CN1317381C - Polysaccharose film of brevibacterium and its use in bacteria preservisation - Google Patents
Polysaccharose film of brevibacterium and its use in bacteria preservisation Download PDFInfo
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- CN1317381C CN1317381C CNB200410061418XA CN200410061418A CN1317381C CN 1317381 C CN1317381 C CN 1317381C CN B200410061418X A CNB200410061418X A CN B200410061418XA CN 200410061418 A CN200410061418 A CN 200410061418A CN 1317381 C CN1317381 C CN 1317381C
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Abstract
The present invention provides a pullulan film and an application in bacteria preservation thereof. The film is prepared according to a flow spreading method, the thickness of the film is from 0.02mm to 0.04mm, the tensile strength of the film is from 27MPa to 32MPa, the right-angle tear strength of the film is from 31 KN/m to 38 KN/m, the light transmission rate of the film is from 95% to 98%, and the oxygen penetrating quantity of the film is from 2.7cm<3>/m<2>. bar. d to 1.6cm<3>/m<2>. bar. d. In the application of the pullulan film in bacteria preservation, glycerol, trehalose dihydrate protecting agent and microbe bacteria are coated in the middle of two pullulan films after bacteria free treatment, after the two films are closely applied, the two films are covered by a plastic film, and environmental elements in the bacteria preservation, such as drying environment, oxygen deficiency, low temperature, nutrition deficiency, protecting agent addition, etc., can be met. The films with the bacteria are formed into albums, and therefore, the classification research of the microbe bacteria can be conveniently carried out, and the pullulan film has the advantages of small occupation area, convenient operation, no need of expensive appliance and convenient posted operation. When the pullulan film is used, a person only needs to shear a small preservation unit of a bacteria preservation piece, and microbes can grow when the small preservation unit is dissolved in a culture medium.
Description
Technical field
The present invention relates to a kind of new microbial preservation method.
Background technology
The microbial strains method for preserving is commonly used inclined plane method, sandpipe method, mineral oil preservation method, freeze-drying and liquid nitrogen cryopreservation etc.Inclined plane method, mineral oil preservation method, preservation overlong time bacterial classifications such as sandpipe method can taper off, and freeze-drying and the culture presevation of liquid nitrogen method are effective, but need the relevant device condition, and cost is higher, and liquid nitrogen also easily sets off an explosion, and is dangerous.The shared space of above-mentioned the whole bag of tricks preservation bacterial classification is big, and mailing is used all inconvenient.Existing various microbial preservation method relatively (Zhou Deqing, microbiology study course, Higher Education Publishing House 2002,5) sees the following form:
| Method | Advantage | Shortcoming |
| Inclined-plane and liquid nutrient medium are regularly transplanted preservation | Simple, cost is little, be convenient to observe to detect suitable all kinds of bacterial classifications | The preservation time is short 3-6 month, and bacterial classification is easily degenerated |
| Mineral oil covers low temperature (refrigerator) | Under given condition, make bacterial strain approach inactive state, suitable all kinds of bacterium, preservation time lengthening 1-2 | It is big to take up space, and easily variation, microbiological contamination chance are also more |
| Distilled water | Simple and practical, effect is better | Suitable face is wideless, and preservation term is short, and water quality might influence the effect of culture presevation |
| Desiccating method: do sand, soil, silica gel | Longer than the mineral oil covering preservation time, be suitable for sporogenic microorganism, preservation term 1-10 | Some bacterium is not suitable for |
| Filter paper | Some little round shape filter paper can be used to shift bacterium colony, and each only the transfer gets final product with a filter paper | Preparation work is than above more trouble, perhaps easier microbiological contamination |
| Dry freezing | Be applicable to most of bacterium, comprise actinomycetes and some fungies, yeast, be extensive use of preservation 5-15 at present | Complex operation, technical requirements is higher, and is not too suitable to the spore fungi of some suitable humidities |
| Very low temperature (making protective material in-75 ℃ Ultralow Temperature Freezer) with 20% glycerine | Be applicable to most of bacterium, yeast.Comprise actinomycetes and fungi, the metabolism state that seizes up, easy to operate, preservation term is long | Cost of equipment is higher, and general grass-roots unit is difficult to reach |
| Very low temperature (in liquid nitrogen-196 ℃) | The metabolism state that seizes up, the preservation time can reach more than 20 years | Initial cost is higher, need stable economic condition to guarantee the supply of liquid nitrogen, if using liquid phase preserves, be exposed in the air and will blast, need well exercise to use |
Summary of the invention
The objective of the invention is to the problem at above-mentioned existence, propose a kind of method of lacking the mould polysaccharide films of stalk and being used for culture presevation, this method for preserving takes up room little, and the preservation cost is low, mailing, easy to use, safe.
