CN101629146B - Zinc-rich yeast with high biomass, breeding selection method and application thereof - Google Patents
Zinc-rich yeast with high biomass, breeding selection method and application thereof Download PDFInfo
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Abstract
The invention relates to a zinc-rich yeast with high biomass, a breeding selection method and application thereof. A zinc-rich yeast bacterial strain with high biomass is Sacharomyces cerevisiae) F-090428X. The bacterial strain is preserved in China General Microbiological Culture Collection Center (CGMCC for short) in July 2nd, 2009 with the preservation number of No.3156. The breeding selection method of the zinc-rich yeast with high biomass adopts microwave irradiation. The zinc-rich yeast with high biomass can be used for preparing high zincic human food or high zincic additives or high zincic medicaments, or can be used for preparing high zincic animal feed or high zincic feed addictive.
Description
Technical field
The present invention relates to the kind of a kind of microorganism, selection of this microorganism and application thereof, the microorganism that the present invention relates to exactly is a kind of high-biomass Zinc-rich saccharomyces cerevisiae.
Background technology
In recent years, having some products that are called " Zinc-rich saccharomyces cerevisiae " comes out, as Li Wenzhen etc. with cereuisiae fermentum (Sacchsromyces cerevisiae) for setting out bacterial classification, utilize the corn saccharification to cross and add suitable nutritive element and zine ion, obtain product through fermentation test, 1994 the 14th 1 phases of volume of JOURNAL OF MICROBIOLOGY; Hu Zhenying etc. cultivate Yeast strain of beer carried out for 50 generations under high zink rod spare adaptability, have obtained the Zinc-rich saccharomyces cerevisiae bacterial strain, 1997 years the 6th phases of Chinese veterinarian's medical magazine; Chen Peilin: with yeast IFO2346 bacterial strain is bacterial classification, adds an amount of zine ion in the substratum, obtains high zinc yeast product through fermentation test, animal medicine progress 2003,24 (3) 62-64; Guo Xuena etc. have used crossbreeding technology to the yeast strain of different genera, go out sieve, multiple sieve, monoploid separation, nitrosoguanidine (NTG) mutagenesis, and seed selection (is numbered ZGH.374, microorganism journal 44 2 phases of volume of April in 2004 to a strain Zinc-rich saccharomyces cerevisiae bacterial strain.
Chinese invention patent application 93119253.6 discloses a kind of medical zinc-rich yeast, production technique and goods thereof, it adopts and selects the high Yeast strain of beer of zinc content for use in the strain culturing process, in seeding tank or commodity jar inoculation culture process, in substratum, add zinc sulfate respectively, through corresponding technological process, obtain Zinc-rich saccharomyces cerevisiae; Zinc-rich saccharomyces cerevisiae and batching are made the medical zinc-rich yeast goods through the production technique of medicinal granule or medicinal tablets.
Chinese invention patent 03119428.1 discloses a strain Zinc-rich saccharomyces cerevisiae and selection and application, and bacterial strain provided by the invention is high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896.Its selection comprises: 1) screen the high barms of biomass respectively and anti-zinc ability reaches the high barms of cell zinc content by force; 2) screen the monoploid mutant strain that high barms of biomass and anti-zinc ability reach the high barms of cell zinc content by force; 3) yeast monoploid mutant strain that biomass is high and anti-zinc ability reach the high yeast monoploid mutant strain of cell zinc content by force and carry out the protoplastis fusion; 4) at the fusion diploid strain excellent of selecting to screen on the substratum high-biomass and high zinc content, i.e. Zinc-rich saccharomyces cerevisiae bacterial classification.
But these products exist obvious defects and deficiency: the research of relevant Zinc-rich saccharomyces cerevisiae both at home and abroad mainly lays particular emphasis on yeast cell and carries out method mutagenesis such as nitrosoguanidine (NTG), cobalt 60, ultraviolet to the transmission of zine ion and regulation and control or to bacterial classification, its major defect is: different barmses have than big-difference the resistance capacity of zinc, thereby the absorption and conversion capability of zine ion is differed greatly; And the zinc content in its yeast cell is lower, and the product deficient in stability is unfavorable for suitability for industrialized production, and referring to Confucian Garden etc., November the 25th in 2006 rolled up for the 6th phase by food and biotechnology journal.
Summary of the invention
The invention provides the high yeast strain of zinc content of a plant height biomass, the invention provides this zymic selection, cultural method and uses thereof simultaneously.
High-biomass Zinc-rich saccharomyces cerevisiae bacterial strain provided by the invention (Saccharomyces cerevisiae) F-090428X.This bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 07 02nd, 2009, preserving number is CGMCC No.3156.
