CN106635851A - Breeding method of high nucleic acid saccharomyces cerevisiae - Google Patents
Breeding method of high nucleic acid saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention relates to a breeding method of high nucleic acid saccharomyces cerevisiae. The breeding method of the high nucleic acid saccharomyces cerevisiae is characterized in that saccharomyces cerevisiae strains are preliminarily screened and secondarily screened, optimized by a culture medium, and then subjected to expanded cultivation through the optimization of a small-scale and pilot-scale fermentation process, thereby obtaining the high nucleic acid saccharomyces cerevisiae with the RNA content of saccharomycetes being 14.6% to 15.2%. The saccharomyces cerevisiae bred by the invention is high in RNA content, and further is a non-transgenic engineering bacterium, thereby being a genetically stable, safe and non-toxic product capable of ensuring the safety of state food and drugs.
Description
Technical field
The invention belongs to technical field of biological fermentation, and in particular to a kind of selection of high nucleic acid saccharomyces cerevisiae.
Background technology
Nucleotide source method is numerous, and so far, the method that extraction nucleic acid is decomposed again from yeast still accounts for leading
Status.All the time, nucleic acid is all to extract to obtain from common bakery yeast, but bakery yeast Nucleic Acid is generally 6-
8%, in terms of economics of mass production benefit, cost accounting is very high, therefore, the high barmses of selection-breeding nucleic acid content just become
Nucleic acid industrial key.
At present for the research of high nucleic acid saccharomyces cerevisiae is one of current study hotspot, Chinese patent application
CN1498264A is disclosed a kind of " rich ribonucleic acid beer yeast strain and its manufacture method ", yeast thalline prepared by the method
In contain equivalent to ribonucleic acid more than 10 weight % of thalline weight, but be no more than 12 weight %；Chinese patent application
CN101760437A is disclosed a kind of " bread yeast with high nucleic acid content and preparation method thereof ", the training that the method passes through control amplification culture
The conditions such as foster time, have prepared containing nucleic acid more than 20 weight % equivalent to bread microzyme body weight, wherein RNA
Reach more than 9.5%；China applies for a patent CN102559522A and discloses " a kind of bread yeast with high nucleic acid content and its preparation side
Method ", contains equivalent to nucleic acid more than 20 weight % of thalline weight in yeast thalline prepared by the method, and RNA therein is big
In 12 weight %.
Nucleic acid purposes is extremely wide, and in food industry, nucleic acid is the requisite raw material of exploitation novel foodstuff flavoring agent.
The condiments such as second filial generation monosodium glutamate (strength monosodium glutamate), third generation monosodium glutamate (local flavor monosodium glutamate), the special delicious sauce in the world in fashion, be all
Nucleic acid and its result of derivant application.On medical industry, nucleic acid is the diseases such as manufacture treatment coronary heart disease, tumor, myocardial infarction
The raw material of medicine.Agriculturally, nucleic acid and its derivant are widely used as crops（Such as Oryza sativa L., melon and fruit, beans）Growth
Promote material.
At present, the domestic high nucleic acid saccharomyces cerevisiae strain performance rich in required for I+G yeast extracts is less than American-European, day
This grade country, and the production technology of high nucleic acid saccharomyces cerevisiae is still immature.Therefore, the fermentation technique of high nucleic acid saccharomyces cerevisiae is studied
It is the only way of China's yeast extract upgrading of industries.
Therefore, if by traditional method select rich in high nucleic acid Wine brewing yeast strain, enter one rich in yeast by this
Yeast extract nucleic acid series product prepared by step, and industrialization is converted it into, so as to prepare the core for meeting the market demand
Acid production, to whole nucleic acid industry great impetus will be brought.
The content of the invention
To solve the above problems, the present invention provides a kind of selection of high nucleic acid saccharomyces cerevisiae, the method selection-breeding
Saccharomyces cerevisiae rna content is high, non-transgenic engineering bacteria, inheritance stability, safe and nontoxic product, it is ensured that state food
Drug safety.
