CN101665771B - Yeast preserving solution and using method thereof - Google Patents
Yeast preserving solution and using method thereof Download PDFInfo
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- CN101665771B CN101665771B CN2008101465602A CN200810146560A CN101665771B CN 101665771 B CN101665771 B CN 101665771B CN 2008101465602 A CN2008101465602 A CN 2008101465602A CN 200810146560 A CN200810146560 A CN 200810146560A CN 101665771 B CN101665771 B CN 101665771B
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Abstract
The invention provides yeast preserving solution and also provides a method for preserving yeasts by using the yeast preserving solution and a fermentation composition containing the yeasts and the yeast preserving solution. The yeast preserving solution contains buffer solution serving as a solvent and mycose and adenine which are dissolved in the solvent and can preserve a bacterial strain for 1 to 2 years under a condition of 4 to 10 DEG C or for 1 to 3 weeks at normal temperature. The fermentation composition can be used as a seed to be directly inoculated in a K tank for production without resurrection propagation in a liquid culture medium in a triangular flask, thereby reducing half of the time of a seed production period of a production line.
Description
Technical field
The present invention relates to a kind of culture presevation with solution and application method thereof, more particularly, relate to a kind of yeast preservation solution and application method thereof.
Background technology
Yeast is human the application relatively early, also is the microorganism that is most widely used, and people often utilize its fermentative action to make various fermentation food and wine brewing.
The development of saccharomycetic research and industrial applications thereof is inevitable to interrelate with safety, long lasting cell preservation technique, for the yeast that important economic worth is arranged, if the physiology of bacterial classification, gene character sustain damage, variation will bring enormous economic loss in preserving process.In the saccharomycetic suitability for industrialized production of needs, often will be on production line (surpass the time in 1 year) steadily in the long term and use the barms of same lot number, sometimes the barms that also needs at room temperature to transport for long-distance uses to offer different production lines, for this reason, need take required measure to improve the stability of bacterial classification, the high reactivity of maintenance bacterial classification.Two class methods below main the employing are carried out the preservation of barms in the prior art, the first kind is to allow the method for preserving of bacterial classification continuous growth, promptly regularly transplant preserving process, mainly comprise inclined-plane transplanting, oil pipe preservation and distilled water preservation etc., though these class methods have easy handling, do not need the advantage of specific installation, but also have the easily shortcoming of variation of cost manpower and the time that exists, Pollution risk, bacterial strain, and needing to be not suitable for the suitability for industrialized production of the same lot number bacterial classification of life-time service to use; Second class methods are to utilize the method that suppresses the bacterial classification metabolic activity to carry out preservation, mainly comprise freeze-drying, sharp freezing method and liquid nitrogen cryopreservation, these class methods are the preservation bacterial classification for a long time, and bacterial classification is difficult for variation, but method is general complicated, need to use expensive equipment, and cost is higher; Therefore, and even but need a kind of low cost and the preservation barms also method of preservation certain hour at room temperature steadily in the long term, especially with the method for liquid form preservation barms.
Existing in the prior art with the method for bacterial classification with the liquid form preservation, for example WO0039281A2 discloses a kind of microbial strains (comprising yeast) culture of liquid, it comprises synthetic relevant compound (comprising VITAMIN B4) and the optional trehalose that adds of biological nucleic acid with metabolic activity stabilization, but the culture presevation temperature of this method is lower, be-20 ℃ to 0 ℃, be not suitable for preservation at room temperature, therefore be not easy to long-distance transport, in addition, the culture presevation time of this method is shorter, be no more than for 16 weeks, therefore also be not suitable on production line, using steadily in the long term the bacterial classification of same lot number above 1 year.
Therefore, need solution above-mentioned with the saccharomycetic shortcoming of liquid form preservation.
Summary of the invention
The present invention is based on a kind of like this discovery and finishes, promptly use and comprise as the buffered soln of solvent and be dissolved in trehalose in this solvent and the yeast preservation of VITAMIN B4 is carried out the barms preservation with solution, can be unexpectedly under higher temperature condition preservation bacterial classification steadily in the long term, or at room temperature the preservation bacterial classification reaches for some time of being convenient to transport for long-distance.
Therefore, the object of the present invention is to provide a kind of yeast preservation solution and application method thereof, by in as the buffered soln of solvent, adding a certain amount of trehalose and VITAMIN B4, thereby can be effectively under 4-10 ℃ condition preservation bacterial classification 1-2 steadily in the long term, therefore when suitability for industrialized production, do not need to change midway and produce bacterial classification, thereby guaranteed to produce the continuous and stable of bacterial classification, or preservation bacterial classification 1-3 week at room temperature, so that offering different production lines, uses the barms of at room temperature transporting for long-distance.In addition, comprising barms and above-mentioned preservation can be used as the seed direct inoculation with the fermentation of solution with composition and produces to the KShi jar, no longer need to spread cultivation, make the seed production cycle of production line reduce the time of half through the resurrection of triangular flask liquid nutrient medium.
