CN112195103A - Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof - Google Patents
Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof Download PDFInfo
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- CN112195103A CN112195103A CN202011087816.4A CN202011087816A CN112195103A CN 112195103 A CN112195103 A CN 112195103A CN 202011087816 A CN202011087816 A CN 202011087816A CN 112195103 A CN112195103 A CN 112195103A
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- protective agent
- drying
- lyoprotectant
- polydextrose
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 64
- 239000003223 protective agent Substances 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 244000234623 Coprinus comatus Species 0.000 title abstract description 5
- 235000004439 Coprinus comatus Nutrition 0.000 title abstract description 5
- 238000004519 manufacturing process Methods 0.000 title description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 72
- DLRVVLDZNNYCBX-UHFFFAOYSA-N Polydextrose Polymers OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(O)O1 DLRVVLDZNNYCBX-UHFFFAOYSA-N 0.000 claims description 72
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 68
- 239000004471 Glycine Substances 0.000 claims description 36
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- 235000013856 polydextrose Nutrition 0.000 claims description 36
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 35
- 229930195725 Mannitol Natural products 0.000 claims description 35
- 239000000594 mannitol Substances 0.000 claims description 35
- 235000010355 mannitol Nutrition 0.000 claims description 35
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 34
- 229930003268 Vitamin C Natural products 0.000 claims description 34
- 239000011718 vitamin C Substances 0.000 claims description 34
- 235000019154 vitamin C Nutrition 0.000 claims description 34
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 32
- 229920000053 polysorbate 80 Polymers 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 26
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 26
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 6
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- 241000894007 species Species 0.000 description 2
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 2
- DBPRUZCKPFOVDV-UHFFFAOYSA-N Clorprenaline hydrochloride Chemical compound O.Cl.CC(C)NCC(O)C1=CC=CC=C1Cl DBPRUZCKPFOVDV-UHFFFAOYSA-N 0.000 description 1
- 241000222511 Coprinus Species 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 241000233866 Fungi Species 0.000 description 1
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- 241001138501 Salmonella enterica Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000004584 Tamarindus indica Species 0.000 description 1
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- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 description 1
- 229940107187 fructooligosaccharide Drugs 0.000 description 1
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- 230000004054 inflammatory process Effects 0.000 description 1
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- 210000000936 intestine Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
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- 235000013923 monosodium glutamate Nutrition 0.000 description 1
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- 230000035764 nutrition Effects 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 229940073490 sodium glutamate Drugs 0.000 description 1
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- 230000002195 synergetic effect Effects 0.000 description 1
- 230000025366 tissue development Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 229940074410 trehalose Drugs 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
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
- C12N1/04—Preserving or maintaining viable microorganisms
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the field of microorganisms, and particularly relates to a freeze-drying protective agent, a coprinus comatus freeze-dried product and a preparation method thereof.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a freeze-drying protective agent, a coprophila freeze-dried product and a preparation method thereof.
Background
The freeze drying technology is a drying technology that freezes wet materials or solutions into solid state at a lower temperature, then directly sublimates the moisture in the solid state into gas state without passing through the liquid state under a low vacuum degree, and finally dehydrates the materials. Since the freeze-drying is performed at a low temperature and a low pressure and the moisture is directly sublimated, there are many advantages in drying the material, such as the ability to dry heat-sensitive material, and the dried material is very stable and convenient for long-term storage. Because the drying of the material is completed in a frozen state, the physical structure and molecular structure of the material are changed little, and the organization structure and appearance form of the material are better preserved compared with other drying methods. In the freeze drying process, the material has no surface hardening problem, and the material is formed into a porous sponge shape inside, so that the material has excellent rehydration property, and can be recovered to the state before drying in a short time.
Based on the above advantages, freeze-drying has been widely used in the food and drug industry, including preservation and production of microorganisms. The human microorganism is called "the second genome of human". In 2016, 13 months, the United states white House initiated the National Microbiome Initiative (NMI), which projected a U.S. investment of $ 5.21 million, bringing Microbiome research to the National strategic position. The intestinal tract is the largest micro-ecological environment in the human body and participates in a plurality of important physiological processes of nutrition absorption, energy metabolism, tissue and organ development, immune defense, endocrine regulation and the like of the human body. In recent ten years, the intestinal micro-ecology has been developed rapidly, and the human knowledge is deepened, and the intestinal micro-ecology is considered as the 'second brain' and 'second genome' of the human body, which are closely related to the human health.