What the present invention was achieved through the following technical solutions：
A kind of selection of high nucleic acid saccharomyces cerevisiae, from saccharomyces cerevisiae strain after primary dcreening operation and secondary screening, Jing culture medium
Optimization, then cultivate through lab scale and being enlarged of pilot scale fermentation process optimization, obtain high nucleic acid saccharomyces cerevisiae, yeast
Rna content be 14.6%-15.2%.
Preferably, the culture medium quality fraction set that primary dcreening operation is adopted with secondary screening strain is into being：The sucrose of 5-10%, 3% ferment
Mother's leaching powder, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1% potassium dihydrogen phosphate, balance of water, adjust PH
To 4.8.
Preferably, the mass fraction of the culture medium that described medium optimization is used is consisted of：10% sucrose, 3%
Yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1% potassium dihydrogen phosphate, balance of water is adjusted
PH to 4.8.
Preferably, the culture medium prescription that the lab scale and pilot scale fermentation technique are adopted is（10L fermentation liquids）For：Molasses
1.5L, ammonium sulfate 180g, ammonium dihydrogen phosphate 28g, magnesium sulfate 5g, zinc sulfate 1g, balance of water；The sugar content of molasses is 28-32
(g/dL)。
It is highly preferred that in described lab scale and pilot scale fermentation technique, the side of adding of its molasses, ammonium sulfate and ammonium dihydrogen phosphate
Formula is fed-batch mode.
In selection described above, lab scale, the fermentation technology of pilot scale：Initial thalline weight in wet base is 40-60g/L；Put tank
Weight in wet base is 180-220g/L；Fermentation period is 5-10 hours；Temperature is 29-31 DEG C；PH is 4.8-5.5.
It is highly preferred that described lab scale, the fermentation technology of pilot scale are：Initial thalline weight in wet base is 50g/L；Putting tank weight in wet base is
200g/L；Fermentation period is 7 hours；Temperature is 30 DEG C；PH is 5.2.
Selection of the invention, for filtering out plant rich in high nucleic acid yeast strain, Jing medium optimizations, send out
After ferment process optimization, the high nucleic acid saccharomyces cerevisiae prepared, its rna content is up to 15.2%.
Bacterial strain has following properties：
1st, colonial morphology feature：
Cultivate in sucrose-yeast extract culture medium, 30 DEG C, 3 days, cell was spherical in shape or avette, size is 3～10 μ ms 4
～20 μm.Bacterium colony is white to cream color, slightly gloss on wort agar.When incubation time is long, bacterium colony is hardened.
2nd, physiology and biochemical characteristic：
The typical amphimicrobian type unicellular fungi of bacterial strain.In the case of aerobic, it carries out aerobic respiration, discharges titanium dioxide
Carbon；And under conditions of anoxia, it carries out anaerobic respiration, decomposing organic matter（Mainly glucose）, produce ethanol and water；The bacterium
Optimum growth temp be 30 DEG C, optimum pH is 5.2.
3rd, nutritional character：
The bacterium can utilize glucose, maltose, sucrose or molasses.
The useful achievement of the present invention：
1. the bacterial strain in the present invention belongs to saccharomyces cerevisiae, and one is filtered out from a large amount of saccharomyces cerevisiaes by traditional selection
Yeast strain of the strain rich in high nucleic acid, compared with traditional radiation method and chemical mutagen, the yeast is non-transgenic engineering bacteria,
Inheritance stability, preferable new varieties are easily obtained, are safe and nontoxic product, can directly in medicine, food, agricultural, cosmetics etc.
Aspect is used.
2. the saccharomyces cerevisiae of present invention screening, is carbon source from molasses, and ammonium sulfate is nitrogen source, and ammonium dihydrogen phosphate is phosphorus
Source, raw material is easy to get, cheap, and industrial production cost is low；The yeast nucleic acid content that it is prepared is high, beneficial to large-scale industry
Metaplasia is produced.
3. the high nucleic acid saccharomyces cerevisiae of present invention production, makes yeast extract nucleic acid series product, or directly by ferment
Mother produces high nucleotide（I+G）Yeast extract and high protein feed, and by single cell protein (feed yeast) production technology
Digestion production process in waste water, belong to environmental friendliness engineering, can energy-saving and emission-reduction realize zero-emission.
