CN102337224B - High-nitrogen yeast and preparation method thereof - Google Patents
High-nitrogen yeast and preparation method thereof Download PDFInfo
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- CN102337224B CN102337224B CN201110250844.8A CN201110250844A CN102337224B CN 102337224 B CN102337224 B CN 102337224B CN 201110250844 A CN201110250844 A CN 201110250844A CN 102337224 B CN102337224 B CN 102337224B
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Abstract
The invention relates to to a high-nitrogen yeast (particularly referring to Saccharomyces cerevisiae) and a preparation method thereof. Saccharomyces cerevisiae is preserved in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.5004; and Saccharomyces cerevisiae is obtained by heavy ion radiation mutation, the content of nitrogen in Saccharomyces cerevisiae is 40-60%. A heavy ion mutation technology has the following main advantages that (1) variation rate is high, generally 1000 times higher than natural variation rate; (2) variation spectrum is wide, namely type of variation is diverse, and a new type of microbe strain can be obtained; (3) variation stability is strong, relatively stable strains (coexistence of positive and negative mutations) can be obtained, and screening period can be shortened; and (4) mutation operability is strong, and the radiation mutation of the microbe can be easily completed.
Description
Technical field
The present invention relates to yeast saccharomyces cerevisiae and preparation method thereof.
Background technology
Bread yeast belongs to yeast saccharomyces cerevisiae on taxonomy.Yeast is a kind of unicellular microorganism, and its form is circular, oval, oval, and size is different with the difference of bacterial classification.
Moisture content 65%-70% in yeast cell, wherein approximately has 15% for free-water; Carbohydrate containing is 25%-35%, exists greatly mainly with polysaccharide form; Containing protein 40%-60%, yeast contains complete amino acid group, comprises 8 seed amino acids of needed by human, the particularly less Methionin of content in com gluten protein, and in yeast, content is higher.In addition, the amino acid ratio in yeast approaches the ideal amino acid composition value that Food and Argriculture OrganizationFAO (FAO) is recommended, therefore its nutritive value is higher.Ash content 5%-10%, large more mainly with the content of phosphorus and potassium.Yeast is also natural source of nutrition and nutrition carrier, its nutritive ingredient has the feature of " three low four excellent ", be low fat, low sugar, do not contain cholesterol, be rich in high-quality complete protein, complete vitamin B group, several mineral materials and the function food fibre existing with life combined form.
At occurring in nature, there is the shortcomings such as oneself protein content is low, biomass accumulation is slow in the yeast of institute's seed selection.Therefore be necessary yeast cell to carry out mutagenesis and screening, finally obtain protein content high, the excellent species that biomass accumulation is fast.For suitability for industrialized production is from now on established solid basis.
The microbial strains breeding technique that uses at present mainly comprises that physical method is as ultraviolet mutagenesis, and chemical process is as, the combine method of mutagenesis of the single mutagenesis such as nitrosoguanidine, lithium chloride, sulfuric acid second diester (DES) or several material.Adopt above-mentioned physics, that chemical process all exists workload is large, the low inferior problems of efficiency of inducing mutation.And can produce resistance in various degree to bacterial classification self.The another kind of main method that adopts genetic engineering breeding, mainly comprises Protoplast Fusion Technique, DNA recombinant technology.Protoplast Fusion Technique be at present the most frequently used be also one of the most effective induction mutation of bacterium means, advantage is that cellularstructure is simple, rapidly, required equipment is simple, easy to operate in growth.Shortcoming is that mutation rate is low, and mutagenic compound effect is unstable.Its advantage of DNA recombinant technology is that purpose is strong, can be according to people's wish transformation goal gene.But shortcoming is large in technical difficulty, mRNA period of storage is short, operating process complexity.
Summary of the invention
The object of the invention is to avoid the deficiencies in the prior art that a kind of high nitrogen yeast is provided.Utilize heavy ion irradiation mutagenesis to obtain high nitrogen yeast, for yeast industry provides excellent species.
Another object of the present invention is to provide a kind of preparation method of high nitrogen yeast.
For achieving the above object, the technical scheme that the present invention takes is: a kind of high nitrogen yeast, is
saccharomyces cerevisiaeyeast saccharomyces cerevisiae, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No 5004, preservation date is on June 29th, 2011; Through heavy ion irradiation mutagenesis, its nitrogen content is 40-60%.
