CN100347286C - Calcium enriched beer yeast strain and use - Google Patents
Calcium enriched beer yeast strain and use Download PDFInfo
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- CN100347286C CN100347286C CNB2004100327185A CN200410032718A CN100347286C CN 100347286 C CN100347286 C CN 100347286C CN B2004100327185 A CNB2004100327185 A CN B2004100327185A CN 200410032718 A CN200410032718 A CN 200410032718A CN 100347286 C CN100347286 C CN 100347286C
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Abstract
The present invention discloses a strain of calcium-enriched beer yeast and application thereof. The present invention aims to provide a strain of calcium-enriched beer yeast with high biomass and a method for producing the calcium-enriched yeast. The calcium-enriched beer yeast strain with high biomass provided in the present invention is beer yeast (Saccharomyces cerevisiae) ZGFH-66 CGMCC No. 1116. The method for producing the calcium-enriched yeast provided in the present invention comprises the following step: fermenting the beer yeast (Saccharomyces cerevisiae) ZGFH-66 CGMCC No. 1116 so as to obtain the calcium-enriched yeast. The fermentation process comprises strain cultivation on an inclined plane, liquid strain cultivation, at least two stages of liquid seed cultivation and fermentation cultivation in a fermentation tank. After the cultivation in the fermentation tank, yeast cells are collected, dried and crushed so as to obtain the calcium-enriched yeast. With the advantages of low production cost and simple operation, the method for producing the calcium-enriched yeast by using the beer yeast (Saccharomyces cerevisiae) ZGFH-66 CGMCC No. 1116 in the present invention has broad prospects on practical application.
Description
Technical field
The present invention relates to rich calcium cereuisiae fermentum of a plant height biomass and application aborning in the microbial fermentation industrial technology field.
Background technology
Calcium is bioelement, plays crucial effect in the human body growth and development process, and especially particularly important to the children, women and the elderly that are growing, calcium deficiency can cause multiple disease.When human body can not obtain the calcium of enough somagenic needs by food, common employing was taken calcium preparation and is replenished.Along with progress of science and technology, calcium preparation develops to calglucon, amino acid calcium chelating calcium preparation from simple lime carbonate.But these calcium preparations normally adopt chemical reaction or chemical synthesis process to obtain, and have following problem in the application: 1) water solubility is little, and human body is lower to its absorption rate; 2) contain the by product that in the chemosynthesis process, generates, usually human body is had certain side effect; 3) production cost is higher.
Yeast cell can be enriched to the calcium of the mineralized in the nutrient solution calcium that changes into organic state in the cell in its breeding process of growth, utilizing yeast is development of new calcium source with the calcium that the calcium of mineralized changes into organic state, improves the effective measure of the utilization ratio of calcium.In addition, yeast 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.
At present, have some and be called the product of " calcic yeast ", the method for producing these products 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 calcium chloride 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 calcium chloride handles certain hour, makes calcium ion be adsorbed in yeast cell surface; The third is a culturing yeast cell in containing the substratum of certain calcium ion.
In the aforesaid method, the major defect of first kind and second kind is that yeast cell fails effectively inorganic calcium to be converted into organic calcium, causes the people low to its calcareous absorption rate of institute.The third method then exists following deficiency and defective:
The one, barms is not screened and breeding, often be with cereuisiae fermentum or bread yeast bacterial classification fermentative production calcic yeast.Because different barmses differ greatly to the resistance of calcium, and are also bigger to the absorption and conversion capability difference of calcium ion, so the product deficient in stability, are unfavorable for suitability for industrialized production.
The 2nd, calcium ion concn, the culture condition to calcic yeast fermentation substratum is not optimized, though the yeast of cultivating is called " calcic yeast ", the organic calcium content of its yeast cell lower (content of every gram dry yeast cell organic calcium is below 20 milligrams), cellular biomass are also lower.
Summary of the invention
The purpose of this invention is to provide the rich calcium cereuisiae fermentum of a plant height biomass.
Rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) ZGFH-66 of high-biomass provided by the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 03 22nd, 2004, preserving number is CGMCC № 1116.
The cell of rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of this high-biomass № 1116 be an ellipse, and colony morphology characteristic is bacterium colony projection, smooth, oyster white, neat in edge.30 ℃ of optimum growth temperatures, pH is 5.5.
Another object of the present invention provides the rich calcium yeast method of a kind of production.
The rich calcium yeast method of production provided by the present invention is that rich calcium cereuisiae fermentum (Saccharomycescerevisiae) ZGFH-66 of fermentation culture CGMCC № 1116 obtains rich calcium yeast.
The fermentation culture process comprises slant strains cultivation, strain cultivation, secondary liquid seeds cultivation at least, ferment tank cultivation.
Described liquid seeds is cultivated and is multistage seed culture, with three grades of-six grades of seed culture for well; After fermentor cultivation is finished, collect yeast cell, carry out drying, pulverizing then, obtain rich calcium yeast.
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 calcium salt of 80-200 mg/ml, pH are 4.0-7.0.The inoculum size of fermentation culture is 10%-40%.Described organism is selected from wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination; Calcium salt adds with the calcium chloride form usually.The fermentation culture conditions of optimization of the present invention is: inoculum size is 10-30%, 25-35 ℃ stir culture 16-30 hour.
The present invention has obtained rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of the high-biomass № 1116 of a strain stabilization characteristics of genetics by screening and cross-breeding.This bacterial strain has the high and high advantage of cell organic calcium content of biomass, and under the fermentation culture conditions of optimizing, the dry cell weight of every liter of nutrient solution can reach more than 20 grams, and every gram stem cell calcic reaches more than 60 milligrams.
The present invention utilizes rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116 to produce rich calcium yeast, used fermention medium is common wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination, only need to regulate its total sugar content, add calcium salt (calcium chloride) and get final product, have advantage 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.
Embodiment
The seed selection of embodiment 1, rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116
The seed selection of rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66CGMCC № 1116 of high-biomass is divided into following step:
1) according to yeast the resistant determination of calcium chloride is carried out primary dcreening operation: the yeast of getting 380 strain different generas, its cultivation is being contained 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, 180 mg/ml, YEPD solid medium (the yeast powder 1% of the gradient calcium ion concn of 200 mg/ml, peptone 1%, glucose 2%, 1% agar powder) on, therefrom selects 32 strains well-grown bacterial strain on the YEPD solid medium that contains 120 mg/ml calcium ion concns.
2) measure 32 strain bacterial strains cultured cells biomass and the cell calcium content in the YEPD liquid nutrient medium that contains 120 mg/ml calcium ion concns that primary dcreening operation obtains.By screening, (dry cell weight of every liter of nutrient solution is more than 15 grams to obtain the higher bacterial strain of 6 strain cellular biomass, but the calcium content of every gram stem cell is below 20 milligrams) and the higher bacterial strain (calcium content of every gram stem cell is more than 40 milligrams, but the dry cell weight of every liter of nutrient solution is below 11 grams) of 7 strain cell calcium contents.
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 bread microzyme ZY-29 of spore (Chinese common micro-organisms DSMZ preservation, preserving number: CGMCC 2.240) and a strain cell calcium content is higher, give birth to the good diploid bread microzyme ZY-242 of spore (Chinese common micro-organisms DSMZ preservation, preserving number: CGMCC 2.558), according to a conventional method it being given birth to the spore cultivation separates with monoploid.Measure the cellular biomass and the cell calcium content of haploid strains respectively, therefrom select higher monoploid ZY-29-22 (a) of biomass and the higher monoploid ZY-242-16 (α) of cell calcium content.
4) respectively ZY-29-22 (a) and ZY-242-16 (α) are carried out mutagenesis with ethyl sulfate and nitrosoguanidine, obtain the nutrient defect mutation strain, from mutant strain, select the mutant strain ZY-29-22-14 (a that has the different aminoacids flaw labeling, his) and ZY-242-16-7 (α, met) as hybridization parental plant.