Realize the mould polysaccharide films of short stalk of the object of the invention, the thickness of its film is 0.02mm～0.04mm, tensile strength 27Mpa～32Mpa, angle tear strength 31KN/m～38KN/m, transmittance 95%～98%, oxygen transit dose 2.7cm
3/ m
2.bar.d～1.6cm
3/ m
2.bar.d
The preparation method of the mould polysaccharide films of described short stalk, be equipped with salivation method, its method is: at first obtain the mould polysaccharide of short stalk by fermentation, fermentation raw material adopts starch, starch obtains glucose and oligosaccharide mixture through hydrolysis, this mixture preparation substratum transforms into the mould polysaccharide of short stalk by the mould fermentation of short stalk; This polysaccharide separates, decolours, filters, is condensed into the mould polysaccharide soln of short stalk that concentration is 15g/100ml～20g/100ml through thalline; The mould polysaccharide soln of spissated short stalk is packed in the sealed vessel, give the air pressure that applies 0.08Mpa～0.15Mpa in the sealed vessel then, the nozzle aperture transfers to 0.1mm～0.2mm, and hot blast temperature transfers to 60～80 ℃, and controlling dried film thickness is 0.02mm～0.04mm.
The mould polysaccharide films of described short stalk is used for the method for preservation bacterial classification, at first its film is cut into the area size and is 8mm * 8mm～20mm * 20mm unit diaphragm, and carry out aseptically process; In the middle of the mould polysaccharide films of short stalk of aseptically process, coat glycerine and trehalose protective material and microbial strains two-layer then, outer after two films are adjacent to other two layers of plastic film sleeve envelope through aseptically process, constitute a polysaccharide films culture presevation unit; Again this culture presevation unit is assembled into the preservation volume, is preserving below-24 ℃, the time can reach 12 months, takes out the preservation unit during use, cuts off the plastics covering, and the polysaccharide membrane that will contain bacterial classification drops in the substratum, and polysaccharide membrane dissolves, and bacterial classification can be grown.
The outer layer plastic film that is adopted is high temperature resistant and heat sealable coextrusion nylon film, or the polyester composite membrane, and tolerable temperature is 121 ℃～125 ℃.
The aseptically process of the mould polysaccharide films of described short stalk, its treatment process is: the mould polysaccharide membrane of short stalk uses 15W ultraviolet lamp 20cm apart from irradiation 20 minutes.
The aseptically process of the plastic film of described covering, its treatment process is: its plastic film in Autoclave 121 ℃ the sterilization 30 minutes.
The microbial strains for the treatment of preservation among the present invention is picking from the inclined-plane and on the plate bacterium colony; coat on the mould polysaccharide membrane of the short stalk of one deck; the mould polysaccharide membrane coating of the short stalk of another layer protective material glycerine and trehalose, then two films are adjacent to, two plastic films of outer cover; and with four banding mouths; form the preservation unit, its operating process is all finished in sterilisable chamber.
Its preservation dice is assembled into the bacterium fascicle that is similar to stamp album, the yeast fascicle, and the mould fascicle, the actinomycetes fascicle, the material of volume can be a plastics polyester film etc., and each page or leaf in the volume can insert many preservation dice, and each volume is formed by some pages.
The present invention specifically is achieved like this
The first, the mould polysaccharide membrane used preparation of aseptic short stalk
At first obtain the mould polysaccharide of short stalk by fermentation, fermentation raw material adopts starch, and starch obtains glucose and oligosaccharide mixture through hydrolysis, and this mixture preparation substratum transforms into the mould polysaccharide of short stalk by the mould fermentation of short stalk.It is 15g/100ml～20g/100ml that this polysaccharide separates, decolours, filters, is condensed into concentration through thalline, must lack the mould polysaccharide films of stalk with salivation method, and the salivation method film is made up of three unit, and first unit is to adorn spissated polysaccharide in the sealed vessel; Second unit is the hydrostomia shower nozzle; The 3rd unit is the loft drier (see figure 1) of being furnished with the stainless steel tape loop, can lead to the hot blast heating in the case.Sealed vessel links to each other with pipeline with the hydrostomia shower nozzle, during the system film, apply the air pressure of 0.08Mpa～0.15Mpa in the sealed vessel, glycocalix is pressed into the hydrostomia shower nozzle and is evenly distributed on the stainless steel tape loop through nozzle, the nozzle aperture transfers to 0.1mm～0.2mm, hot blast temperature transfers to 60～80 ℃, and controlling dried film thickness is 0.02mm～0.04mm.The film that makes is cut to the area size of 8mm * 8mm～20mm * 20mm, and this polysaccharide membrane is water white, its tensile strength 27Mpa～32Mpa, angle tear strength 31KN/m～38KN/m, transmittance 95%～98%, oxygen transit dose 2.7cm
3/ m
2.bar.d～1.6cm
3/ m
2.bar.d
Place in the sterilisable chamber with upper film, irradiation-sterilize under the 15W ultraviolet lamp, irradiation distance is 20cm, and irradiation time is 20 minutes, and it is standby to become aseptic polysaccharide membrane after radiation treatment.