Of the present invention aspect first in, a kind of yeast preservation solution is provided, its pH that comprises as solvent is the VITAMIN B4 of the buffered soln of 6.2-6.4 and trehalose and the 6-10mg/L that is dissolved in the 10-20g/L in the described solvent.Wherein said buffered soln is spawn culture weak acidic buffer commonly used, include but not limited to phosphate buffered saline buffer, citrate buffer solution and Sodium phosphate dibasic-citrate buffer solution, the concentration of described damping fluid is that yeast is cultivated acceptable concentration, preferred 5-1000mM, more preferably 10-500mM, most preferably 15-200mM.
In a preferred embodiment of the invention, described yeast preservation further comprises the reduced glutathion that is dissolved in the 5-20g/L in the described solvent with solution.
In another preferred embodiment of the present invention, described yeast preservation further comprises the gum arabic that is dissolved in the 1-5g/L in the described solvent with solution.
In another preferred embodiment of the present invention, described yeast preservation further comprises the vitamin-E that is dissolved in the 10-60mg/L in the described solvent with solution.
In another preferred embodiment of the present invention, described yeast preservation further comprises the glycine that is dissolved in the 0.5-1g/L in the described solvent with solution.
In a preferred embodiment of the present invention, described yeast preservation is 8mg/L with the content of VITAMIN B4 described in the solution.
In the most preferred embodiment of the present invention, described yeast preservation is the buffered soln of 6.2-6.4 and the trehalose that is dissolved in the 10-20g/L in the described solvent, the VITAMIN B4 of 8mg/L, the reduced glutathion of 5-20g/L, the gum arabic of 1-5g/L, the vitamin-E of 10-60mg/L and the glycine of 0.5-1g/L with the pH that solution comprises as solvent.
In aspect second of the present invention, provide a kind of saccharomycetic method for preserving, it comprises the steps:
(1) obtains yeast by purebred cultivation;
(2) with resulting yeast in the step (1) through aseptic washing and centrifugal after add the invention described above the yeast preservation with in the solution, making viable bacteria concentration is 1 * 10
10-1 * 10
14Cfu/ml, preferred 1 * 10
10-1 * 10
12The bacteria suspension of cfu/ml, the cell age of viable bacteria are 12-24 hour, preferred 16-20 hour; With
(3) described bacteria suspension and seal preservation above the packing.
Described yeast can pass through the fermentor tank mass production in the aseptic experiment chamber, and described bacteria suspension can divide and installs in the cillin bottle and seal preservation.
In aspect the 3rd of the present invention, provide a kind of fermentation composition, comprised the yeast preservation solution of yeast and the invention described above.
Wherein above-mentioned saccharomycetic method for preserving or fermentation comprise the yeast of yeast belong (Saccharomyces), mycocandida (Candida) and Rhodotorula (Rhodotorula) with the described yeast in the composition.
Compared with prior art, yeast preservation of the present invention is with solution and use this yeast preservation to have following beneficial effect with the saccharomycetic method of solution preservation: after adopting yeast preservation of the present invention with solution and yeast method for preserving, for production line provides a kind of preservation time long (1-2), the centre does not need transferred species, preservation is stable, convenient transportation (can be transported for long-distance under the room temperature, the preservation time reaches 1-3 week), the fermentation composition that can on producing, directly use, this fermentation can be produced to the KShi jar as the seed direct inoculation with composition, therefore no longer need to spread cultivation, thereby make the seed production cycle of production line reduce the time of half through the resurrection of triangular flask liquid nutrient medium.
Embodiment
The present invention will carry out more specific description according to the following example.Yet protection scope of the present invention is not limited to following embodiment.
Embodiment 1: preparation yeast preservation solution
Press the yeast preservation solution that the prescription 1-3 of tabulation in 1 prepares tool different components content respectively:
Table 1: yeast preservation solution formula table
Compound method: be solvent at room temperature, various solutes added in the solvent, stir, leave standstill, filter, sterilize, promptly get yeast preservation solution of the present invention by the amount in the above-mentioned prescription with the buffered soln in the above-mentioned prescription.