4. the present invention solves that current yeast nucleic acid content is low, and the high difficult problem of industrial production cost can by the present invention
To realize that large-scale industrial production prepares high nucleic acid saccharomyces cerevisiae, the characteristics of stablizing with high-yield character, ribonucleic acid is produced
Property retention stablize, meet the growing life requirement of the people nucleic acid production, pole will be brought to whole nucleic acid industry
Big impetus.
Specific embodiment
Essence for a better understanding of the present invention, enters to advance below by specific embodiment to technical scheme
The elaboration of one step.
Embodiment 1
The breeding high-nucleic acid Wine brewing yeast strain from saccharomyces cerevisiae：Adopt the culture medium after optimization for（It is mass fraction）：10%
Sucrose, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1% potassium dihydrogen phosphate,
Balance of water, adjusts PH to 4.8.
Culture medium is sub-packed in 500mL triangular flasks, liquid amount 100mL, 121 DEG C of Jing, after 20min high pressure steam sterilizations, will
Bacterial strain to be screened is placed in above-mentioned aseptic culture medium, at 30 DEG C, under the conditions of rotating speed 120rpm 48h is cultivated, and carries out primary dcreening operation.Its result
It is as shown in table 1 below.
Table 1：Each bacterial strain nucleic acid content（It is converted into content mg of RNA in 1g over dry yeast）
HN2, HN4, HN14, HN16, HN25, HN26 and HN31 are selected by primary dcreening operation result, secondary screening, secondary screening are carried out to this 7 plants of bacterial strains
Used medium and the same primary dcreening operation of cultural method.Its result is as shown in table 2 below.
Table 2：Each bacterial strain nucleic acid content（It is converted into content mg of RNA in 1g over dry yeast）
Shown by primary dcreening operation and secondary screening result, rna content highest bacterial strain is HN2.
Embodiment 2
High nucleic acid Wine brewing yeast strain HN2 obtained by above-mentioned Jing primary dcreening operations and secondary screening is carried out into Shake flask medium optimization：Using difference
Carbon source, nitrogen source（Percentage composition is mass fraction）.
（1）10% sucrose, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and
0.1% potassium dihydrogen phosphate, PH4.8；
（2）10% glucose, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1%
Potassium dihydrogen phosphate, PH4.8；
（3）10% maltose, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1%
Potassium dihydrogen phosphate, PH4.8；
（4）10% sucrose, 3% ammonium sulfate, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1% phosphorus
Acid dihydride potassium, PH4.8.
Jing is tested, and yeast rna content results show that optimization culture medium is：（1）：10% sucrose, 3% yeast extract,
0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and 0.1% potassium dihydrogen phosphate, PH4.8.
Embodiment 3
30L fermentation tank pilot plant tests
Strain spreads cultivation：The high nucleic acid saccharomyces cerevisiae HN2 in inclined-plane, in being inoculated in the aseptic culture medium of the above-mentioned optimizations of 1L, in 30 DEG C, rotating speed
120rpm, shaking table culture 24h is stand-by.First carry out seed in 30L fermentation tanks to spread cultivation, seed is spread cultivation and fermented using traditional yeast
Technique is carried out, and the strain of gained is carried out into commodity fermentation.
Fermentation technology is as follows：
Strain：It is above-mentioned spread cultivation after strain 1L（Weight in wet base 500g/L）；Fermentation tank bed material：9L water, 15g magnesium sulfate, 3g zinc sulfate；Carbon
Source：Molasses（30g/dL containing reducing sugar）4.5L；Nitrogen source：Ammonium sulfate 540g；Phosphorus source：Ammonium dihydrogen phosphate 84g, nitrogen source, phosphorus source is molten
Solution is in 800mL water.25% sodium carbonate liquor is used in PH controls.Carbon, nitrogen, phosphorus source and sodium carbonate liquor at 121 DEG C, 20min high pressure
Use after steam sterilization, feed supplement adopts fed-batch mode.Its technological parameter is as shown in table 3 below：
Table 3：Fermentation technology control table
Respectively in 5,6,7,8,9,10 hours sampling detection rna contents, its result is as shown in table 4 below：
Table 4：Different fermentations time rna content
From the data result of table 4, the high nucleic acid saccharomyces cerevisiae of present invention screening, in lab scale（30L fermentation tanks）In, fermentation 7 is little
When, rna content is up to 15.22%.
