CN100375636C - Saccharide composition with protection on microecologic prepn - Google Patents
Saccharide composition with protection on microecologic prepn Download PDFInfo
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- CN100375636C CN100375636C CNB2005101055750A CN200510105575A CN100375636C CN 100375636 C CN100375636 C CN 100375636C CN B2005101055750 A CNB2005101055750 A CN B2005101055750A CN 200510105575 A CN200510105575 A CN 200510105575A CN 100375636 C CN100375636 C CN 100375636C
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- trehalose
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Abstract
The present invention discloses a saccharide composition for protecting a microecologic preparation, which belongs to the technical field of medicine. Microecologic preparation is an important biological preparation for maintaining health. The microecologic preparation of the present invention comprises bacillus bifidus, bacterium lacticum, etc. Since has strict requirements to life conditions, the bacillus bifidus is difficult to store in a liquid state or a dry state. Presently, microecologic products on markets has the disadvantages of low survival rate of microbe, low temperature storage, high circulating cost and limited popularization and application. The present invention provides the saccharide composition (a protective agent) for protecting the microecologic preparation during the processing of drying and storage, and uses the double and cooperative protecting function of hyaluronic acid and trehalose dihydrate to improve the survival rate of micro organisms, prolong survival time and make the microecologic preparation stored for long time at normal temperature. Experiments prove that the saccharide composition can be applied to the microecologic preparation to obviously improve the survival rate, the storing stability and the activity of the micro organisms during a freeze drying process. The saccharide composition is compared with single protecting agents and conventional protecting agents, and the protecting effect is obviously improved.
Description
Technical field:
The present invention is a kind of saccharide combination (protective agent) that microbial ecological agent is had protective effect in dry and storage, belongs to medical technical field.
Background technology:
Along with the develop rapidly of microecology, or form industry over nearly 10 years, become the important biomolecule preparation of people's disease preventing and treating, maintaining healthy based on a new quasi drugs, the health food of microbial ecological agent in China.The short-term effect of its existing instant effect, as: prevention and treatment acute and chronic diarrhea, constipation, improve gastrointestinal function etc.Also have significant long-term effect, as: by reducing the toxins in human body level, improve nutriture, promote hepatic cell growth, improving body immunity etc. and play protective effect to liver, auxiliary improvement hepatopathy symptom; For degradation under the symptom that causes dysbacteriosis with the untoward reaction in chemistry, radiation, the immunosuppressant treatment such as appetite depression, weak, the numeration of leukocyte, microbial ecological agent can improve aforementioned symptom, disease resistance enhancemen.In addition, normal intestinal flora is participated in digesting and assimilating of food directly, and multivitamin and enzyme are arranged in its metabolite, can be used as nutritional health food old, young, patient.Therefore the maximum characteristics of microbial ecological agent are " ill curing the disease, not sick diseases prevention, anosis health cares ".China microecology founder professor Wei Xi once foretold: " epoch after the antibiotic will be the epoch of ecological agent ".21 century, little ecological industry will form the industry of creating huge social benefit and economic benefit.
The microbial ecological agent that the present invention protected comprises bacillus bifidus (Bifidobacterium) and lactobacillus (Lactobacillus) etc.The bacillus bifidus that is usually used in the enteral microecological formulation is the G of more difficult Long-term Storage
+, no spore, the specificity anaerobe of not moving, the conditions of existence is required very harsh, the slight variations of external environment just easily causes the death of this bacterium.In its vital movement process, also can produce metabolism products such as some lactic acid that jeopardize the breeding of self normal growth, acetic acid in addition, thus not only under the liquid condition, be difficult to preserve, even under drying regime, also be difficult for Long-term Storage.Therefore, how improving the survival rate of thalline, the shelf life of assurance microbial ecological agent, is one of technical barrier that perplexs at present the active bacteria formulation industry.Little ecological product thalline survival rate of Chu Xianing is low in the market, and all needs long-term storage at low temperatures, and circulation cost is increased, and applies being restricted.Utilize the dual of hyaluronic acid and trehalose and coordinating protection effect, the microbial survival rate is improved, survival period prolongs, and can be in the room temperature long-term storage.Also can in microbial ecological agent, add bifidus factor, as dextrinosan, oligofructose, oligomeric galactose, soybean oligo saccharide, oligomeric lactulose, oligomeric isomaltulose, oligomeric xylose, oligomeric lactulose, Oligomeric manna sugar, isomerized lactose, oligomeric dragon gallbladder sugar, Raffinose, stachyose, xylitol, mannitol, sorbitol, caseic hydrolysate, alpha lactalbumin, lactoferrin, tea polyphenols, ginsenoside etc., promote bacillus bifidus propagation.