The preparation method of high nitrogen yeast, its principal feature is to comprise the following steps:
(1) prepare bacterial classification:
saccharomyces cerevisiaeyeast saccharomyces cerevisiae:
(2) prepare solid medium glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, agar 0.5%-1.5%, all the other are water;
(3) prepare liquid nutrient medium: glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, all the other are water;
(4) slant culture: will be linked in the described culture medium slant of the bacterium of having gone out after the single bacterium colony picking in culture dish, 25-35 DEG C leaves standstill cultivation;
(5) seed culture: picking slant strains is linked in the described liquid nutrient medium of the bacterium of having gone out, and liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 8-12 hour, and shaking speed is 100-300r/min, and inoculum size is 5%-10%;
(6) heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out 1 than 98-102 dilution proportion, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 20Gy-100Gy;
(7) if the barms preservation time, length need to be-80 DEG C of preservations, if slant preservation is 4 DEG C of preservations.
A preparation method for high nitrogen yeast, its principal feature is to comprise the following steps:
(1) prepare bacterial classification:
saccharomyces cerevisiaeyeast saccharomyces cerevisiae:
(2) prepare solid medium glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, agar 0.5%-1.5%, all the other are water;
(3) prepare liquid nutrient medium: glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, all the other are water;
(4) slant culture: will be linked in the described culture medium slant of the bacterium of having gone out after the single bacterium colony picking in culture dish, 25-35 DEG C leaves standstill cultivation;
(5) seed culture: picking slant strains is linked in the described liquid nutrient medium of the bacterium of having gone out, and liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 8-12 hour, and shaking speed is 100-300r/min, and inoculum size is 5%-10%;
(6) heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out 1 than 98-102 dilution proportion, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 20Gy-100Gy;
(7) if the barms preservation time, length need to be-80 DEG C of preservations, if slant preservation is 4 DEG C of preservations.
The preparation method of described high nitrogen yeast, further comprising the steps of: the barms of step (6) is linked into and in 500ml or 1000ml triangular flask, in described liquid nutrient medium, carries out enlarged culturing, liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 6-12 hour, and shaking speed is 100-300r/min, inoculum size: 5%-10%.
The preparation method of described high nitrogen yeast, further comprising the steps of:
The screening of described high nitrogen bacterial classification:
(1) cultivation of yeast and separation:
Under claim 2 step 1-6 the same terms, respectively the bacterial classification under various dose is carried out to liquid culture, described yeast suspension is obtained to wet thallus in 3000-4000r/min centrifugation, distilled water wash, separate, repeat aforesaid operations 2-4 time, add the hot distilled water of 80-100 DEG C of 1:10 to carry out broken wall, maintain after the about 20-30min of this temperature, centrifugation collection supernatant liquor is for subsequent use;
(2) mensuration of protein content: a primary dcreening operation, the above-mentioned supernatant liquor of collecting is measured to its spectrophotometric value under different spectrophotometrics, at 260nm and 280nm place, measuring respectively spectrophotometric value has linear relationship with the protein concentration of surveying, and brings formula protein concentration=1.45 × A into
260-0.74 × A
280wherein A
260, A
280represent respectively the absorbance at 260nm and 280nm place; Carry out successively preliminary screening; B Accurate Measurement, utilizes the determining the protein quantity method specifying in GB/T 5009.5-2003 to carry out quantitative mensuration to the bacterial classification of primary dcreening operation.
Beneficial effect of the present invention: utilize heavy ion irradiation mutagenic treatment microbial strains, embody its practicality and operability thereof for fermentation industry provides good object bacterial strain and makes it industrialization.Enrich the method for induction mutation of bacterium, and for to have opened up the new world with heavy ion mutagenesis microorganism from now on.The major advantage of heavy ion induced-mutation technique shows: (1) aberration rate is high, generally high more than 1000 times than natural variation rate; (2) variation spectrum width, the type of variation is many, can obtain the microbial strains of novel type; (3) variation stability is strong, can obtain metastable bacterial classification (positive and negative sudden change exists simultaneously) and can shorten the screening cycle; (4) suddenly change workablely, can complete relatively easily the irradiation mutagenesis to microorganism.
1 has obtained the barms of high nitrogen by heavy ion irradiation mutagenic treatment.Utilize heavy ion induced-mutation technique can effectively filter out required object bacterial classification, there is original mutagenesis superiority compared with existing induced-mutation technique.
2 utilize simple wall-breaking method: utilize hot distilled water to carry out quick-acting broken walls, saved the time of yeast autolysis, compared saving cost of equipment with ultrasonication with high-pressure homogeneous broken wall, reduce screening cost.
3 have improved screening efficiency and accuracy, and screening is divided into 2 stages, have shortened the needed time of strain screening, have improved screening efficiency.Meanwhile, utilize spectrophotometer to carry out determining the protein quantity, reduced error, improved the accuracy of data.