5) with the colony hybridization method with ZY-29-22-14 (a, his) and ZY-242-16-7 (α, cell met) is hybridized, it is sub to go up screening hybridization at minimum medium (YNB substratum).
6) measure hybridization cultured cells biomass and the cell calcium content in the YEPD substratum that contains 120 mg/ml calcium ion concns that obtains, therefrom select the high hybrid strain ZGFH-66 of cellular biomass and cell calcium content, i.e. rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116.
The dry cell weight of every liter of nutrient solution of this hybrid strain ZGFH-66 is more than 20 grams, and the calcium content of every gram stem cell all apparently higher than parental plant ZY-29 and ZY-242, is the good rich calcium yeast strain of a strain more than 45 milligrams.
The genetic stability analysis of rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116: rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66CGMCC № 1116 of high-biomass gone down to posterity at the YEPD solid medium cultivate 30 times, getting cell cultivates on the plate of coating YEPD, then at random 100 single bacterium colonies of picking 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+HIS substratum and the YNB+MET substratum that contains 120 mg/ml calcium 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 10 single bacterium colonies at random and cultivate in the YEPD substratum that contains 120 mg/ml calcium ions, cultivate the back and measure cellular biomass and cell calcium content, the result shows that cellular biomass and cell calcium content all do not have considerable change.Rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66CGMCC № 1116 of The above results proof high-biomass has good genetic stability.
The optimization culture condition of embodiment 2, rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116
The culture condition experiment of adopting single-factor fermenting experiment and multiplefactor orthogonal experiment to carry out the rich calcium cereuisiae fermentum of high-biomass ZGFH-66 (comprises nutrient media components, calcium concentration, the pH value of substratum, air flow, inoculum size and fermentation time etc.), determined the optimization culture condition: total sugar content is the malt extract medium (or molasses culture medium or amylum hydrolysate of the sugar substratum) of 8%-20%, calcium salt in the substratum (calcium chloride) concentration is 80 milligrams-200 mg/ml, pH is 4.5-7.0, air flow is 0.8v.v.m, the cultivation inoculum size is 10%-40%, 25 ℃-35 ℃ stir culture 16-30 hour.
Under the culture condition of this optimization, the dry cell weight of every liter of nutrient solution can reach more than 20 grams, and the calcium content of every gram stem cell reaches more than 60 milligrams.
Embodiment 3, rich calcium zymic are produced
The production technique of the rich calcium yeast product of high-biomass and high calcium content comprises:
(1) slant strains: rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66CGMCC № 1116 of high-biomass is inoculated in contains that total sugar content is 8%, calcium chloride concentration is on the wort solid inclined-plane of 80 mg/ml, under 25-35 ℃ condition, cultivate after 48 hours, put into 4 ℃ of refrigerators and preserve.
(2) liquid spawn: after rich calcium cereuisiae fermentum (Saccharomyces cerevisiae) the ZGFH-66 CGMCC of high-biomass № 1116 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%, calcium chloride concentration is 100 mg/ml is housed, under 25-35 ℃ condition, shaking culture 16-20 hour, be 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%, calcium chloride concentration is 100 mg/ml is housed, under 25-35 ℃ condition, shaking culture 16-20 hour, be 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%, calcium chloride concentration is the Xiao Kashi fermentor tank of the malt extract medium of 100 mg/ml, under 25-35 ℃ condition, stir culture 16-20 hour, be 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%, calcium chloride concentration is the Da Kashi fermentor tank of the malt extract medium of 120 mg/ml, under 25-35 ℃ condition, stir culture 12-16 hour, be 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%, calcium chloride concentration is the fermentor tank of the malt extract medium of 140 mg/ml, under 25-35 ℃ condition, stir culture 12-18 hour.
(7) collect yeast cell and drying: adopt sheet frame squeezing or the rich calcium yeast cell of centrifugal collection.Under this culture condition, the dry cell weight of every liter of nutrient solution is the 20-22 gram, and the calcium content of every gram stem cell is the 60-64 milligram.