The second, the plastic film of covering choosing and sterilizing
The mould polysaccharide membrane as carrier conserving microorganism of short stalk, must covering one deck plastic film, the microorganism of preservation diaphragm in just can polluted air like this, the plastic film of covering also must be aseptic, so just can guarantee to seal the bacterial classification of preserving on the polysaccharide membrane of Tibetan is not have assorted bacterium, and the plastic film of covering is chosen resistant to elevated temperatures nylon membrane, polyester or similar matrix material, with the 121 ℃ of sterilizations 30 minutes in Autoclave of this plastic film wrapping back, taking-up is put in the sterilisable chamber, and is standby.
Three, treat the cultivation of conserving microorganism bacterial
The microbial strains of preservation of the present invention mainly refers to bacterium, yeast, mould and actinomycetes, the microorganism of institute's preservation can be the bacterial classification of DSMZ's collection, the also bacterial classification that gets of other any approach, treat that the cultured microorganism bacterial classification cultivates respectively with its corresponding substratum on plate and test tube slant, obtain single bacterium colony and pure slant strains.
Four, make the culture presevation unit
In sterilisable chamber, the pure strain that it is good that picking is grown, coat ready short stalk mould polysaccharide membrane used on, on another is polysaccharide membrane used, be coated with the glycerine of the bacterium of going out, then two diaphragms are close to, glycerine and bacterial classification extruding mixing are sealed up for safekeeping between two films, get two layers of plastic film of the bacterium of going out, area is slightly larger than above-mentioned polysaccharide membrane used, so that heat sealing machine four banding mouths.Two layers of plastic film upper and lower sleeve envelope is loaded with the polysaccharide membrane used of bacterial classification, with portable heat sealing machine along the edge four banding mouths, form the culture presevation unit of sealing.
Five, make the culture presevation volume
Be prepared into the culture presevation volume of stamp album form with general plastic material such as polyester etc., the front cover of volume can be put on the bacterium fascicle, mould fascicle, yeast fascicle, actinomycetes fascicle.Page or leaf has a lot of grids that are similar to stamp album in the volume, inserts the above-mentioned culture presevation dice that has prepared in the grid disaggregatedly.Above-mentioned preservation volume places-24 ℃ of following prolonged preservation in the refrigerator.
Six, survey preservation unit viable count
With the culture presevation unit of above-mentioned preservation volume, got a bacterium and survey its viable count in every 3-6 month.
With the mould polysaccharide membrane of short stalk is that carrier carries out culture presevation; because the mould polysaccharide good film-forming property of short stalk; the film that is become is oxygen flow not; have with plastic film similarly heat-sealable; can satisfy culture presevation low temperature, anaerobic, drying, lack environmental elements such as nutrition and interpolation protective material, because the basic structure of the mould polysaccharide of short stalk is glucose, microorganism is not had toxic action.
The present invention obstructs mould polysaccharide membrane as carrier preservation bacterial classification with weak point, and it is little to take up room, and the preservation cost is low, mailing, easy to use, safe.