Utilize carbon heavy ion to carry out radiation treatment to yeast suspension, the best irradiation dose of having determined by many experiments to irradiation dose, bacteria suspension concentration, semilethal rate and positive mutation rate.And obtained by screening operation the high nitrogen barms that nitrogen content is 50%-60%, improve 10%-15% compared with onset bacterial classification nitrogen content.Heavy ion irradiation has broad application prospects as a kind of novel mutagenesis method for microorganism, and the high nitrogen barms simultaneously obtaining has been established solid basis for producing from now on yeast series product.
Embodiment
Below principle of the present invention and feature are described, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1: a kind of high nitrogen yeast, its principal feature is
saccharomyces cerevisiaeyeast saccharomyces cerevisiae, by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No 5004;
The preparation method of high nitrogen yeast, its principal feature is to comprise the following steps:
(1) prepare bacterial classification:
saccharomyces cerevisiaeyeast saccharomyces cerevisiae: yeast saccharomyces cerevisiae 2399, this yeast is purchased from Institute of Microorganism, Academia Sinica.
(2) prepare solid medium glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, agar 0.5%-1.5%, all the other are water;
(3) prepare liquid nutrient medium: glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH by mass percentage
4sO
40.2%-3%, MgSO
40.03%-1.5%, KH
2pO
40.2%-1.5%, all the other are water;
(4) slant culture: will be linked in the described culture medium slant of the bacterium of having gone out after the single bacterium colony picking in culture dish, 25-35 DEG C leaves standstill cultivation;
(5) seed culture: picking slant strains is linked in the described liquid nutrient medium of the bacterium of having gone out, and liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 8-12 hour, and shaking speed is 100-300r/min, and inoculum size is 5%-10%;
(6) heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out 1 to 98 dilution proportion, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 100Gy;
(7) if the barms preservation time, length need to be-80 DEG C of preservations, if slant preservation is 4 DEG C of preservations.
Experimental example 1;
1, experiment content
1.1 bacterial classifications: Saccharomyces cerevisiae yeast saccharomyces cerevisiae; Yeast saccharomyces cerevisiae 2399, this yeast is purchased from Institute of Microorganism, Academia Sinica.
1.2 substratum: glucose 3.0%-5.0%, peptone 0.5%-1.0%, yeast extract powder 0.2-1.0%, NH4SO40.2%-3%, MgSO40.03%-1.5%, KH2PO40.2%-1.5%, all the other are water.
1.3 experiment equipment
Triangular flask, electric furnace, Autoclave, shaking table, Bechtop, batch cultur ware
1.4 seed culture
Picking 3 encircles in the liquid nutrient medium that slant strains is linked into the bacterium of having gone out, and at suitable temperature, shaking table is cultivated 8-12 hour, treats that bacteria suspension visual inspection muddiness carries out a certain proportion of dilution, as the onset bacteria suspension of heavy ion mutagenesis pre-irradiation.
Culture condition: seed culture medium pH4-6, leavening temperature: 25-35 DEG C, shaking speed: 100-300r/min, inoculum size: 5%-10%
1.5 heavy ion irradiation processing
Bacteria suspension is distributed in batch cultur ware, chooses different irradiation dose points and carry out respectively heavy ion irradiation processing, irradiation dose is: 20Gy-100Gy.According to predetermined irradiation metering, actual irradiation dose is corrected, the data of surveying are used for instructing and completing follow-up experiment.
The screening of 1.6 high nitrogen bacterial classifications
1.6.1 the cultivation of yeast and separation
Under the same conditions, respectively the bacterial classification under various dose is carried out to liquid culture, yeast suspension after cultivating is obtained to wet thallus in 3000-4000r/min centrifugation, utilize distilled water wash, separate and repeat aforesaid operations 3 times, add that a certain proportion of hot distilled water dilutes, broken wall, that supernatant liquor is collected in centrifugation is for subsequent use.
1.6.2 the mensuration of protein content
Be divided into primary dcreening operation and Accurate Measurement two portions, because how complicated screening operation is tired, need to carry out preliminary screening, by above-mentioned collected on to clear liquid under different spectrophotometrics, measure its spectrophotometric value, in certain scope, spectrophotometric value has linear relationship with the protein concentration of surveying, and therefore can be used as out the standard of sieve, and the larger spectrophotometric value of concentration is larger.Carry out successively preliminary screening.
Accurately the object of screening is to utilize the determining the protein quantity method specifying in GB/T 5009.5-2003 to carry out quantitative mensuration to the bacterial classification of primary dcreening operation.Determine concrete protein content after radiation treatment.
By the screening of aforesaid method, laboratory has obtained the high nitrogen barms that protein content is greater than 55% at present.
Embodiment 2: a kind of high nitrogen yeast, except step (6) heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out 1 to 100 dilution proportion, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 60Gy; All the other are with embodiment 1.