1 hyaluronic structure, character and main effect
The straight chain mucopolysaccharide that hyaluronic acid is made up of for disaccharidase unit glucuronic acid and N-n acetylglucosamine n.In aqueous solution, hyaluronan molecule interacts to form and has certain inflexible macromolecular network structure, and this structure can be embedded in microorganism in the mesh and play a protective role; Hyaluronic acid vitrification under the higher concentration, the molecule of material that is in glassy state is very inactive, and hyaluronic acid forms glassy state in that microorganism is outside, thereby plays a protective role.
Hyaluronic acid has no side effect for organism normal group composition.
The structure of 2 trehaloses, character and main effect
Trehalose is the disaccharidase that is formed by the condensation of hemiacetal hydroxyl by two glucose molecules.Trehalose has three kinds of isomers, i.e. α, α-type, α, β-type and β, β-type.Trehalose exists two crystalline lenss, noncrystal and three kinds of forms of anhydrous crystalline.Trehalose character is highly stable.
Trehalose extensively is present in nature, waits in pteridophyta, algae, antibacterial, fungus, yeast, insecticide and the invertebrates as low.Being the material that a kind of stress state that forms for environmental change has high resistance, is biological intravital a kind of typical stress metabolite.Under various adverse circumstances, trehalose shows the good protective action such as biomembrane, protein and nucleic acid to species.Current research shows that ectogenic trehalose has good non-specific protective effect.
Trehalose is nontoxic, and human body is had no adverse reaction.
The biological protection mechanism of trehalose:
" water substitutes " hypothesis: wrapping water membrane around the biomolecule, when losing moisture film under the condition such as dry, freezing, trehalose molecule can be connected with hydrogen bond with biomolecule at the dehydration position, with the structure moisture film that replaces losing, and does not cause the biomolecule loss of activity.
" glassy state " hypothesis: when biotic component is dry, trehalose closely encases adjacent molecule, form a kind of structurally with the similar sugared vitreous body of glass ice, its diffusion coefficient is very low, molecular motion and molecule degeneration are very faint, can make biomolecule keep certain space structure.
" preferential exclusion " hypothesis: the water molecules of the preferential and protein surface of trehalose, the proteinic solvated layer radius of result reduces, and molecular structure is tightr, and conformation is more stable, helps resisting the influence of extraneous extreme environment.
As seen from the above, in the organism dehydration, trehalose plays unique protective effect to biomembrane, protein etc., improves the survival rate of microorganism.
3 hyaluronic acids and trehalose combination are as protectant advantage of microbial ecological agent
Because hyaluronic acid is a macromolecular compound, can not enter microbial cell inside, but can form tridimensional network microorganism is embedded in the mesh.Trehalose is a micromolecular compound, can penetrate into cell interior and play a role.We are used in combination hyaluronic acid and trehalose, all have protective agent like this inside and outside microbial cell, and protective effect is better than single protective agent and GPF (General Protection False agent.
Summary of the invention
The purpose of this invention is to provide a kind of saccharide combination (protective agent) that in dry and storage, microbial ecological agent is had protective effect.
The present invention has the saccharide of protective effect to be combined as hyaluronic acid and trehalose to microbial ecological agent, and both concentration are respectively 0.01%～1.5%, 0.1%～50%.
Hyaluronic acid of the present invention is meant the hyaluronic acid in any source, comprise by animal tissue's extraction separation get, microbial fermentation and must reach genetic engineering preparation and must wait.