Description of drawings
Short mould polysaccharide hydrostomia film forming of stalk of Fig. 1 and forced air drying synoptic diagram
The face synoptic diagram is conferred titles of nobility in Fig. 2 culture presevation
Sequence number 1 flow liquid mouth among the figure, 2 polysaccharide membrane feed liquids, 3 switchboards, 4 stainless steel tape loops, 5 loft drier, 6 gas blowers, 7 nichrome wire, 8 active machines, 9 volume membranous discs
Embodiment
Embodiment 1
Starch rice obtains glucose and oligosaccharide mixture through hydrolysis, and this mixture preparation substratum transforms into the mould polysaccharide of short stalk by the mould fermentation of short stalk.It is 20g/100ml that this polysaccharide separates, decolours, filters, makes with extra care, is concentrated into concentration through thalline, squeeze in the spray film head, nozzle aperture 0.1mm, with constant pressure 0.09MPa spray film, the circulation Stainless Steel Band evenly connects film, 80 ℃ of warm air dryings obtain thick transparent exsiccant polysaccharide films one volume of 0.03mm, and it is as follows to record its performance index:
Tensile strength 32Mpa angle tear strength 38KN/m
Transmittance 96% oxygen transit dose 1.6cm
3/ m
2.bar.d
Embodiment 2
The polysaccharide membrane that embodiment 1 is made is cut into the dice of 10mm * 10mm, place in the sterilisable chamber, with the 15W ultraviolet lamp, the 20cm distance is shone 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes to the diaphragm sterilization respectively, after sterilization finishes, with a small amount of physiological saline solution diaphragm, then separate application on the plate of perfect medium and broth culture 30 ℃ cultivated 6 days, observations is as follows:
Irradiation time (minute) 5 10 15 20 30
Colony number 17 3000 on the perfect medium
Colony number 13 1000 on the broth culture
Polysaccharide membrane usedly can reach perfectly sterile in 15 minutes through uv irradiating.For the purpose of the safety, irradiation time can be extended down to 20 minutes
Embodiment 3
The some polysaccharide membrane used uv irradiating that embodiment 1 is made 20 minutes is standby respectively, and commercially available coextrusion nylon film sheet is cut into 15mm * 15mm size, and 121 ℃, sterilization in 30 minutes is standby.
Get intestinal bacteria (AS1.751), Beijing rod stalk bacterium (AS1.299), subtilis (AS1.339) and brevibacterium flavum (AB 91058) inclined-plane bacterial strain respectively, be coated with on the plate broth culture, 32 ℃ of cultivations grow into single bacterium colony;
Get yeast (AY91014) and on plate wort agar substratum, be coated with, cultivate for 30 ℃ and generate single bacterium colony; Get Aureobasidium pullulans AS3.3693 and be coated with on the plate perfect medium, 30 ℃ of incubation growth become single bacterium colony.In the sterilisable chamber, the single bacterium colony (each bacterium colony size of each strain bacterium is equal substantially) of the above-mentioned 6 strain bacterium of difference picking from culture dish, be coated on above-mentioned uviolizing aseptic polysaccharide membrane used, being wiped with the aseptic polysaccharide membrane used of thin layer glycerine and micro-trehalose with another is adjacent to it, with the coextrusion nylon film of above-mentioned two bacterium of going out that this polysaccharide membrane is topped up and down then, with plastic sealing machine its four limit is sealed, form a covering plastic film, in the culture presevation unit of interlayer polysaccharide membrane is arranged, more than operation is all carried out in sterilisable chamber.The preservation unit diaphragm of above-mentioned 6 kinds of bacterium was-24 ℃ of following preservations 6 months, 9 months, 12 months, 18 months, each bacterium is respectively taken out three at every turn, dissolve dilution method with physiological saline respectively and be coated onto on the corresponding plate substratum and cultivate, survey its viable count following (each three are averaged):
The preservation time (moon) 69 12 18
Intestinal bacteria AS1.751 (10
6Individual) 87 83 81 83
Beijing rod bacillus AS1.299 (10
6Individual) 134 135 134 133
Subtilis AS1.339 (10
6Individual) 119 117 116 116
Brevibacterium flavum AB 91058 (10
6Individual) 97 93 93 92
Yeast AY91014 (10
6Individual) 106 104 103 105
Aureobasidium pullulans AS3.3693 (10
6Individual) 168 166 169 166
More than each bacterium plate culture all find assorted bacterium.
Claims (6)
1, a kind ofly lack the mould polysaccharide films of stalk and be used for the method for preservation bacterial classification, its film is thickness 0.02mm～0.04mm, tensile strength 27Mpa～32Mpa, angle tear strength 31KN/m～38KN/m, transmittance 95%～98%, oxygen transit dose 2.7cm
3/ m
2.bar.d～1.6cm
3/ m
2.bar.d, it is characterized in that, at first its film is cut into the area size and is 8mm * 8mm～20mm * 20mm unit diaphragm, and carry out aseptically process; In the middle of the mould polysaccharide films of short stalk of aseptically process, coat glycerine and trehalose protective material and microbial strains two-layer then, outer after two films are adjacent to other two layers of plastic film sleeve envelope through aseptically process, constitute a polysaccharide films culture presevation unit; Again this culture presevation unit is assembled into the preservation volume, is preserving below-24 ℃, take out the preservation unit during use, cut off the plastics covering, the polysaccharide membrane that will contain bacterial classification drops in the substratum, and polysaccharide membrane dissolves, and bacterial classification can be grown.