The selection of the Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention be with 2000 μ g/l～10000 μ g/l zinc ion concentrations the wort solid medium in filter out the biomass height in the saccharomyces cerevisiae of taming, anti-zinc ability reaches the high barms of cell zinc content by force as the mutagenesis starting strain, a bacterial strain access of getting a transfering loop fills the 30ml liquid nutrient medium and is prepared into bacteria suspension, at 29 ℃, shaking table jolting 24h, getting 2ml bacterium liquid again changes in the stroke-physiological saline solution that fills 18ml band bead, again with the bacteria suspension microwave exposure that is not higher than 900W, irradiation time is not more than 65 minutes, to be coated in the wort solid medium flat board that zinc sulfate concentration is not higher than 15000 μ g/ml through the yeast zinc bacteria suspension of microwave exposure, through repeatedly sieving and go down to posterity test again, obtain the Zinc-rich saccharomyces cerevisiae bacterial strain of desired high-biomass, more than used wort solid medium be the 8Bx wort, weight by volume is than the urea and 1.2% agar that are 0.3%, pH5.0, used liquid nutrient medium are the 8Bx wort, weight by volume is than the urea that is 0.3%, pH5.0.
Used microwave frequency is 2450MHz in the seed selection of the Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention.
The culture condition of the Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention is inoculation high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X on malt extract medium, inoculum size is volume ratio 10%-30%, 28 ℃～30 ℃ stir culture 16～48 hours, used substratum is 8Bx wort, weight by volume than the wort-urea medium that is 0.3% urea, total sugar content is 5%～20%, zinc salt concentration is 10000～15000 μ g/ml in the substratum, and the pH of substratum is 4.5～5.5.
The Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention is in the human high zinc-containing food of preparation or high zinc-containing food additive or the high application that contains in the zinc medicine.
The Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention contains zinc feed or the high application that contains in the zinc feed additive at preparation animal height.
High-biomass Zinc-rich saccharomyces cerevisiae (Sacchsromyces cerevisiae) F-090428X that the present invention uses is the biomass height not only, and the organic zinc content of cell is higher, detection shows, its every gram stem cell contains the organic zinc amount up to 50mg, biomass 12g/L, and the every gram of existing high-biomass Zinc-rich saccharomyces cerevisiae ZGH-374 bacterial strain stem cell contains the organic zinc amount only for 14.3mg, and biomass 9.2g/L was referring to microorganism journal " seed selection of a Zinc-rich saccharomyces cerevisiae and culture condition research " literary composition in 2004.
The method that the present invention produces the Zinc-rich saccharomyces cerevisiae product is a fermentation substrate with common wort, 0.3% urea medium, only needs to regulate its total sugar content, and interpolation zinc salt amount (as zinc sulfate) etc. can be produced, and method is simple, and is with low cost.Employed best microwave radiation frequency is 2450MHz in its seed selection, can adopt common household microwave oven to realize by the irradiation mutagenesis of seed selection bacterial classification; The preferred culture condition of the Zinc-rich saccharomyces cerevisiae bacterial strain of the high-biomass that the present invention provides can make it that maximum productive rate is arranged; Therefore method of the present invention is practical, easy and simple to handle, fermentation equipment and working condition there is not particular requirement, utilize the equipment and the working condition of general fermentation plant to produce, less investment, instant effect, high efficiency, not only be suitable for the production that large-scale production also is suitable for short run, have wide actual application prospect.
Description of drawings
Fig. 1 is the technological process of production synoptic diagram of Zinc-rich saccharomyces cerevisiae product.
Embodiment
The seed selection of embodiment 1, high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X
High-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) seed selection (F-090428X) is divided into following step:
1) according to yeast the patience mensuration of zinc sulfate is carried out primary dcreening operation, measured the growing state of saccharomyces cerevisiae on the wort solid medium of the gradient zinc ion concentration that contains 5000 μ g/ml, 6000 μ g/ml, 8000 μ g/ml, 10000 μ g/ml, therefrom selected the bacterial strain that it is good that 2 strains are grown on the wort solid medium that contains 10000 μ g/ml zine ions.
2) measure 1 strain bacterial strain cultured cells biomass and the cell zinc content in the malt juice liquid medium that contains 10000 μ g/ml zine ions that primary dcreening operation obtains.By domestication, screening, obtain 1 strain (F-090415) stem cell zinc content 35.5mg/g, the bacterial strain of dry cell weight 9.2g/L.