Hyaluronic acid of the present invention comprises that hyaluronic acid and its esters are as: hyaluronate sodium (claim not only hyaluronic acid sodium), Curiosin (but also claiming glass acid zinc) etc.
Hyaluronic acid of the present invention is meant the hyaluronic acid of any relative molecular mass.
Trehalose of the present invention is meant the trehalose in any source, comprises by plant, animal tissue's extraction separation getting microbial fermentation and must reach the genetic engineering preparation and must wait.
Trehalose of the present invention is meant the trehalose of any existence form, comprises α, α-type, α, β-type and β, β-type, two crystalline lenss, noncrystal and anhydrous crystalline.
The microbial ecological agent that the present invention protected comprises bacillus bifidus and lactobacillus.
The bacillus bifidus that the present invention protected comprises one or more in bifidobacterium bifidum (B.bifidum), bifidobacterium adolescentis (B.adolescentis), bifidobacteria infantis (B.infants), bifidobacterium longum (B.longum) and the bifidobacterium breve (B.breve).Lactobacillus comprises bacillus acidophilus (L.acidophilus), Lactobacillus bulgaricus (L.bulgaricus), lactobacillus casei (L.casei), Lactobacillus gasseri (L.gasseri)) and Lactobacillusreuteri (L.reuteri) in one or more.
The microbial ecological agent that the present invention protected also can add bifidus factor, promotes bacillus bifidus propagation.Bifidus factor comprises dextrinosan, oligofructose, oligomeric galactose, soybean oligo saccharide, oligomeric lactulose, oligomeric isomaltulose, oligomeric xylose, oligomeric lactulose, Oligomeric manna sugar, isomerized lactose, oligomeric dragon gallbladder sugar, Raffinose, stachyose, xylitol, mannitol, sorbitol, caseic hydrolysate, alpha lactalbumin, lactoferrin, tea polyphenols, ginsenoside etc.
The microbial ecological agent that the present invention protected has the intestinal microecology of keeping balance, intestinal tract diseases such as the constipation that treatment causes because of dysbacteriosis, diarrhoea; The enhancing human body immunity function; Reduce cholesterolemia and triglyceride; Prevention, inhibition tumor take place; The anti-ageing effect of waiting for a long time.
The present invention has the saccharide combination (protective agent) of protective effect to fill a prescription to microbial ecological agent in dry and storage: in 100mL protective agent solution, and hyaluronic acid 0.01～1.5g wherein, trehalose 0.1～50g, all the other are deionized water.
The present invention has saccharide combination (protective agent) preparation method of protective effect to be to microbial ecological agent in dry and storage: the saccharide combination formula and the consumption that microbial ecological agent are had protective effect by the present invention; after a certain amount of hyaluronic acid, trehalose placed the small amount of deionized water stirring and dissolving; add deionized water to the certain volume standardize solution; obtain containing the solution of hyaluronic acid and trehalose; with the filtering with microporous membrane degerming of this solution, be protective agent of the present invention.
The present invention has the application process of the saccharide combination of protective effect to be to microbial ecological agent in dry and storage: bacillus bifidus, lactobacillus strain activation, cultivate and CO respectively at 37 ℃ of constant temperature anaerobism
2Cultivate 16～18h, make cell concentration all reach 1 * 10
11More than the cfu/mL, with above-mentioned bacterium liquid in 4 ℃ respectively with 4000r-min
-1Centrifugal 20min collects thalline, and with cold sterile deionized water washed twice, recentrifuge is collected, and two kinds of thalline are suspended in the protective agent solution with 1: 1 mixed, and protectant dosage makes cell concentration greatly about l * 10
11Cfu/mL.Can add bifidus factor, bifidus factor is handled through degerming in advance.Mixed bacteria liquid is carried out dried.Drying means comprises lyophilization, vacuum drying and spray drying.
The specific embodiment:
The embodiment of the invention 1:
Get hyaluronic acid 0.01g, trehalose 1g, use the deionized water dissolving mixing, be settled to 10ml,, get protective agent of the present invention with 0.22 μ m filtering with microporous membrane degerming.Get above-mentioned protective agent 0.5mL, take by weighing the wet thallus 0.1mg after centrifugal, be suspended in the protective agent.Add the bifidus factor of the prior degerming of a certain amount of warp more as required, get mixed bacteria liquid.
