CN1530436A - A high-biomass iron-rich yeast and its breeding method and application - Google Patents
A high-biomass iron-rich yeast and its breeding method and application Download PDFInfo
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Abstract
A high-biomass iron-enriched saccharomyces cerevisiae ZYGF-15 (CGMCC No.0895) is selectively cultured through respectively screening high-biomass yeast strain and the yeast strain with high resistance to iron and high iron content in its cell, screening their haplontic mutant strains, hybridizing, selective culturing, and screening the excellent hydridized diploid strain.
Description
Technical field
The present invention relates to a strain ferrum-rich saccharomyces cerevisiae and selection and application aborning in the microbial fermentation industrial technology field, particularly relate to a plant height biomass ferrum-rich saccharomyces cerevisiae and selection thereof and application aborning.
Background technology
Iron is that humans and animals earns a bare living and grows necessary nutritive substance, is indispensable trace element in the body, participates in the translocation of oxygen in the blood.The shortage of iron can cause people and animal hypoferric anemia and some other clinical symptom.In the daily life, replenish iron in the inorganic salt mode usually, but this benefit iron mode is unfavorable for people and animal absorbing iron.
Yeast cell can change into the iron of mineralized the iron of organic state, can improve the utilization ratio of iron.Utilizing ferrum-rich saccharomyces cerevisiae is the development of new source of iron with the iron that the iron of mineralized changes into organic state, improves the effective measure of the utilization ratio of iron.Ferrum-rich saccharomyces cerevisiae will brought into play significant effect aspect prevention and treatment people and animal hypoferric anemia and some other iron deficiency diseases.In addition, the ferrum-rich saccharomyces cerevisiae cell also contains tens kinds of essential trace elements of rich in protein, nucleic acid, VITAMIN and body, is a kind of rich nutrient substances.
In recent years, be called the product of " ferrum-rich saccharomyces cerevisiae " although have some, these products exist obvious defects and deficiency:
The one, barms is not screened and breeding, often be with the barms fermentative production ferrum-rich saccharomyces cerevisiae product that obtains at random.But owing to the resistance of different barmses to iron differs greatly, also bigger to the absorption and conversion capability difference of iron ion, so the product deficient in stability, be unfavorable for suitability for industrialized production.
The 2nd, iron concentration, the culture condition to the ferrum-rich saccharomyces cerevisiae fermention medium is not optimized, though the yeast of cultivating is called " ferrum-rich saccharomyces cerevisiae ", the iron-holder of its yeast cell is lower, and cellular biomass is also lower.
At present, producing the method that is called " ferrum-rich saccharomyces cerevisiae " cell has three kinds substantially: first kind is to adopt mixed processing method, and the bread yeast or the cerevisiae that are about on a certain amount of ironic citrate, ferric sulfate or ferrous sulfate and the market mix; Second kind is to adopt simple adsorption method, the bread yeast on the market or cerevisiae is suspended in the aqueous solution that contains certain density ironic citrate, ferric sulfate or ferrous sulfate handles certain hour, makes iron ion be adsorbed in yeast cell surface; The third is a culturing yeast cell in containing the substratum of certain iron ion, but barms is not carried out any screening and breeding, and barms obtains at random.
In the aforesaid method, the major defect of first kind and second kind is that yeast cell fails effectively inorganic iron to be converted into organic iron, causes the human or animal low to its ferruginous absorption rate of institute.The major defect of the third method is that this technology is not optimized iron concentration, the culture condition of substratum, therefore adopt in the yeast that this technology cultivates, organic iron level lower (the organoferric content of every gram dry yeast cell is below 10 milligrams), cellular biomass are also lower.
Summary of the invention
The ferrum-rich saccharomyces cerevisiae bacterial strain that the purpose of this invention is to provide a plant height biomass.
High-biomass ferrum-rich saccharomyces cerevisiae provided by the invention (Saccharomyces cerevisiae) ZYGF-15, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 02 26th, 2003, preserving number is CGMCC № 0895.