(8) air-dry: the air-dry water content of rich calcium yeast cell that makes is less than 5% under 45～85 ℃ of conditions.
(9) pulverize: air-dry rich calcium yeast cell is pulverized with pulverizer.
(10) packing: use air-locked packaging bags, obtain finished product.
Embodiment 4, rich calcium zymic are produced
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 calcium chloride of 80-200 mg/ml, the pH of substratum are 4-6, and inoculum size is 10-40%, under 28-35 ℃ condition, stir culture 12-20 hour.
The production technique of present embodiment increases the level Four liquid seeds cultivates, and it is identical with three grades of liquid seeds cultivations that its inoculum size and substratum are formed, and other processing condition are identical with embodiment 3.Under the condition of present embodiment, the rich calcium yeast cell dry weight of every liter of nutrient solution is the 20-23 gram, and the calcium content of every gram stem cell is the 60-65 milligram.
Embodiment 5, rich calcium zymic are produced
With molasses is substratum, its fermentative medium formula and culture condition are: the beet sirup of total reducing sugar amount 8-20% and sucrose molasses, its ratio of weight and number is 1: 3,0.8% corn hydrolyzed solution, 0.8% ammonium sulfate, 0.10% phosphoric acid, the calcium chloride of 80-200 mg/ml, the pH of substratum was 46, and inoculum size is 10-40%, at 25-35 ℃ of stir culture 12-20 hour.
Its production technique comprises: 1) liquid spawn; 2) level liquid seed culture; 3) the secondary liquid seeds is cultivated; 4) three grades of liquid seeds are cultivated; 5) the level Four liquid seeds is cultivated; 6) the Pyatyi liquid seeds is cultivated; 7) ferment tank is cultivated; 8) collect yeast cell and drying; 9) pulverize; 10) rich calcium yeast.
Present embodiment increases the Pyatyi liquid seeds on the basis of embodiment 4 cultivates, and it is identical with the cultivation of level Four liquid seeds that its inoculum size and substratum are formed, and other processing condition are identical with embodiment 4.Under the condition of present embodiment, the rich calcium yeast cell dry weight of every liter of nutrient solution is the 20-25 gram, and the calcium content of every gram stem cell is the 60-63 milligram.
Claims (4)
1, cereuisiae fermentum (Saccharomyces cerevisiae) ZGFH-66 CGMCC № 1116.
2, the rich calcium yeast method of a kind of production is that fermentation beer yeast (Saccharomyces cerevisiae) ZGFH-66 CGMCC № 1116 obtains rich calcium yeast; Wherein, fermenting process comprises slant strains cultivation, strain cultivation, the secondary liquid seeds is cultivated and the ferment tank cultivation at least; The substratum that described slant strains cultivation, strain cultivation, liquid seeds cultivation and ferment tank are cultivated comprises wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination of 8%-20% total reducing sugar amount, the calcium chloride of 80-200 mg/ml, pH are 4.0-7.0.
3, the rich calcium yeast method of production according to claim 2 is characterized in that: it is three grades or level Four or Pyatyi or six grades of seed culture that described liquid seeds is cultivated; After described fermentor cultivation is finished, collect yeast cell and carry out drying, pulverizing, obtain rich calcium yeast.
4, according to claim 2 or the rich calcium yeast method of 3 described productions, it is characterized in that: the inoculum size of described fermentor cultivation is 10%-40%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1309177A (en) * | 2001-01-17 | 2001-08-22 | 中国科学院微生物研究所 | Se-enriched high-biomass yeast and its preparing process |
| CN1366037A (en) * | 2001-01-17 | 2002-08-28 | 中国科学院微生物研究所 | Cr-enriched yeast containing high biomass and its preparing process |
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| CN1309177A (en) * | 2001-01-17 | 2001-08-22 | 中国科学院微生物研究所 | Se-enriched high-biomass yeast and its preparing process |
| CN1366037A (en) * | 2001-01-17 | 2002-08-28 | 中国科学院微生物研究所 | Cr-enriched yeast containing high biomass and its preparing process |
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