3) the superior strain F-090415 (zinc 10000 μ g/ml) that obtains with screening of the investigation of mutagenesis dosage is as starting strain, get the bacteria suspension for preparing in a triangular flask, be equipped with bacteria suspension triangular flask put into 2450MHz 900W household microwave oven and carry out the irradiation of different time.Irradiation time totally is respectively 0,10,20,30,40,60 and 80s, and when irradiation 80s, lethality rate has reached 100%.
4) the mutagenesis bacterial screening is according to the relation of microwave irradiation dosage and mortality ratio, is coated in the culture medium flat plate that zinc sulfate concentration is 10000 μ g/ml, 12000 μ g/ml, 15000 μ g/ml from the yeast zinc bacteria suspension of 0s, 10s, 20s, 30s, 40s, 60s.After treating that bacterium colony grows, each dosage group is chosen single bacterium colony switching slant culture respectively.With long good bacterium inclined-plane, serve as to contrast the shaker test of fermenting with starting strain F-090415, measure each bacterial strain zinc content and dry cell weight.The result is with respect to starting strain, and the bacterial strain percentage that throughput exceeds more than 20% in the 40s dosage group significantly improves.Illustrate that microwave irradiation irradiation 40s has positive-effect to the bacterial classification that yeast zinc produces bacterium.The bacterial strain of choosing above starting strain throughput 20% from each group carries out multiple sieve.Through repeatedly sieving and go down to posterity test again, obtain 40s dosage group F-090428X bacterial strain, the yeast zinc throughput in shaking bottle (dry cell weight 12g/L) improves 30% than starting strain F-090415, and zinc content 50.0mg/g improves 40.8% than starting strain.
The optimization culture condition of embodiment 2, high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X
1) culture condition that adopts single-factor fermenting experiment and multiplefactor orthogonal experiment to carry out high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X (comprises nutrient media components, zinc salt concentration, the pH value of substratum, air flow, inoculum size and fermentation time etc.) experiment, test by culture condition, determined the optimization culture condition: wort+0.3% urea medium, total sugar content is 5%-20%, zinc salt in the substratum (as zinc sulfate) concentration is 10000-15000 μ g/ml, the pH of substratum is 4.5-5.5, inoculum size is 10%-30%, 28 ℃ of-30 ℃ of stir culture 16-48h.
2) at the fermentation culture conditions bottom fermentation cultivation high-biomass of optimizing and the Zinc-rich saccharomyces cerevisiae of high zinc content: optimizing under the culture condition, the dry cell weight of every liter of nutrient solution reaches more than the 12g, and the zinc content of every gram stem cell reaches more than the 50mg.
The production of embodiment 3, high-biomass Zinc-rich saccharomyces cerevisiae
The production technique of the Zinc-rich saccharomyces cerevisiae product of high-biomass and high zinc content comprises:
Slant strains → one-level is shaken rich zinc → packing → preparation (fodder additives or capsule, tablet etc. the) → packing of a bottle spawn culture → secondary shake-flask seed cultivation → seeding tank → secondary liquid seeds jar → fermentor tank → storage tank → solid-liquid separation → freeze-drying or spraying drying → (or adopting sheet frame squeezing or centrifugal collection Zinc-rich saccharomyces cerevisiae cell → pulverizing) → high-biomass, high rich zinc content.
Below production technique is further specified as follows:
(1) slant strains is cultivated
High-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X is inoculated in and contains that total sugar content is 16%, zinc salt (as zinc sulfate) concentration is on the wort solid inclined-plane of 15000 μ g/ml,, put into 4 ℃ of refrigerators and preserve after three days 28-30 ℃ of cultivation:
(2) one-level is shaken a bottle spawn culture
Behind high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) the F-090428X actication of culture of preserving, 150 milliliters of total sugar contents are 10% in being equipped with, zinc salt (as zinc sulfate) concentration is in the 1-3 * 500ml Erlenmeyer flask of the malt extract medium of 12000 μ g/ml to connect a garland cells, are liquid spawn in 28-30 ℃ of stir culture 18h;
(3) the secondary shake-flask seed is cultivated
Inoculum size by 10% inserts liquid spawn and is equipped with in 150 milliliters of total sugar contents are 12%, zinc salt (as zinc sulfate) concentration is the malt extract medium of 12000 μ g/ml 20-30 * 500ml Erlenmeyer flask, at 28-30 ℃ of stir culture 20h, be the level liquid inoculum:
(4) seed tank culture
Inoculum size by 15% inserts the first order seed nutrient solution and is equipped with that 100 liter total sugar contents are 12%, zinc salt (zinc sulfate) concentration is the fermentor tank of the malt extract medium of 12000 μ g/ml, at 28-30 ℃ of stir culture 18h, is secondary liquid seeds culture;
(5) the secondary liquid seeds is cultivated
Inoculum size by 20% inserts the secondary seed nutrient solution and is equipped with that 500 liter total sugar contents are 16%, zinc salt (zinc sulfate) concentration is the fermentor tank of the malt extract medium of 15000 μ g/ml, at 28-30 ℃ of stir culture 20h, is three grades of liquid seeds cultures:
(6) ferment tank is cultivated
Inoculum size by 30% inserts three grades of seed culture fluids and is equipped with that 30000 liter total sugar contents are 14%, zinc salt (zinc sulfate) concentration is the fermentor tank of the malt extract medium of 15000 μ g/ml, at 28-30 ℃ of stir culture 24h;
(7) collect yeast cell and drying
Adopt lyophilize or spraying drying (or sheet frame squeezing or centrifugal collection Zinc-rich saccharomyces cerevisiae cell, 45-85 ℃ of air-dry its water content that makes less than 5%);
(8) preparation
Fodder additives or capsule, tablet etc.