Embodiment 4
30L fermentation tanks lab scale craft optimizes
By changing method of the conditions such as different inoculum concentration, fermentation period, fermentation temperature, PH using orthogonal experiment, carry out little
Examination fermentation technology optimization experiment, by the rna content for detecting fermented yeast, show that optimal lab scale fermentation technology is：Initial bacterium
Body weight in wet base is 40-60g/L（It is preferred that 50g/L）；Tank weight in wet base is put for 180-220g/L（It is preferred that 200g/L）；Fermentation period is that 6-8 is little
When（It is preferred that 7 hours）；29-31 DEG C of fermentation temperature（It is preferred that 30 DEG C）；Fermentation pH value is 4.8-5.5（It is preferred that 5.2）.
Embodiment 5
High nucleic acid saccharomyces cerevisiae industrialized manufacturing technique（300m3）
Strain spreads cultivation：The high nucleic acid saccharomyces cerevisiae HN2 in inclined-plane, is inoculated in 10L rustless steel culture tank, and culture is finished and is followed by 10M3Send out
Fermentation tank, its culture is linked into again 300m after finishing3Seed is carried out in big fermentation tank to spread cultivation, seed is spread cultivation and sent out using traditional yeast
Ferment technique is carried out, and the strain of gained is centrifuged, laggard product fermentation of doing business is dissolved.Carbon source：Molasses（30g/dL containing reducing sugar）
45m3；Nitrogen source：The kg of ammonium sulfate 5400；Phosphorus source：Ammonium dihydrogen phosphate 840kg, by nitrogen source, phosphorus source 8m is dissolved in3In hot water.Commodity are sent out
Ferment technique is as shown in table 5 below：
Table 5：Commodity fermentation parameter table
Respectively in 5,6,7,8,9,10 hours sampling detection rna contents, its result is as shown in table 6 below：
Table 6：Different fermentations time rna content
From the data result of table 6, the high nucleic acid saccharomyces cerevisiae of present invention screening, in big production, ferment 7 hours, rna content
It is up to 14.62%.
Embodiment 6
Saccharomyces cerevisiae HN2 is continuously cultivated into the strain in 1-5 generations, the performance for producing ribonucleic acid is shown in Table 7
It is more than the embodiment to a kind of selection-breeding rich in high nucleic acid saccharomyces cerevisiae provided by the present invention and its industrialization technology
It is discussed in detail.One plant of barms rich in high nucleic acid is obtained by traditional breeding method in a large amount of saccharomyces cerevisiae strains, then
The optimization of Jing culture medium is stablized, the commercial production barmses rich in nucleic acid.On this basis, then through lab scale, in
Examination fermentation technology optimization, is translated into industrialized production, above-mentioned optimization process, and rna content is up to 15.22% in lab scale, greatly
RNA reaches 14.62% in production.For this purpose, a kind of selection-breeding rich in high nucleic acid saccharomyces cerevisiae provided by the present invention and its industrialization
Technology is suitable to popularize in an all-round way aborning.
Claims (7)
1. a kind of selection of high nucleic acid saccharomyces cerevisiae, it is characterised in that：By primary dcreening operation and again from saccharomyces cerevisiae strain
After sieve, the optimization of Jing culture medium, then cultivate through lab scale and being enlarged of pilot scale fermentation process optimization, obtain high nucleic acid wine brewing
Yeast, saccharomycetic rna content is 14.6%-15.2%.
2. selection according to claim 1, it is characterised in that：The culture medium quality point that primary dcreening operation and secondary screening strain are adopted
Array is into being：The sucrose of 5-10%, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate and
0.1% potassium dihydrogen phosphate, balance of water adjusts PH to 4.8.
3. selection according to claim 1, it is characterised in that：The culture medium that described medium optimization is used
Mass fraction is consisted of：10% sucrose, 3% yeast extract, 0.1% magnesium sulfate, 0.02% zinc sulfate, 0.01% ferrous sulfate
Potassium dihydrogen phosphate with 0.1%, balance of water adjusts PH to 4.8.
