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A method of preserving yeast

A technology for yeast and yeast liquid is applied in the field of yeast preservation, which can solve the problems affecting the expansion of yeast culture, the death of yeast, and the decrease of the viable rate of yeast, so as to improve the survival rate, improve the growth state, and increase the protein content. Effect

Active Publication Date: 2018-03-27
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional storage method will cause a large number of yeasts to die, especially as the storage time increases, the viable rate of yeasts shows a clear downward trend, which affects the expansion of yeast culture after recovery.

Method used

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  • A method of preserving yeast
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 preserves the method for yeast

[0030] 1. Streak and inoculate the identified engineering strains of Saccharomyces cerevisiae, Pichia pastoris and Hansenula on YPD agar solid medium, and after culturing at 33°C for 48 hours, pick a single colony and inoculate it in YPD liquid medium, 33 ℃, shaking culture for 24 hours, spectrophotometrically measure the density of yeast:

[0031] Table 1

[0032]

[0033] 2. Transfer 5ml of the bacterium solution cultivated in step 1 to 95ml of new YPD liquid medium (inoculation volume ratio 5%), continue to vibrate at 33°C for 24 hours, and measure the density of the yeast by spectrophotometry:

[0034] Table 2

[0035]

[0036] 3. Take 1 ml × 2 parts of each of the Saccharomyces cerevisiae, Pichia pastoris and Hansenula yeast engineering bacteria liquid obtained in step 2 (6 parts in total for the three kinds of yeasts), and add sterilized glycerin to a final concentration of 15% (mass percentage) , add steriliz...

Embodiment 2

[0037] Embodiment 2 Different storage time live bacteria rate compares

[0038] After each saccharomycete in embodiment 1 is preserved 3 months, 6 months, 9 months, 12 months, take out, detect its viable bacteria rate (%), the result is as follows:

[0039] Table 3 3-month live bacteria rate detection rate (%)

[0040]

[0041] Table 4 6-month live bacteria rate detection rate (%)

[0042]

[0043] Table 5 9-month live bacteria rate detection rate (%)

[0044]

[0045] Table 6 12-month live bacteria rate detection rate (%)

[0046]

[0047] The above results show that after adding low concentration of liquid agar, the survival rate of yeast strains at -20°C and -70°C and at different storage times is significantly higher than that without adding agar.

Embodiment 3

[0048] Example 3 The influence of the method for preserving yeast of the present invention on the expression of yeast protein

[0049] At the different storage time of Saccharomyces cerevisiae, Pichia pastoris and Hansenula bacterial classification, get the bacterial classification that does not add agar (traditional method) and add agar (method of the embodiment of the present invention 1) respectively, cultivate according to the method for embodiment 1 , after 24 hours of culture, the same dose of methanol was added to induce the expression of the target antigen. After 72 hours of induced expression, the antigen expression levels of Saccharomyces cerevisiae, Pichia pastoris and Hansenula were measured by ELISA method, and the antigen expression rate (%) was calculated, as shown in Table 7. "No addition" in the table refers to the traditional method of adding only glycerin to a final concentration of 15% and storing the yeast in liquid nitrogen, and "adding" refers to the yea...

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Abstract

The invention provides a method for preserving yeast, and belongs to the technical field of strain preservation. The method comprises the steps of adding glycerol and agar into a yeast liquid, rapidly freezing in liquid nitrogen and then preserving at 20 DEG C below zero for a long term. The preservation method can greatly improve the live strain rate when the yeast revives, improve the growth situation of the yeast, improve the expression rate of target protein, and increase the yield of a target product. The method can be applied to the field of gene engineering, can preserve yeast hosts capable of expressing foreign genes, can also be applied to preservation of common yeast, greatly improves the production efficiency, lowers the production cost, and has a better market application prospect and economic value.

Description

technical field [0001] The invention relates to a method for preserving yeast, which belongs to the technical field of yeast preservation. Background technique [0002] Yeast is a single-celled fungus, which is a general term for several families of single-celled fungi such as Ascomycetes and Basidiomycetes. Yeast is harmless and easy to grow. It can decompose macromolecules into small molecules that can be easily utilized by cell metabolism. It belongs to heterotrophs. Yeast is the earliest microorganism used in the history of human civilization. It can be used not only in brewing production, but also as a model organism for genetic engineering and cell cycle research, and is favored by many scientific researchers. [0003] Saccharomyces cerevisiae is the earliest known in molecular genetics, and it is also the first yeast host for exogenous gene expression. The exogenous gene expression system of Pichia pastoris as a host has developed rapidly in recent years and is the m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/04C12R1/865C12R1/84C12R1/78
CPCC12N1/04C12N1/16
Inventor 张星星董高峰高强尹卫东
Owner SINOVAC RES & DEV