Mixed bacteria liquid is sub-packed in the aseptic tool plug vial, and first pre-freeze is put into the about 25h of freeze dryer lyophilization again to-35 ℃ approximately, gets the lyophilizing mycopowder.Residual moisture reaches 2%～3% in the requirement product.
The embodiment of the invention 2;
Get hyaluronic acid 0.01g, trehalose 1g, use the deionized water dissolving mixing, be settled to 10ml,, get protective agent of the present invention with 0.22 μ m filtering with microporous membrane degerming.Get above-mentioned protective agent 0.5mL, take by weighing the wet thallus 0.1mg after centrifugal, be suspended in the protective agent.Add the bifidus factor of the prior degerming of a certain amount of warp more as required, get mixed bacteria liquid.
Mixed bacteria liquid is sub-packed in the aseptic tool plug vial, puts in the vacuum drier in vacuum 20～30Pa, 30 ℃ of about 48h of following vacuum drying of product temperature, dry mycopowder.Residual moisture reaches 2%～3% in the requirement product.
The embodiment of the invention 3:
Get hyaluronic acid 0.01g, trehalose 1g, use the deionized water dissolving mixing, be settled to 10ml,, get protective agent of the present invention with 0.22 μ m filtering with microporous membrane degerming.Get above-mentioned protective agent 0.5mL, take by weighing the wet thallus 0.1mg after centrifugal, be suspended in the protective agent.Add the bifidus factor of the prior degerming of a certain amount of warp more as required, get mixed bacteria liquid.
Mixed bacteria liquid is sub-packed in the aseptic tool plug vial, puts and carry out spray drying in the centrifugal spray dryer, inlet temperature is 120 ℃, and outlet temperature is 80 ℃, gets dry mycopowder.Residual moisture reaches 2%～3% in the requirement product.
Experimentation data of the present invention
This experiment is not to add the negative contrast after the same manner lyophilizing of protectant bacterium liquid; be respectively the positive contrast of protectant lyophilizing mycopowder group with 10% sucrose, 10% lactose, 0.1% hyaluronic acid and 10% trehalose; investigated in the embodiment of the invention protective agent to the protection effect of lyophilizing microbial ecological agent, investigating index is that thalline survival rate and thalline produce sour power.Experimental result is shown in table 1～table 3.
Protective agent is to the influence of lyophilizing thalline survival rate in table 1 embodiment of the invention
| Group | Survival rate (%) |
| Unprotect agent 10% sucrose, 10% lactose 0.1% hyaluronic acid, 10% trehalose protective agent of the present invention | 55.2 75.5 80.9 71.4 85.6 95.3 |
Protective agent produces the influence of sour power in table 2 embodiment of the invention to the lyophilizing thalline
| Group | Produce sour power (%) | |
| Before the lyophilizing | After the lyophilizing | |
| Unprotect agent 10% sucrose, 10% lactose 0.1% hyaluronic acid, 10% trehalose protective agent of the present invention | 0.20 0.21 0.23 0.22 0.21 0.23 | 0.08 0.10 0.12 0.09 0.15 0.19 |
Protective agent is to the influence of lyophilizing thalline storage-stable in table 3 embodiment of the invention
| Group | Per 1 gram lyophilizing mycopowder viable count (1gN) | |
| 4℃ | 25℃ | |
| 0d 30d 60d | 0d 30d 60d | |
| Unprotect agent 10% sucrose, 10% lactose 0.1% hyaluronic acid, 10% trehalose protective agent of the present invention | 10.11 9.10 8.07 10.59 10.08 9.14 10.82 10.32 9.37 10.56 10.10 9.04 10.92 10.37 10.16 11.59 10.94 10.59 | 10.11 8.76 7.24 10.59 9.17 7.52 10.82 9.34 7.73 10.56 9.02 7.17 10.92 9.98 9.05 11.59 10.71 10.11 |
Above result shows; hyaluronic acid and trehalose applied in any combination are in the protective agent of microbial ecological agent; can significantly improve survival rate, vigor and the storage-stable of freeze-drying process microorganism; use hyaluronic acid and trehalose all to have protective effect separately, both are used in combination but effect is not as in the embodiment of the invention.Protective agent of the present invention obviously improves than the protection effect of GPF (General Protection False agent (as sucrose, lactose).