The individual cells shape of high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) is oval, and colony morphology characteristic is bacterium colony projection, smooth, oyster white, neat in edge.30 ℃ of optimum growth temperatures.The growth optimal pH is 5.5.
Second purpose of the present invention provides a kind of selection of ferrum-rich saccharomyces cerevisiae bacterial strain of high-biomass.
A kind of selection of ferrum-rich saccharomyces cerevisiae bacterial strain of high-biomass comprises the steps:
1), in the substratum that contains the gradient iron concentration, screens the high barms of biomass respectively and anti-iron ability reaches the high barms of cell iron-holder by force according to the physio-biochemical characteristics of different barmses;
2) high barms of biomass and anti-iron ability are reached the high barms of cell iron-holder by force and give birth to that spore is cultivated, monoploid separates, mutagenesis, screen monoploid mutant strain with different mating types and different genetic markers;
3) yeast monoploid mutant strain that the biomass that is separated to is high and anti-iron ability reach the high yeast monoploid mutant strain of cell iron-holder by force and hybridize;
4) selecting screening high-biomass and high Fe contained diploid hybrid strain excellent, i.e. high-biomass ferrum-rich saccharomyces cerevisiae bacterial strain on the substratum.
The 3rd purpose of the present invention provides a kind of method of utilizing high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) to produce the ferrum-rich saccharomyces cerevisiae product of high-biomass.
A kind of method of utilizing high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) to produce the ferrum-rich saccharomyces cerevisiae product comprises slant strains cultivation, strain cultivation, secondary liquid seeds cultivation at least, ferment tank cultivation, obtains product.
Described liquid seeds is cultivated and is secondary to six grade seed culture.After described fermentor cultivation was finished, also drying, pulverizing obtained product to collect yeast cell.The substratum that described slant strains cultivation, strain cultivation, liquid seeds cultivation and ferment tank are cultivated comprises the organism of 8%-20% total reducing sugar amount, and the wustite of 200-800 mcg/ml, pH are 4.0-8.0; The inoculum size of described fermentor cultivation is 5%-30%.Described wustite is ironic citrate, ferric sulfate or ferrous sulfate.Described organism is selected from wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination.The fermentation culture conditions of optimization of the present invention is: inoculum size 5-30%, 25-35 ℃ stir culture 16-30 hour.
Organic iron level of the not only biomass height, and cell of high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) that the present invention uses is higher, and every gram stem cell contains organic iron amount up to 25 milligrams.
The fermentation substrate that the method that the present invention utilizes high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) to produce the ferrum-rich saccharomyces cerevisiae product of high-biomass is used is common wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination, only need to regulate its total sugar content, interpolation wustite (as ironic citrate, ferric sulfate or ferrous sulfate) etc. can be produced, method is simple, and is with low cost.Production 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 high-biomass ferrum-rich saccharomyces cerevisiae
Embodiment
The seed selection of embodiment 1, high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895)
The seed selection of high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) is divided into following step:
1) according to yeast the resistant determination of ferrous sulfate is carried out primary dcreening operation, the yeast of having measured 402 strain different generas is containing 200 mcg/ml respectively, 400 mcg/ml, 600 mcg/ml, 800 mcg/ml, 1000 mcg/ml, 1200 mcg/ml, 1400 mcg/ml, 1600 mcg/ml, 1800 mcg/ml, YEPD solid medium (the yeast powder 1% of the gradient iron concentration of 2000 mcg/ml, peptone 1%, glucose 4%, 1% agar powder) growing state on is therefrom selected 28 strains well-grown bacterial strain on the YEPD solid medium that contains 1600 mcg/ml iron concentrations.