4. selection according to claim 1, it is characterised in that：What the lab scale and pilot scale fermentation technique were adopted
Culture medium prescription is（10L fermentation liquids）For：Molasses 1.5L, ammonium sulfate 180g, ammonium dihydrogen phosphate 28g, magnesium sulfate 5g, zinc sulfate
1g, balance of water；The sugar content of molasses is 28-32 (g/dL).
5. selection according to claim 4, it is characterised in that：In described lab scale and pilot scale fermentation technique, its sugar
The additional way of honey, ammonium sulfate and ammonium dihydrogen phosphate is fed-batch mode.
6. selection according to claim 1, it is characterised in that：Described lab scale, the fermentation technology of pilot scale：Initial bacterium
Body weight in wet base is 40-60g/L；Tank weight in wet base is put for 180-220g/L；Fermentation period is 5-10 hours；Temperature is 29-31 DEG C；PH is
4.8-5.5。
7. selection according to claim 6, it is characterised in that described lab scale, the fermentation technology of pilot scale are：Initially
Thalline weight in wet base is 50g/L；Tank weight in wet base is put for 200g/L；Fermentation period is 7 hours；Temperature is 30 DEG C；PH is 5.2.
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Cited By (6)
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| CN109207386A (en) * | 2017-06-30 | 2019-01-15 | 安琪酵母股份有限公司 | Wine brewing yeast strain and the methods and applications for producing high-nucleic acid yeast |
| CN110184203A (en) * | 2019-06-19 | 2019-08-30 | 乐斯福(明光)有限公司 | A kind of bread yeast with high nucleic acid content and preparation method thereof |
| CN112680370A (en) * | 2021-03-16 | 2021-04-20 | 广东海天创新技术有限公司 | High-nucleic-acid saccharomyces cerevisiae and application thereof |
| CN115478024A (en) * | 2022-10-31 | 2022-12-16 | 齐鲁工业大学 | Non-transgenic high-nucleic-acid saccharomyces cerevisiae strain and application thereof |
| CN116286939A (en) * | 2023-02-03 | 2023-06-23 | 齐鲁工业大学(山东省科学院) | Method for improving nucleic acid yield of saccharomyces cerevisiae and application |
| CN117947015A (en) * | 2023-04-19 | 2024-04-30 | 安琪酵母股份有限公司 | Method for high-throughput rapid breeding of high ribonucleic acid yeast |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207386A (en) * | 2017-06-30 | 2019-01-15 | 安琪酵母股份有限公司 | Wine brewing yeast strain and the methods and applications for producing high-nucleic acid yeast |
| CN109207386B (en) * | 2017-06-30 | 2021-09-28 | 安琪酵母股份有限公司 | Saccharomyces cerevisiae strain, and method and application for producing high-nucleic-acid yeast |
| CN110184203A (en) * | 2019-06-19 | 2019-08-30 | 乐斯福(明光)有限公司 | A kind of bread yeast with high nucleic acid content and preparation method thereof |
| CN112680370A (en) * | 2021-03-16 | 2021-04-20 | 广东海天创新技术有限公司 | High-nucleic-acid saccharomyces cerevisiae and application thereof |
| CN115478024A (en) * | 2022-10-31 | 2022-12-16 | 齐鲁工业大学 | Non-transgenic high-nucleic-acid saccharomyces cerevisiae strain and application thereof |
| CN115478024B (en) * | 2022-10-31 | 2023-03-17 | 齐鲁工业大学 | Non-transgenic high-nucleic-acid saccharomyces cerevisiae strain and application thereof |
| CN116286939A (en) * | 2023-02-03 | 2023-06-23 | 齐鲁工业大学(山东省科学院) | Method for improving nucleic acid yield of saccharomyces cerevisiae and application |
| CN116286939B (en) * | 2023-02-03 | 2023-09-01 | 齐鲁工业大学(山东省科学院) | Method for improving nucleic acid yield of saccharomyces cerevisiae and application |
| CN117947015A (en) * | 2023-04-19 | 2024-04-30 | 安琪酵母股份有限公司 | Method for high-throughput rapid breeding of high ribonucleic acid yeast |
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Application publication date: 20170510 |