Fecal bacteria transplantation (FMT) is defined as transplanting functional flora in feces of healthy people into gastrointestinal tract of patients to reconstruct new intestinal flora and realize treatment of intestinal tract and intestinal tract diseases
Since the drying object is an active microorganism, the microorganism is more fragile during the drying process and more sensitive to the drying conditions than other non-living active substances (such as proteins, enzymes, etc.). Therefore, the freeze drying process of the microorganisms is more difficult, and the most common freeze drying of the lactic acid bacteria and the probiotics requires a protective agent to be added in the formula of the freeze drying microorganism except common filling agents and excipients.
Common microbial freeze-drying protectants rely primarily on trehalose and maltodextrin. The freeze-drying protection of bacteria by trehalose and maltodextrin has been reported in a large number of studies, and particularly trehalose shows excellent protection to bacterial cells during freeze-drying. Trehalose and maltodextrin have been used in the food industry for the production of freeze-dried powders of lactic acid bacteria and probiotics. However, recent studies have shown that trehalose and maltodextrin pose potential risks. Collins j. et al published a research paper in 2018, Nature, and found that trehalose can be metabolized by some strains of clostridium difficile in the human intestinal tract, thereby improving the virulence of clostridium difficile. Maltodextrin is polysaccharide generated after partial hydrolysis of starch, and in recent years, more researches report the negative effect of edible maltodextrin on intestinal health. For example, Laudisi F et al reported on Cellular and Molecular biology and Hepatology that consumption of foods rich in maltodextrin promotes mucosal loss and exacerbates the development of intestinal inflammation. It has also been found that maltodextrin promotes the survival rate of Salmonella enterica and increases its colonization ability in the intestine (PLoS ONE 9(7): e 101789.). Another study found that maltodextrin potentiates intestinal viscosity of E.coli, which is associated with Crohn's disease (PLoS one.2012; 7(12): e 52132.). Maltodextrin impairs the antibacterial response of cells and thus inhibits the bacteriostatic mechanisms of the intestinal tract.
Chinese application patent 201610191460 discloses a protective agent for storing lactobacillus lyophilized powder at room temperature, which comprises skimmed milk powder 2-4 wt%, sodium glutamate 2-4 wt%, trehalose 5-8 wt%, maltitol 2-5 wt% and polydextrose 12-18 wt%.
Chinese patent application 201911307346 discloses a freeze-drying protective agent for protecting thallus of fermentation strain, which comprises skimmed milk powder, tamarind seed polysaccharide, polydextrose and water.
Unlike traditional lactic acid bacteria and probiotics, the flora from feces is not a single well-defined strain or species, but a complex group of a large variety of flora. Compared to lactic acid bacteria and probiotics, which are now mature and commercially available, the microorganisms in the faecal flora are more anaerobic, many of which are sensitive to environmental conditions and difficult to survive in a laboratory environment for a long time. Therefore, freeze-drying is an important means for preserving and producing fecal flora, and the search for suitable freeze-drying protective agents to protect the stability of flora in feces is an important technical barrier to the realization of human intestinal flora industrialization.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a freeze-dried powder protective agent composition which is used for preserving the activity of faecal microorganisms to the maximum extent, does not need long-term ultralow-temperature cryopreservation and widens the preservation condition of products.
The above purpose of the invention is realized by the following technical scheme:
a lyophilized protectant comprises polydextrose, mannitol, glycine, vitamin C and Tween 80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 1-60% of polydextrose, 0.1-10% of mannitol, 0.1-10% of glycine, 0.01-1% of vitamin C and 0.01-5% of Tween-80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 10-30% of polydextrose, 0.1-5% of mannitol, 0.1-5% of glycine, 0.1-0.5% of vitamin C and 0.01-5% of Tween 80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 10% polydextrose, 1% mannitol, 1% glycine, 0.3% vitamin C and 0.01% Tween 80, the balance being water; or 30% polydextrose, 0.1% mannitol, 0.1% glycine, 0.01% vitamin C and 0.05% Tween 80, and the balance water.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: polydextrose, trehalose, mannitol, glycine, vitamin C and Tween 80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 1-60% of polydextrose, 1-60% of trehalose, 0.1-10% of mannitol, 0.1-10% of glycine, 0.01-1% of vitamin C and 0.01-5% of Tween-80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 10-30% of polydextrose, 10-30% of trehalose, 0.1-5% of mannitol, 0.1-5% of glycine, 0.1-0.5% of vitamin C and 0.01-5% of Tween 80.