2. the method that is used for the preservation bacterial classification as claimed in claim 1 is characterized in that the outer layer plastic film that is adopted is high temperature resistant and heat sealable coextrusion nylon film, or the polyester composite membrane, and tolerable temperature is 121 ℃～125 ℃.
3. the method that is used for the preservation bacterial classification as claimed in claim 1 is characterized in that, the aseptically process of the mould polysaccharide films of described short stalk, and its treatment process is: the mould polysaccharide membrane of short stalk uses 15W ultraviolet lamp 20cm apart from irradiation 20 minutes.
4. the method that is used for the preservation bacterial classification as claimed in claim 1 is characterized in that, the aseptically process of the plastic film of described covering, its treatment process is: its plastic film in Autoclave 121 ℃ the sterilization 30 minutes.
5. the method that is used for the preservation bacterial classification as claimed in claim 1; it is characterized in that; the microbial strains for the treatment of preservation is picking from the inclined-plane and on the plate bacterium colony; coat on the mould polysaccharide membrane of the short stalk of one deck; the mould polysaccharide membrane coating of the short stalk of another layer protective material glycerine and trehalose, then two films are adjacent to, two plastic films of outer cover; and, forming the preservation unit with four banding mouths, its operating process is all finished in sterilisable chamber.
6. the method that is used for the preservation bacterial classification as claimed in claim 1, it is characterized in that the preservation dice is assembled into the bacterium fascicle of stamp album formula, the yeast fascicle, the mould fascicle, the actinomycetes fascicle, the material of volume is the plastics polyester films, and each page or leaf in the volume inserts many preservation dice, and each volume is formed by some pages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410061418XA CN1317381C (en) | 2004-12-23 | 2004-12-23 | Polysaccharose film of brevibacterium and its use in bacteria preservisation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410061418XA CN1317381C (en) | 2004-12-23 | 2004-12-23 | Polysaccharose film of brevibacterium and its use in bacteria preservisation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1644675A CN1644675A (en) | 2005-07-27 |
| CN1317381C true CN1317381C (en) | 2007-05-23 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB200410061418XA Expired - Fee Related CN1317381C (en) | 2004-12-23 | 2004-12-23 | Polysaccharose film of brevibacterium and its use in bacteria preservisation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1317381C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100465191C (en) * | 2006-11-03 | 2009-03-04 | 天津市工业微生物研究所 | Drying method of bearing blue polysaccharide |
| US10130587B2 (en) | 2011-01-11 | 2018-11-20 | Capsugel Belgium Nv | Hard capsules |
| CN103667061A (en) * | 2013-11-21 | 2014-03-26 | 宁夏启元药业有限公司 | Strain preservation method |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| WO2018189587A1 (en) | 2017-04-14 | 2018-10-18 | Capsugel Belgium Nv | Process for making pullulan |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09275896A (en) * | 1996-04-11 | 1997-10-28 | Tokushima Pref Gov | Preservation and packaging of food |
| CN1295125A (en) * | 1999-11-03 | 2001-05-16 | 中国科学院沈阳应用生态研究所 | Filamentous fungus passing, culturing, coating and preserving method |
| US6387666B1 (en) * | 1991-10-16 | 2002-05-14 | Shin-Etsu Bio, Inc. | High molecular weight pullulan and method for its production |
-
2004
- 2004-12-23 CN CNB200410061418XA patent/CN1317381C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6387666B1 (en) * | 1991-10-16 | 2002-05-14 | Shin-Etsu Bio, Inc. | High molecular weight pullulan and method for its production |
| JPH09275896A (en) * | 1996-04-11 | 1997-10-28 | Tokushima Pref Gov | Preservation and packaging of food |
| CN1295125A (en) * | 1999-11-03 | 2001-05-16 | 中国科学院沈阳应用生态研究所 | Filamentous fungus passing, culturing, coating and preserving method |
Non-Patent Citations (1)
| Title |
|---|
| 一种短梗霉多糖及其薄膜性能研究 李士杰等，高分子材料科学与工程，第17卷第3期 2001 * |
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