(9) packing
Use air-locked packaging bags, be the Zinc-rich saccharomyces cerevisiae of high-biomass, high rich zinc content, the dried Zinc-rich saccharomyces cerevisiae cell of every gram contains organic zinc and is higher than 50mg).
The Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass of the present invention can be in the human high zinc-containing food of preparation or high zinc-containing food additive or the high application that contains in the zinc medicine; Prop can contain zinc feed or the high application that contains in the zinc feed additive at preparation animal height.
Claims (4)
1. a Zinc-rich yeast with high biomass (Saccharomyces cerevisiae) F-090428X, bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 07 02nd, 2009, and preserving number is CGMCC No.3156.
2. the cultural method of the Zinc-rich saccharomyces cerevisiae bacterial strain of the described high-biomass of claim 1, it is characterized in that inoculation high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) F-090428X on malt extract medium, inoculum size is volume ratio 10%-30%, 28 ℃～30 ℃ stir culture 16～48 hours, used substratum is 8Bx wort, weight by volume than the wort-urea medium that is 0.3% urea, total sugar content is 5%～20%, zinc salt concentration is 10000～15000 μ g/ml in the substratum, and the pH of substratum is 4.5～5.5.
3. the Zinc-rich saccharomyces cerevisiae bacterial strain of the described high-biomass of claim 1 is in the human high zinc-containing food of preparation or high zinc-containing food additive or the high application that contains in the zinc medicine.
4. the Zinc-rich saccharomyces cerevisiae bacterial strain of the described high-biomass of claim 1 contains zinc feed or the high application that contains in the zinc feed additive at preparation animal height.
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| CN108192854A (en) * | 2018-01-31 | 2018-06-22 | 浙江大学 | A kind of richness manganese yeast product and its production method |
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| CN103126968B (en) * | 2011-11-30 | 2014-09-03 | 安琪酵母股份有限公司 | Zinc-rich yeast refined extract and preparation method thereof |
| CN103478406A (en) * | 2013-08-27 | 2014-01-01 | 格特生物制药（天津）有限公司 | Fermented mineral feed and preparation method thereof |
| CN104372061B (en) * | 2014-10-21 | 2018-04-06 | 浙江省林业科学研究院 | A kind of preparation method of zinc-rich Phellinus liquid fermentation production |
| CN104543401B (en) * | 2015-01-20 | 2018-04-20 | 河南科技大学 | A kind of preparation method for the state manganese feed addictive that ferments |
| CN104543400B (en) * | 2015-01-20 | 2018-04-20 | 河南科技大学 | A kind of preparation method for the state zinc feed additive that ferments |
| CN107418904A (en) * | 2017-08-30 | 2017-12-01 | 广州富诺营养科技有限公司 | A kind of preparation method of Zinc-rich saccharomyces cerevisiae |
| CN112251366B (en) * | 2020-11-06 | 2022-07-15 | 河南省科学院生物研究所有限责任公司 | Zinc-rich candida tropicalis strain and application thereof |
| CN113621669B (en) * | 2021-09-02 | 2022-12-23 | 广东丸美生物技术股份有限公司 | Zinc-rich yeast polypeptide and preparation method and application thereof |
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| CN1530435A (en) * | 2003-03-12 | 2004-09-22 | 中国科学院微生物研究所 | High-biomass zinc-riched yeast, breeding method and use thereof |
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