Preferably, the freeze-dried powder protective agent comprises the following components in percentage by mass: 10% polydextrose, 15% trehalose, 1% mannitol, 1% glycine, 0.3% vitamin C and 0.01% Tween 80, the balance being water.
The invention also provides a preparation method of the freeze-drying protective agent, which comprises the following steps: dissolving the components of the freeze-drying protective agent in water according to the proportion, stirring for 10-30 minutes, heating to 120-122 ℃, preserving heat, sterilizing for 15-30 minutes, and then cooling to room temperature to obtain the freeze-drying protective agent.
Has the advantages that:
(1) the components of the lyoprotectant provided by the invention are polydextrose, mannitol, glycine, vitamin C and Tween 80, wherein the characteristics of the high molecular polysaccharide promote the outflow of water from cells during the sample freezing stage, so as to limit the formation of ice crystals in the cells. And by increasing the viscosity of the extracellular medium, an extracellular scaffold is formed in the drying process, and the growth of extracellular crystals is reduced, so that the survival of cells is protected finally.
(2) Glycine in the freeze-drying protective agent is easy to combine with water molecules in the solution, so that the viscosity of the solution is increased, the crystallization process of water is weakened, and the purpose of protecting cells is achieved; mannitol is used as an excellent framework in the formula, and mannitol and glycine can also improve the disintegration temperature of a freeze-dried product in the composition, so that the product is easy to freeze and synergistically protect the freezing effect of microorganisms; the glycine and the vitamin C can also synergistically eliminate free radicals, improve the storage stability of the coprinus comatus freeze-dried product, improve the storage temperature and prolong the storage time. The raw materials interact with each other to preserve the activity of the mixed flora of the sample to the maximum extent.
(3) The freeze-dried fecal strain product prepared by the freeze-drying protective agent has high total fecal strain survival rate and better effect than trehalose; and can specifically improve the protection of beneficial bacteria in the fecal strain, especially bifidobacteria.
(4) Polydextrose is added into the freeze-drying protective agent, so that the freeze-drying protective agent not only can effectively protect cells, but also has the function of prebiotics, can be used as fermentable fibers by probiotics (especially bifidobacteria and lactobacillus) in human intestinal tracts, can promote the proliferation of the probiotics in the intestinal tracts, and simultaneously improves the generation of beneficial metabolites such as short-chain fatty acid (SCFA); in addition, polydextrose also produces a sweetness flavor, which can partially mask unpleasant odor of fecal bacteria;
(5) the freeze-drying protective agent can protect mixed bacteria to the maximum extent, and can treat intestinal tract and parenteral diseases by improving the quantity and activity of the beneficial bacteria and the associated metabolites;
(6) the freeze-drying protective agent can reduce potential risks brought by trehalose, maltodextrin and the like, and has potential for improving the final curative effect of a coprophilous fungi transplant product.
Detailed Description
The invention may be further understood by reference to the following examples, which illustrate some methods of making or using. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.
The freeze-drying protective agents in the examples and the comparative examples are prepared by the following method: dissolving the components of the freeze-drying protective agent in water according to the proportion, stirring for 20 minutes, heating to 120 ℃, preserving heat, sterilizing for 20 minutes, and then cooling to room temperature to obtain the freeze-drying protective agent.
Example 1
A lyoprotectant composition comprising the following components: 10% polydextrose, 1% mannitol, 1% glycine, 0.3% vitamin C and 0.01% Tween 80, the balance being water.
Example 2
A lyoprotectant composition comprising the following components: 50% of polydextrose, 5% of mannitol, 5% of glycine, 0.05% of vitamin C, 1% of Tween 80 and the balance of water.
Example 3
A lyoprotectant composition comprising the following components: 30% of polydextrose, 0.1% of mannitol, 0.1% of glycine, 0.01% of vitamin C, 0.05% of Tween 80 and the balance of water.
Example 4
A lyoprotectant composition comprising the following components: 10% of polydextrose, 0.5% of mannitol, 0.5% of glycine, 0.3% of vitamin C, 0.01% of Tween 80 and the balance of water.