2) measure 28 strain bacterial strains cultured cells biomass and the cell iron-holder in the YEPD liquid nutrient medium that contains 600 mcg/ml iron concentrations that primary dcreening operation obtains.By screening, (dry cell weight of every liter of nutrient solution is more than 17 grams to obtain the higher bacterial strain of 7 strain cellular biomass, but the iron-holder of every gram stem cell is below 8 milligrams) and the higher bacterial strain (iron-holder of every gram stem cell is more than 16 milligrams, but the dry cell weight of every liter of nutrient solution is below 10 grams) of 5 strain cell iron-holder.
3) by giving birth to the spore experiment, from above-mentioned bacterial strains, pick out a strain biomass higher, give birth to the good diploid ZY-46 (saccharomyces cerevisiae) of spore and a strain cell iron-holder is higher, give birth to the good diploid ZY-173 (bread microzyme) of spore, according to a conventional method it is given birth to the spore cultivation and separates with monoploid.Measure the cellular biomass and the cell iron-holder of haploid strains respectively, therefrom select higher monoploid ZY-46-12 (α) of biomass and the higher monoploid ZY-173-29 (a) of cell iron-holder.
4) respectively ZY-46-12 (α) and ZY-173-29 (a) are carried out mutagenesis with nitrosoguanidine and ethyl sulfate, obtain the nutrient defect mutation strain, from mutant strain, select the mutant strain ZY-46-12-30 (α that has the different aminoacids flaw labeling, leu) and ZY-173-29-5 (a, trp) as hybridization parental plant.
5) with the colony hybridization method with ZY-46-12-30 (α, leu) and ZY-173-29-5 (a, cell trp) is hybridized, it is sub to go up screening hybridization at minimum medium (YNB substratum).
6) measure hybridization cultured cells biomass and the cell iron-holder in the YEPD substratum that contains 600 mcg/ml iron ions that obtains.Therefrom select cellular biomass and cell iron-holder and obviously be better than hybridizing the hybrid strain ZYGF-15 of parental plant.Hybrid strain ZYGF-15 has the good character of hybridization parental plant.The cellular biomass of hybrid strain ZYGF-15 and cell iron level are apparently higher than parental plant ZY-46 and ZY-173, the dry cell weight of every liter of nutrient solution reaches more than 25 grams, the iron-holder of every gram stem cell is more than 20 milligrams, the result shows that hybrid strain ZYGF-15 is the good ferrum-rich saccharomyces cerevisiae of a strain, i.e. high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895).
7) the genetic stability analysis of high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895): high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) gone down to posterity at the YEPD solid medium cultivate 30 times, get an amount of cell after suitably diluting, coating YEPD plate, after the cultivation, distinguish 100 single bacterium colonies of picking at random in sterilized water, hungry at ambient temperature 4-6 hour.The bacterium liquid of getting after the hunger is inoculated in YEPD substratum, YNB substratum, YNB+LEU substratum and the YNB+TRP substratum that contains 600 mcg/ml iron ions respectively, 100 single bacterium colonies of cultivation results proof picking are all grown at these four kinds of substratum, separation phenomenon do not occur.Choose at random 10 singly drop on contain 600 mcg/ml iron ions the YEPD substratum in cultivate to cultivate the back and measure cellular biomass and cell iron-holder, the result shows that cellular biomass and cell iron-holder do not have considerable change.The above results proof high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) be a strain inheritance stability, cellular biomass height, good ferrum-rich saccharomyces cerevisiae bacterium that the cell iron-holder is high.
The optimization culture condition of embodiment 2, high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895)
1) culture condition that adopts single-factor fermenting experiment and multiplefactor orthogonal experiment to carry out high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) (comprises nutrient media components, wustite concentration, the pH value of substratum, air flow, inoculum size and fermentation time etc.) experiment, determined the optimization culture condition: malt extract medium (or molasses culture medium or amylum hydrolysate of the sugar substratum), total sugar content is 8%-20%, wustite is (as ironic citrate in the substratum, ferric sulfate or ferrous sulfate) concentration is 200 micrograms-800 mcg/ml, the pH of substratum is 4.5-7.0, inoculum size is 5%-30%, 25 ℃-35 ℃ stir culture 16-30 hour.