Embodiment 2: use yeast preservation solution preservation yeast
(culture presevation is numbered the rich chromium yeast saccharomyces cerevisiae Z1.3 (Saccharomycescerevisiae Hansen Z1.3) (referring to CN101045906A) of yeast belong of CCTCC No:M205125 at first to produce yeast in the aseptic experiment chamber by a large amount of purebred cultivations of fermentor tank, culture presevation is numbered the yeast belong yeast saccharomyces cerevisiae Z2.3 (Saccharomyces cerevisiae Hansen Z2.3) (referring to CN101050430A) of CCTCCNo:M205129, culture presevation is numbered the mycocandida Candida utilis (Candida utilis) (referring to US4720457A) of ATCC9950 or the Rhodotorula rhodotorula glutinis (Rhodotorula glutinis) (referring to CN1415738A) that culture presevation is numbered CGMCC No.2.703, then will by aseptic washing and centrifugal after the above-mentioned yeast that obtains add the yeast preservation of the present invention described in the embodiment 1 respectively with solution (prescription 1,2 or 3) in, making viable bacteria concentration is 1 * 10
10, 1 * 10
12Or 1 * 10
14The bacteria suspension of cfu/ml, the cell age of viable bacteria are 12,16,20 or 24 hours, divide then to install in the cillin bottle and sealing, place 4-10 ℃ or room temperature (25 ℃) preservation down at last.
Embodiment 3: stability experiment
Experimental technique:
Various bacteria suspensions prepared in the foregoing description 2, are detected living cell rate and carry out the production performance test down during preservation for some time as described below in 4-10 ℃ or room temperature (25 ℃) under opticmicroscope.
Experimental result:
(1) prepared various bacteria suspensions use yeast preservation of the present invention to use solution 4-10 ℃ of following preservation in the foregoing description 2, living cell rate is all more than 80% in the time of 1 year, living cell rate is all more than 75% in the time of 2 years, and production performance is not less than 4-10 ℃ of solid slant strains of preserving month down.
(2) prepared various bacteria suspensions use yeast preservation of the present invention to use solution in room temperature (25 ℃) preservation down in the foregoing description 2, living cell rate is all more than 75% during one week, living cell rate is all more than 50% during 3 weeks, and production performance is not less than 4-10 ℃ of solid slant strains of preserving month down.
Claims (8)
1. yeast preservation solution, its pH that comprises as solvent are the buffered soln of 6.2-6.4 and the trehalose that is dissolved in the 10-20g/L in the described solvent, the VITAMIN B4 of 6-10mg/L, the reduced glutathion of 5-20g/L, the gum arabic of 1-5g/L, the vitamin-E of 10-60mg/L, the glycine of 0.5-1g/L.
2. yeast preservation solution according to claim 1, the content of wherein said VITAMIN B4 is 8mg/L.
3. saccharomycetic method for preserving, it comprises the steps:
(1) obtains yeast by purebred cultivation;
(2) with resulting yeast in the step (1) through aseptic washing and centrifugal after add claim 1 or 2 described yeast preservations with in the solution, making viable bacteria concentration is 1 * 10
10-1 * 10
14The bacteria suspension of cfu/ml, the cell age of viable bacteria are 12-24 hour; With
(3) the described bacteria suspension of packing and seal preservation.
4. saccharomycetic method for preserving according to claim 3, wherein making viable bacteria concentration in the step (2) is 1 * 10
10-1 * 10
12The bacteria suspension of cfu/ml.
5. saccharomycetic method for preserving according to claim 3, wherein the cell age of viable bacteria is 16-20 hour in the step (2).
6. according to each described saccharomycetic method for preserving of claim 3-5, wherein said yeast comprises the yeast of yeast belong (Saccharomyces), mycocandida (Candida) and Rhodotorula (Rhodotorula).
7. a fermentation composition comprises yeast and claim 1 or 2 described yeast preservation solution.
8. fermentation composition according to claim 7, wherein said yeast comprise the yeast of yeast belong (Saccharomyces), mycocandida (Candida) and Rhodotorula (Rhodotorula).
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| CN2008101465602A CN101665771B (en) | 2008-09-03 | 2008-09-03 | Yeast preserving solution and using method thereof |
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| CN101665771B true CN101665771B (en) | 2011-04-06 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100342005C (en) * | 2005-11-07 | 2007-10-10 | 浙江工商大学 | Process for preparing high actived alctic acid bacteria product |
| CN101148652A (en) * | 2007-09-12 | 2008-03-26 | 北京联合大学 | Bacillus pumilus mutant and alkaline proteinase produced from the same by fermenting |
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2008
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100342005C (en) * | 2005-11-07 | 2007-10-10 | 浙江工商大学 | Process for preparing high actived alctic acid bacteria product |
| CN101148652A (en) * | 2007-09-12 | 2008-03-26 | 北京联合大学 | Bacillus pumilus mutant and alkaline proteinase produced from the same by fermenting |
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