Example 5
A lyoprotectant composition comprising the following components: the components of the freeze-drying protective agent comprise 3% of polydextrose, 15% of trehalose, 1% of mannitol, 1% of glycine, 0.3% of vitamin C, 0.01% of Tween 80 and the balance of water.
Example 6
A lyoprotectant composition comprising the following components: the components of the freeze-drying protective agent comprise 5% of polydextrose, 15% of trehalose, 1% of mannitol, 1% of glycine, 0.3% of vitamin C, 0.01% of Tween 80 and the balance of water.
Example 7
A lyoprotectant composition comprising the following components: 30% of polydextrose, 5% of mannitol, 5% of glycine, 0.01% of vitamin C, 5% of Tween 80, 30% of trehalose and the balance of water.
Comparative example 1
The freeze-drying protective agent comprises the following components: 5% trehalose, 0.9% sodium chloride and 0.01% tween 80, the balance being water.
Comparative example 2
The components of the freeze-drying protective agent comprise 10% of trehalose, 1% of mannitol, 1% of glycine, 0.3% of vitamin C, 0.01% of Tween 80 and the balance of water.
Comparative example 3
The freeze-drying protective agent comprises 10% of fructo-oligosaccharide, 1% of mannitol, 1% of glycine, 0.3% of vitamin C, 0.01% of Tween 8 and the balance of water.
Comparative example 4
A lyoprotectant composition comprising the following components: 10% of polydextrose, 1% of mannitol, 0.3% of vitamin C, 0.01% of Tween 80 and the balance of water.
Comparative example 5
A lyoprotectant composition comprising the following components: 10% of polydextrose, 1% of mannitol, 1% of glycine and 0.01% of tween 80, and the balance of water.
Comparative example 6
A lyoprotectant composition comprising the following components: 10% of polydextrose, 1% of glycine, 0.3% of vitamin C, 0.01% of Tween 80, 15% of trehalose and the balance of water.
Comparative example 7
A lyoprotectant composition comprising the following components: 1% mannitol, 1% glycine, 0.3% vitamin C, 0.01% Tween 80, 15% trehalose, and the balance water.
Effect example 1
Study on the influence of different formulations of the freeze-drying protective agent on the survival rate of the flora after freeze-drying
1. The fecal strain liquid is obtained by the following preparation method (the detailed preparation method is shown in Chinese patent application 2020101493463): adding sterile deoxidized normal saline into the feces of the healthy people according to the mass volume ratio of 1:3-5, stirring and homogenizing the sample, and then filtering step by step; and centrifuging the filtrate, and then resuspending the filtrate by using 50mL of sterile deoxygenated physiological saline to obtain suspension, namely the fecal bacteria liquid.
The preparation method of the coprinus freezing and drying product comprises the following steps:
(1) the fecal strain liquid and the freeze-drying protective agent solutions of the examples 1-7 and the comparative examples 1-7 are mixed according to the volume ratio of 1: vortex mixing is carried out by vortex to obtain a mixed solution of the bacterial liquid and the protective agent;
(2) and (3) sampling the bacterial liquid-protective agent in the step (1) to obtain a bacterial liquid-protective agent mixed liquid before freeze-drying, wherein the bacterial liquid-protective agent mixed liquid is used for comparing samples after freeze-drying. Freezing the rest mixed solution in a refrigerator at-80 deg.C for 30 min;
(3) and (3) putting the frozen mixed bacterium liquid-protective agent in the step (2) into a freeze dryer, freeze-drying for 48 hours, subpackaging, and refrigerating at 4 ℃ to obtain a coprinus comatus freeze-dried product.
2. Detection of sample before freeze-drying of fecal bacteria liquid
Flow cytometry of the sample from the lyophilisationAnd (5) analyzing to determine the effect of different freeze-drying protective agents on diluting the concentrated fecal bacteria liquid. Using ThermoFisher staining kit (LIVE/DEAD Baclight)TMBacterial viatility Kit) the bacteria in the pre-lyophilized Bacterial fluid-protectant mixtures of examples 1-7 and comparative examples 1-7 were stained, counted by flow cytometry, and counted. Similarly, the pre-lyophilized bacterial liquid-protective agent mixtures of examples 1 to 7 and comparative examples 1 to 7 were subjected to anaerobic culture of microorganisms using Columbia broth agar medium, counted and counted after 72 hours.
