, dividing the phosphate buffer into three parts according to the volume ratio of 1:1:1, dissolving the skimmed milk powder and the glycerol in one part of the phosphate buffer, and sterilizing at 107 ℃ for 15min to obtain solution A;
s2, dissolving trehalose and inulin in another phosphoric acid buffer solution, and sterilizing at 110 deg.C for 15min to obtain solution B;
s3, dissolving the amino acid and the salt thereof in the residual phosphate buffer solution, and filtering and sterilizing by using a 0.22 micron filter membrane to obtain solution C;
s4, mixing the solution A, the solution B and the solution C under the aseptic condition to obtain the comparative example 9.
Comparative example 10 the preparation method was as follows:
dissolving vitamin C in appropriate amount of water, filtering with 0.22 micrometer filter membrane for sterilization to obtain solution A; dissolving trehalose, maltodextrin, sucrose, glycerol and L-arginine with the balance of water, stirring uniformly, and sterilizing at 110 ℃ for 15min to obtain a solution B; comparative example 10 was obtained by mixing solution a and solution B uniformly.
Evaluation of the effects:
the compositions of examples 1 to 32 and comparative examples 1 to 10 were mixed with fecal bacteria at a mass ratio of 1:1, mixing.
The used fecal strain is obtained by adopting the following preparation method:
s1, collecting a fresh excrement sample by using a sterile excrement collecting box, covering a cover after sampling, conveying to a laboratory for weighing, and recording the mass;
s2, placing the sampling box in a transfer window of an anaerobic operating platform, opening a box cover to ensure gas exchange, covering the transfer window cover, and starting a deoxidation program; after 5 minutes, transferring the sampling box to the interior of an operation table;
s3, adding sterile deoxidized physiological saline according to the mass-volume ratio of 1:3-5, stirring the sample, and ensuring that the excrement is not excessively adhered to the sampling box;
s4, homogenizing the sample feces;
s5, performing step-by-step filtration on the homogenized liquid, wherein the step-by-step filtration is 1-2 mm, 400-600m and 20-80 m;
s6, transferring the filtrate into a centrifugal tube, centrifuging for 2 times, wherein the first centrifugation condition is 100g, the centrifugation time is 5-7 minutes, the second centrifugation condition is 5000g, the centrifugation time is 5-7 minutes, and collecting thallus precipitates;
s7, resuspending the suspension in 50mL sterile deoxygenated physiological saline, and obtaining the suspension, namely the fecal bacteria.
After the prepared composition and the fecal bacteria are uniformly mixed at room temperature, the freezing and freeze-drying treatment is carried out under the following conditions:
freezing: 1mL of the sample is stored at-80 ℃ for 12 hours and then taken out, and the sample is placed at room temperature for 30 minutes to melt and then subjected to activity test.
Freeze-drying: 1mL of sample is pre-frozen at-50 ℃ for 2 hours, dried at 0.05mbar for 24 hours, and after the freeze drying is finished, the freeze-dried powder is redissolved by water to the weight of the sample before freeze drying, and activity test is carried out.
The test data in the present invention are from two biological replicates, i.e. from fecal faecal bacteria supplied from two donors.
The proliferation capacity of microorganisms in the samples was examined by the spread plate method: columbia broth agar medium was plated in an anaerobic chamber, the plate was incubated at 37 ℃ for 72 hours in an anaerobic environment, and the proliferation potency preservation ratio was calculated from the ratio of the frozen or lyophilized sample to the original sample, and the results are shown in FIG. 1 and FIG. 4.
By using a ThermoFisher staining kit (LIVE/DEAD Baclight)TMBacterial Viability Kit) were stained for samples before and after freezing or lyophilization and counted by flow cytometry to calculate the membrane integrity preservation rate, the results are shown in fig. 2 and 5.
The samples before and after freezing or freeze-drying are cracked by an ATP detection kit (Biyun day), the ATP reaction luminescence intensity is detected by a fluorescence microplate reader, and the energy conservation rate is calculated, and the results are shown in figures 3 and 6.
Samples before and after freezing or freeze-drying are subjected to the activity detection, and the preservation rate of the fecal microorganisms of each composition is calculated by comparing the difference before and after the detection.
The storage ratio indexes of examples 1 to 6 were averaged to obtain the total storage ratio (FIG. 7).
From the data in the figure, the most preferred scheme, in terms of this retention rate ranking, is example 30, followed by example 11, followed by example 12 and example 28.
Claims (15)
1. A microbial protection composition comprising a combination of a polysaccharide, an acidic amino acid and a basic amino acid.
2. The microbial protection composition of claim 1, wherein the total content of acidic amino acids and basic amino acids is 0.1-10% by weight.
3. The microbial protection composition of claim 2, wherein the total content of acidic amino acids and basic amino acids is 1.05-5% by weight.
4. A microbial protection composition according to any one of claims 1 to 3 wherein the acidic amino acid is one or more of aspartic acid, cysteine, glutamic acid, glycine, glutamine and the corresponding salt forms of the five amino acids.
5. A microbial protection composition according to any one of claims 1 to 3 wherein the basic amino acid is at least one of histidine, arginine, lysine and the corresponding salt forms of the three amino acids.
6. A microbial protection composition according to any one of claims 1 to 5 wherein the polysaccharide is at least one of stachyose, arabinogalactan, polydextrose, xylan, alginic acid, alginate, microcrystalline cellulose, inulin, fructo-oligosaccharide, maltodextrin and starch.
7. The microbial protection composition of claim 6, wherein the polysaccharide is present in an amount of 1-60% by weight.
8. A microbial protection composition according to claim 6 or 7, wherein the polysaccharide is present in an amount of from 8 to 30% by weight.
9. A microbial protection composition according to any one of claims 1 to 8, wherein the combination of the acidic amino acid and the basic amino acid is such that the pH of the microbial protection composition is from 7 to 10.
10. The microbial protection composition of claim 1, further comprising a pharmaceutical filler; the medicinal filler is one or more of glucose, fructose, lactose, trehalose, sucrose, sorbitol, mannitol, xylitol, silicon dioxide, zinc oxide, calcium phosphate and calcium carbonate.
11. A cytoprotective preparation comprising the microbe-protecting composition of any of claims 1-10.
12. Use of a microbial protection composition according to any one of claims 1 to 10 in microbial treatment.
13. The use according to claim 12, wherein the microbial treatment is microbial preservation, microbial preservation or microbial transport.
14. Use according to any one of claims 12 to 13, wherein the microorganisms are bacteria and fungi.
15. A process for the preparation of a microbial protection composition according to any one of claims 1 to 10, comprising the steps of:
s1, dissolving the polysaccharide in water, stirring or high shearing, and sterilizing for later use;
s2, dissolving the acidic amino acid and the basic amino acid in water, stirring, and sterilizing for later use to obtain a composition of the acidic amino acid and the basic amino acid;
s3, mixing the polysaccharide solution obtained in the step S1 and the composition of the acidic amino acid and the basic amino acid obtained in the step S2 to obtain the microbial protection composition.
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Application publication date: 20220311 |