2) cultivate high-biomass and high Fe contained ferrum-rich saccharomyces cerevisiae at the fermentation culture conditions bottom fermentation of optimizing: under the culture condition of optimizing, the dry cell weight of every liter of nutrient solution reaches more than 30 grams, and the iron-holder of every gram stem cell reaches more than 25 milligrams.
The production of embodiment 3, ferrum-rich saccharomyces cerevisiae
The production technique of the ferrum-rich saccharomyces cerevisiae product of high-biomass and high Fe content comprises
Slant strains → liquid spawn → level liquid seed culture → secondary liquid seeds is cultivated the ferrum-rich saccharomyces cerevisiae of → three grades of liquid seeds cultivation → ferment tank cultivation → collection yeast cell and drying → pulverizing → high-biomass and high Fe content.Further specify as follows to each production technique below:
(1) slant strains: high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (CGMCC № 0895) is inoculated in contains that total sugar content is 8%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is on the wort solid inclined-plane of 400 mcg/ml, under 25-35 ℃ condition, cultivate after 48 hours, put into 4 ℃ of refrigerators and preserve.
(2) liquid spawn: after high-biomass ferrum-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain ZYGF-15 (the CGMCC № 0895) activation of preserving, connect a garland cells in the triangular flask that the malt extract medium that 150 milliliters of total sugar contents are 10%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is 400 mcg/ml is housed, under 25-35 ℃ condition, stir culture 18 hours is liquid spawn.
(3) level liquid seed culture: the inoculum size by 15% inserts liquid spawn in the triangular flask that the malt extract medium that 150 milliliters of total sugar contents are 18%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is 600 mcg/ml is housed, under 25-35 ℃ condition, stir culture 16 hours is the level liquid inoculum.
(4) the secondary liquid seeds is cultivated: the inoculum size by 15% inserts the first order seed nutrient solution and is equipped with that 45 liters of total sugar contents are 20%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is the Xiao Kashi fermentor tank of the malt extract medium of 600 mcg/ml, under 25-35 ℃ condition, stir culture 16 hours is secondary liquid seeds culture.
(5) three grades of liquid seeds are cultivated: the inoculum size by 15% inserts the secondary seed nutrient solution and is equipped with that 450 liters of total sugar contents are 20%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is the Da Kashi fermentor tank of the malt extract medium of 600 mcg/ml, under 25-35 ℃ condition, stir culture 16 hours is three grades of liquid seeds cultures.
(6) ferment tank is cultivated: the inoculum size by 40% inserts three grades of seed culture fluids and is equipped with that 2250 liters of total sugar contents are 20%, wustite (as ironic citrate, ferric sulfate or ferrous sulfate) concentration is the fermentor tank of the malt extract medium of 600 mcg/ml, under 25-35 ℃ condition, stir culture 14 hours.
(7) collect yeast cell and drying: adopt sheet frame squeezing or centrifugal collection ferrum-rich saccharomyces cerevisiae cell.Air-dry its water content that makes is less than 5% under 45～85 ℃ of conditions.
(8) pulverize: with pulverizer with air-dry ferrum-rich saccharomyces cerevisiae cell pulverization.
(9) packing: use air-locked packaging bags, be the ferrum-rich saccharomyces cerevisiae of the high rich iron level of high-biomass.
The production of embodiment 4, ferrum-rich saccharomyces cerevisiae
With the amylum hydrolysate of the sugar is substratum, its fermentative medium formula and culture condition are: the total reducing sugar amount is the amylum hydrolysate of the sugar of 8-20%, 0.5% corn hydrolyzed solution, 0.4% ammonium sulfate, the wustite of 400-600 mcg/ml (as ironic citrate, ferric sulfate or ferrous sulfate), the pH of substratum is 6, inoculum size is 15%, under 28-35 ℃ condition, stir culture 20 hours.