3. Detecting the freeze-dried fecal strain liquid sample
3.1 influence of Freeze-drying protective agent on Total viable count and survival rate of lyophilized fecal bacteria
The prepared fecal bacteria freeze-dried products of examples 1-7 and comparative examples 1-7 in one week are subjected to total viable count detection and counting by using a flow cytometer matching kit. Similarly, the freeze-dried products of the fecal bacteria of examples 1 to 7 and comparative examples 1 to 7 were put under anaerobic conditions and subjected to anaerobic culture of microorganisms using Columbia broth agar medium, and counted after 72 hours.
Results of the experiment
(1) Effect of different cryoprotectants on microbial population survival before lyophilization
In the evaluation of different cryoprotectants, the influence of different cryoprotectants on the total viable count in a sample is limited, and the influence results of different cryoprotectants on the total viable count are close to each other and have no significant difference. See table 1.
(2) Effect of different protectants on microbial population freeze-drying survival rates
In the evaluation of the freeze-drying effect of different cryoprotectants, the protective effect of different cryoprotectants on the total viable count in a sample is obviously different. Compared with the comparative example, the samples prepared in the examples can achieve obviously better protection effect, wherein compared with the comparative example 1, the freeze-drying protection effect of the sample prepared in the comparative example 1 is far inferior to that of the sample prepared in the example 1, the sample does not contain polydextrose, mannitol, glycine and vitamin C, and contains Tween 80, sodium chloride and trehalose; compared with the example 1, the freeze-drying effect is 64% when the trehalose is added in the case of the comparative example 2 which does not contain polydextrose, and the freeze-drying survival rate is reduced by 22% compared with the example 1; comparative example 3 compared to example 1, without polydextrose, was less effective than comparative example 2 and example 1; comparative example 4 reduced glycine compared to example 1, comparative example 5 reduced vitamin C compared to example 1, and comparative example 6 reduced mannitol compared to example 1, with the freeze-drying protection reduced to different extents, with the absence of vitamin C being most pronounced, and further example 4 being comparable to comparative examples 4 and 6 in total but with significantly better results than comparative examples 4 and 6, well demonstrating the theory that mannitol and glycine have a synergistic freeze-protection effect. See table 1 for details.
Table 1: viable count and freeze-drying survival rate of fecal bacteria protected by different formulas before and after freeze-drying
3.2 protective detection of lyophilized protectant for anaerobe, enterobacter, enterococcus, bacteroides, bifidobacterium, lactobacillus, and clostridium after fecal bacteria lyophilization
The detection method is to use culture mediums aiming at different families of the class to carry out anaerobic plate-coating culture on the fecal strain freeze-dried product samples of the example 6, the example 6 and the comparative example 7 for 72 hours, and carry out statistical analysis after counting. The media used for the different individual bacteria are shown in the table below, all of which were anaerobically cultured at 37 ℃ for 72 hours.
3. Results of the experiment
For 6 different types of bacteria (enterobacter, enterococcus, bacteroides, bifidobacterium, lactobacillus and clostridium) and anaerobic bacteria, the freeze-drying protective agent formulas of example 5 and example 6 have good protective effects on anaerobic bacteria, enterobacter, bifidobacterium, lactobacillus, enterococcus, bacteroides and other types of bacteria. The results are shown in Table 2.
Table 2: viable count and survival rate of freeze-dried different bacteria samples protected by different formulas
Survival rate greater than 100% due to plating operation error is expressed as 100% herein
Effect example 2
Research on the influence of different freeze-drying protective agent formulas on 5 strains of single strains of enterococcus faecium, bacteroides fragilis, lactobacillus salivarius, Weissella confusa and bifidobacterium respectively
1. The preparation method of the 5 strains of single strain freeze-dried product comprises the following steps:
(1) the enterococcus faecium, the lactobacillus salivarius, the weissella confusa and the bifidobacterium 4 are respectively mixed with the freeze-drying protective agent solution of the example 5, the example 6 and the comparative example 7 according to the volume ratio of 1: vortex mixing is carried out by vortex to obtain a mixed solution of the bacterial liquid and the protective agent;
(2) and (3) sampling the bacterial liquid-protective agent in the step (1) to obtain a bacterial liquid-protective agent mixed liquid before freeze-drying, wherein the bacterial liquid-protective agent mixed liquid is used for comparing samples after freeze-drying. Freezing the rest mixed solution in a refrigerator at-80 deg.C for 30 min;
(3) and (3) putting the frozen mixed bacterium liquid-protective agent in the step (2) into a freeze dryer, freeze-drying for 48 hours, subpackaging, and refrigerating at 4 ℃ to obtain freeze-dried products of each single bacterium.