Embodiment 3: a kind of high nitrogen yeast, except step (6) heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out 1 to 102 dilution proportion, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 20Gy; All the other are with embodiment 1.
Embodiment 4: the preparation method of described high nitrogen yeast, step 1-5 is identical with embodiment 1, step 6 is heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out the dilution of 1 to 98 ratio, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 80Gy-100Gy.
Embodiment 5: the preparation method of described high nitrogen yeast, step 1-5 is identical with embodiment 1, step 6 is heavy ion irradiation processing: treat that bacteria suspension visual inspection muddiness carries out the dilution of 1 to 100 ratio, bacteria suspension is carried out to heavy ion irradiation mutagenesis, irradiation dose is: 20Gy-80Gy.
Embodiment 6: the preparation method of described high nitrogen yeast, further comprising the steps of: the barms of step (6) is linked into and in 500ml triangular flask, in described liquid nutrient medium, carries out enlarged culturing, liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 6-8 hour, and shaking speed is 100-300r/min, inoculum size: 5%-10%.
Embodiment 7: the preparation method of described high nitrogen yeast, further comprising the steps of: the barms of step (6) is linked into and in 1000ml triangular flask, in described liquid nutrient medium, carries out further enlarged culturing, liquid nutrient medium is pH4-6, at the temperature of 25-35 DEG C, shaking table is cultivated 6-8 hour, and shaking speed is 100-300r/min, inoculum size: 5%-10%.
Embodiment 8: described high nitrogen yeast preparation method, further comprising the steps of:
The screening of described high nitrogen bacterial classification:
(1) cultivation of yeast and separation:
Under claim 2 step 1-6 the same terms, respectively the bacterial classification under various dose is carried out to liquid culture, described yeast suspension is obtained to wet thallus in 3000-4000r/min centrifugation, distilled water wash, separate, repeat aforesaid operations 2-4 time, add the 5-15 DEG C of distilled water of 1:5 to dilute, broken wall, centrifugation collection supernatant liquor be for subsequent use;
(2) mensuration of protein content: a primary dcreening operation, the above-mentioned supernatant liquor of collecting is measured to its spectrophotometric value under different spectrophotometrics, at 400nm place, spectrophotometric value has linear relationship with the protein concentration of surveying, and the larger spectrophotometric value of protein concentration is larger.Carry out successively preliminary screening; B Accurate Measurement, utilizes the determining the protein quantity method specifying in GB to carry out quantitative mensuration to the bacterial classification of primary dcreening operation.
By the screening of aforesaid method, laboratory has obtained the high nitrogen barms that protein content is greater than 55% at present.
The application of high nitrogen barms, high nitrogen yeast extract is a kind of natural flavouring, has advantages of pure natural, nutritious, delicious flavour, strong and brisk in taste, therefore can be widely used in all conglomeraties such as food-processing, protective foods.Due to the difference of standard of living and food habits, the research of yeast extract and production are in the existing longer history of the western developed countries such as America and Europe, and be widely used, China is in the ground zero stage, but along with developing rapidly of economic level, there is very large raising and variation to food safety, nutrition, the pursuit of health and the people's standard of living and food habits in human consumer, yeast extract is as a kind of natural, safe, healthy food ingredients, and the market requirement will constantly expand.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (1)
1. a high nitrogen yeast, is Saccharomyces cerevisiae yeast saccharomyces cerevisiae, and by the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No.5004.
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| CN102978115B (en) * | 2012-09-21 | 2014-10-01 | 中国科学院青岛生物能源与过程研究所 | Nannochloropsis sp.OZ-1 mutant strain and heavy ion irradiation selection method for the same |
| FR3014900B1 (en) | 2013-12-16 | 2017-10-27 | Lesaffre & Cie | NEW PERFORMING BREAD YEAST STRAINS ON NON-SUGAR OR SLIGHTLY SWEET PULP |
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Non-Patent Citations (5)
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| E ALBERS ET AL.Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.《APPL. ENVIRON. MICROBIOL.》.1996,第62卷(第9期),3187-3195. |
| Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation;E ALBERS ET AL;《APPL. ENVIRON. MICROBIOL.》;19960930;第62卷(第9期);3187-3195 * |
| 何志平.利用啤酒酵母制备复合氨基酸的研究.《中国优秀硕士学位论文全文数据库》.2003,工程科技Ⅰ辑. |
| 利用啤酒酵母制备复合氨基酸的研究;何志平;《中国优秀硕士学位论文全文数据库》;20030915;工程科技Ⅰ辑 * |
| 田三德等.超声波对酿酒酵母诱变作用的初探.《北京工商大学学报(自然科学版)》.2004,第22卷(第1期),12-15. * |
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