The production technique of present embodiment increases the level Four liquid seeds cultivates, and its condition is identical substantially with three grades of liquid seeds cultivations, and other processing condition are identical with embodiment 3.
The production of embodiment 5, ferrum-rich saccharomyces cerevisiae
With amylum hydrolysate of the sugar and beet sirup is substratum, its fermentative medium formula and culture condition are: the amylum hydrolysate of the sugar of total reducing sugar amount 20% and beet sirup, its ratio of weight and number is 1: 3,0.8% corn hydrolyzed solution, 0.8% ammonium sulfate, 0.10% phosphoric acid, the wustite of 600 mcg/ml (as ironic citrate, ferric sulfate or ferrous sulfate), the pH of substratum was 5, and inoculum size is 30%, 25-35 ℃ of stir culture 16 hours.
Its production technique comprises: liquid spawn → level liquid seed culture → secondary liquid seeds is cultivated the ferrum-rich saccharomyces cerevisiae of → three grades of liquid seeds cultivation → level Four liquid seeds cultivation → Pyatyi liquid seeds cultivation → ferment tank cultivation → collection yeast cell and drying → pulverizing → high-biomass high Fe content.
The production technique of present embodiment increases the Pyatyi liquid seeds cultivates, and its condition is identical substantially with three grades of liquid seeds cultivations, and other processing condition are identical with embodiment 4.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629145B (en) * | 2009-07-21 | 2011-05-18 | 中国科学院微生物研究所 | Magnesium-rich saccharomyces cerevisiae as well as cultivation method and application thereof |
| CN101575579B (en) * | 2008-05-06 | 2012-01-04 | 安琪酵母股份有限公司 | Ferrum-rich saccharomyces cerevisiae and production method thereof |
| CN107236677A (en) * | 2017-05-15 | 2017-10-10 | 浙江大学 | A kind of ferrum-rich saccharomyces cerevisiae product and its production method |
| CN111434767A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院微生物研究所 | Copper-rich yeast and preparation method of yeast copper |
| CN113025510A (en) * | 2019-12-25 | 2021-06-25 | 中国科学院微生物研究所 | Function-enhanced yeast culture rich in organic trace elements and preparation method thereof |
| CN113897298A (en) * | 2021-10-15 | 2022-01-07 | 江苏爸爸糖食品科技有限公司 | Optimal control method of natural yeast selection and fermentation process |
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2003
- 2003-03-12 CN CN 03119430 patent/CN1271200C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575579B (en) * | 2008-05-06 | 2012-01-04 | 安琪酵母股份有限公司 | Ferrum-rich saccharomyces cerevisiae and production method thereof |
| CN101629145B (en) * | 2009-07-21 | 2011-05-18 | 中国科学院微生物研究所 | Magnesium-rich saccharomyces cerevisiae as well as cultivation method and application thereof |
| CN107236677A (en) * | 2017-05-15 | 2017-10-10 | 浙江大学 | A kind of ferrum-rich saccharomyces cerevisiae product and its production method |
| CN111434767A (en) * | 2019-01-15 | 2020-07-21 | 中国科学院微生物研究所 | Copper-rich yeast and preparation method of yeast copper |
| CN111434767B (en) * | 2019-01-15 | 2021-10-15 | 中国科学院微生物研究所 | A strain of copper-rich yeast and preparation method of yeast copper |
| CN113025510A (en) * | 2019-12-25 | 2021-06-25 | 中国科学院微生物研究所 | Function-enhanced yeast culture rich in organic trace elements and preparation method thereof |
| CN113025510B (en) * | 2019-12-25 | 2022-09-16 | 中国科学院微生物研究所 | Function-enhanced yeast culture rich in organic trace elements and preparation method thereof |
| CN113897298A (en) * | 2021-10-15 | 2022-01-07 | 江苏爸爸糖食品科技有限公司 | Optimal control method of natural yeast selection and fermentation process |
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