2. Protective effect of freeze-drying protective agent on 5 single bacteria of enterococcus faecium, bacteroides fragilis, lactobacillus salivarius, weissella fusiformis and bifidobacterium
The detection method is to use a specific obligate culture medium for detection. The media used for the different individual bacteria are shown in the table below, and the samples of the freeze-dried products of the faecal bacteria of example 5, example 6 and comparative example 7 were incubated anaerobically at 37 ℃ for 72 hours.
3. Results of the experiment
The freeze-drying protective agent containing polydextrose can achieve better protection effect on 5 single strains of enterococcus faecium, bacteroides fragilis, lactobacillus salivarius, weissella fusca, bifidobacterium and the like. Of these, examples 5 and 6 are superior to comparative example 7 in the protective effect against enterococcus faecium, bacteroides fragilis, lactobacillus salivarius, weissella fusiformis, and bifidobacterium. See table 3 for details.
TABLE 3 Log viable count of lyophilized bacteria of different species protected by different formulations10 CFU
| Group of formulas | Enterococcus faecium | Bacteroides fragilis | Lactobacillus salivarius | Weissella fusiformis | Bifidobacterium |
| Comparative example 7 | 7.06 | 6.12 | 7.55 | 8.19 | 5.91 |
| Example 5 | 7.50 | 6.71 | 7.81 | 8.62 | 6.49 |
| Example 6 | 7.46 | 6.73 | 8.03 | 8.57 | 6.61 |
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A freeze-drying protective agent is characterized by comprising polydextrose, mannitol, glycine, vitamin C and Tween 80.
2. The lyoprotectant of claim 1, wherein said lyoprotectant comprises, in mass percent: 1-60% of polydextrose, 0.1-10% of mannitol, 0.1-10% of glycine, 0.01-1% of vitamin C and 0.01-5% of Tween-80.
3. The lyoprotectant of claim 2, wherein said lyoprotectant comprises, in mass percent: 10-30% of polydextrose, 0.1-5% of mannitol, 0.1-5% of glycine, 0.1-0.5% of vitamin C and 0.01-5% of Tween 80.
4. The lyoprotectant of claim 3, wherein said lyoprotectant comprises, in mass percent: 10% polydextrose, 1% mannitol, 1% glycine, 0.3% vitamin C and 0.01% Tween 80, the balance being water; or 30% polydextrose, 0.1% mannitol, 0.1% glycine, 0.01% vitamin C and 0.05% Tween 80, the balance being water.
5. The lyoprotectant of claim 2, further comprising trehalose.
6. The lyoprotectant of claim 5, wherein said lyoprotectant comprises, in mass percent: 1-60% of polydextrose, 1-60% of trehalose, 0.1-10% of mannitol, 0.1-10% of glycine, 0.01-1% of vitamin C and 0.01-5% of Tween-80.
7. The lyoprotectant of claim 6, wherein said lyoprotectant comprises, in mass percent: 10-30% of polydextrose, 10-30% of trehalose, 0.1-5% of mannitol, 0.1-5% of glycine, 0.1-0.5% of vitamin C and 0.01-5% of Tween 80.
8. The lyoprotectant of claim 7, wherein said lyoprotectant comprises, in mass percent: 10% polydextrose, 15% trehalose, 1% mannitol, 1% glycine, 0.3% vitamin C and 0.01% Tween 80, the balance being water.
9. A method for preparing a lyoprotectant according to any of claims 1-8, comprising the steps of: dissolving the components of the freeze-drying protective agent in water according to the proportion, stirring for 10-30 minutes, heating to 120-122 ℃, preserving heat, sterilizing for 15-30 minutes, and then cooling to room temperature to obtain the freeze-drying protective agent.
10. Use of the lyoprotectant according to claims 1-8 in a freeze-dried product of coprophila.
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Denomination of invention: Freeze dried protectants, fecal bacteria freeze-drying products and their preparation methods Granted publication date: 20210423 Pledgee: Shenzhen Branch of Guoren Property Insurance Co.,Ltd. Pledgor: SHENZHEN WEIZHIJUN BIOLOGICAL TECHNOLOGY Co.,Ltd. Registration number: Y2024980018073 |