Claims (5)
- Hyaluronic acid and trehalose compositions at microbial ecological agent in the dry and storage as protectant purposes.
- 2. claims 1 described purposes, wherein hyaluronic concentration is 0.01%～1.5% in the compositions, and the concentration of trehalose is 0.1%～50%, and all the other are deionized water.
- 3. the described purposes of claim 1, wherein microbial ecological agent is bacillus bifidus and lactobacillus.
- 4. the described purposes of claim 3, wherein bacillus bifidus is one or more in bifidobacterium bifidum, bifidobacterium adolescentis, bifidobacteria infantis, bifidobacterium longum and the bifidobacterium breve; Lactobacillus is one or more in bacillus acidophilus, Lactobacillus bulgaricus, lactobacillus casei, Lactobacillus gasseri and the Lactobacillusreuteri.
- 5. claims 1 or 2 described purposes, wherein hyaluronic acid is hyaluronic acid and salt thereof: hyaluronate sodium, Curiosin.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005101055750A CN100375636C (en) | 2005-09-29 | 2005-09-29 | Saccharide composition with protection on microecologic prepn |
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| CNB2005101055750A CN100375636C (en) | 2005-09-29 | 2005-09-29 | Saccharide composition with protection on microecologic prepn |
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| CN100375636C true CN100375636C (en) | 2008-03-19 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952771B (en) * | 2012-11-19 | 2013-12-25 | 陕西科技大学 | Streptococcus thermophilus freeze-drying protective agent and preparation method thereof |
| CN108486048A (en) * | 2018-03-05 | 2018-09-04 | 贵州泛特尔细胞生物技术有限公司 | A kind of protein stabiliser, its application method and preparation method thereof |
| CN111728937B (en) * | 2020-07-21 | 2022-09-06 | 华熙生物科技股份有限公司 | Private protective suppository filling containing zinc hyaluronate and preparation method thereof |
| CN112189850A (en) * | 2020-09-17 | 2021-01-08 | 合生元（广州）健康产品有限公司 | Use of prebiotics to promote survival of probiotic bacteria in the gastrointestinal tract |
| CN112940942A (en) * | 2021-04-09 | 2021-06-11 | 江苏省农业科学院 | High-heat-resistance heat stabilizer, microbial preparation, and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357037A (en) * | 1999-06-22 | 2002-07-03 | 埃里克·爱德华·沃勒尔 | Method for preserving biologically active material |
| CN1507878A (en) * | 2002-12-15 | 2004-06-30 | 东北农业大学 | Method for preparing lactobacillus dry-powder product |
| CN1651090A (en) * | 2004-11-30 | 2005-08-10 | 中国海洋大学 | Transmission system of medicine containing trehalose and hyaluronic acid for eye part and its preparation method |
| CN1836731A (en) * | 2005-08-30 | 2006-09-27 | 山东省生物药物研究院 | Saccharide combination possessing protective function for life active compound |
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- 2005-09-29 CN CNB2005101055750A patent/CN100375636C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357037A (en) * | 1999-06-22 | 2002-07-03 | 埃里克·爱德华·沃勒尔 | Method for preserving biologically active material |
| CN1507878A (en) * | 2002-12-15 | 2004-06-30 | 东北农业大学 | Method for preparing lactobacillus dry-powder product |
| CN1651090A (en) * | 2004-11-30 | 2005-08-10 | 中国海洋大学 | Transmission system of medicine containing trehalose and hyaluronic acid for eye part and its preparation method |
| CN1836731A (en) * | 2005-08-30 | 2006-09-27 | 山东省生物药物研究院 | Saccharide combination possessing protective function for life active compound |
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