Background technology
Zinc is the essential nutritive substance that humans and animals earns a bare living and grows, and is one of trace element essential in the body.Scarce zinc can influence dna replication dna and protein synthesis in the body, has a strong impact on the normal development of skin, bone and other organ.Children growth phase, women's gestation, lactation, wound, zinc deficiency symptom and the lower immune function of all easily causing such as hemorrhage repeatedly.China preschool children ubiquity lacks the zinc phenomenon.Though certain effect is arranged, absorption rate is low, and reactions such as gastrointestinal upset are often arranged to take inorganic zinc salt (as zinc sulfate etc.), and severe patient can cause enterorrhagia.
Zinc-rich saccharomyces cerevisiae can change into the zinc of mineralized the zinc of organic state, can improve the utilization ratio of zinc.Utilizing Zinc-rich saccharomyces cerevisiae is development of new zinc source with the zinc that the zinc of mineralized changes into organic state, improves the major measure of the utilization ratio of zinc.Zinc-rich saccharomyces cerevisiae will brought into play significant effect aspect prevention and treatment people and the scarce zinc disease of animal.In addition, the Zinc-rich saccharomyces cerevisiae cell also contains abundant nucleic acid, protein, amino acid, VITAMIN etc., is a kind of zinc supplementation protein food product and fodder additives of high nutritive value.
In recent years, come out although have some products that are called " Zinc-rich saccharomyces cerevisiae ", these products exist obvious defects and deficiency:
The one, barms is not screened and breeding, often be with the barms fermentative production Zinc-rich saccharomyces cerevisiae product that obtains at random.But owing to the resistance of different barmses to zinc differs greatly, also bigger to the absorption and conversion capability difference of zine ion, so the product deficient in stability, be unfavorable for suitability for industrialized production.
The 2nd, zinc ion concentration, the culture condition to the substratum of fermentative production Zinc-rich saccharomyces cerevisiae is not optimized, though therefore the yeast of cultivating is called " Zinc-rich saccharomyces cerevisiae ", the zinc content of its yeast cell is lower, and cellular biomass is also lower.
At present, the method that production is called " Zinc-rich saccharomyces cerevisiae " 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 zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) and the market mix.Its major defect is inorganic zinc not to be transformed into organic zinc, and the biological action of this what is called " Zinc-rich saccharomyces cerevisiae " that zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) and yeast cell are mixed is relatively poor, and absorption rate is low.
Second kind is to adopt simple adsorption method, bread yeast on the market or cerevisiae be suspended in the aqueous solution that contains certain density zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) handle certain hour, make zine ion be adsorbed in yeast cell surface, its major defect is that this processing still can not transform into organic zinc with inorganic zinc.The yeast cell of this absorption zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) and above-mentioned first method do not have the difference of essence, and the human or animal is low to its specific absorption, and the biological action of generation is relatively poor.
The third is a culturing yeast cell in containing the substratum of certain zine ion, but barms is not screened and breeding, select for use a kind of barms promptly to ferment at random, its major defect is: owing to the resistance capacity of different barmses to zinc has than big-difference, thereby the absorption and conversion capability of zine ion is differed greatly.In addition, this technology is not carried out system optimization to zinc ion concentration, the culture condition of substratum, therefore adopt in the yeast that this technology cultivates, organic zinc content lower (content of every gram dry yeast cell organic zinc is below 10 milligrams), cellular biomass are also lower.
Summary of the invention
The Zinc-rich saccharomyces cerevisiae bacterial strain that the purpose of this invention is to provide a plant height biomass.
Zinc-rich saccharomyces cerevisiae bacterial strain high-biomass Zinc-rich saccharomyces cerevisiae bacterial strain provided by the invention (Saccharomyces cerevisiae) ZGFH-94, 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 № 0896.
The individual cells shape of high-biomass Zinc-rich saccharomyces cerevisiae bacterial strain (Saccharomyces cerevisiae) ZGFH-94 (CGMCC № 0896) 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 Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass.
A kind of selection of Zinc-rich saccharomyces cerevisiae bacterial strain of high-biomass comprises the steps:
1), in the substratum that contains the gradient zinc ion concentration, screens the high barms of biomass respectively and anti-zinc ability reaches the high barms of cell zinc content by force according to the physio-biochemical characteristics of different barmses;
2) high barms of biomass and anti-zinc ability are reached the high barms of cell zinc content 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-zinc ability reach the high yeast monoploid mutant strain of cell zinc content by force and carry out protoplastis and merge;
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.
Wherein, described gradient zinc ion concentration is 200 micrograms/ml zinc-2000 μ g/ml.The protoplastis integration percentage that barms that described biomass is high and anti-zinc ability reach the high barms of cell zinc content by force is 1: 1-1: 2.Described selection substratum is the YEPD substratum that contains 200 μ g/mL-2000 μ g/mL zine ions.
The 3rd purpose of the present invention provides a kind of method of utilizing high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomycescerevisiae) ZGFH-94 CGMCC № 0896 to produce the Zinc-rich saccharomyces cerevisiae product.
A kind of method of utilizing high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 to produce the Zinc-rich saccharomyces cerevisiae product comprises slant strains cultivation, strain cultivation, secondary liquid seeds cultivation at least, ferment tank cultivation, obtains product.
It is three grades, level Four, Pyatyi or six grades of seed culture that described liquid seeds is cultivated; 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 zinc salt of 200-1000 mcg/ml, the pH of substratum are 3.5-8.5; The inoculum size of described fermentor cultivation is 5%-35%.Described zinc salt is zinc sulfate, acetify zinc, zinc chloride or zinc oxide.Described organism is selected from wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination.
High-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) the ZGFH-94 CGMCC № 0896 that the present invention uses, the organic zinc content of not only biomass height, and cell is higher, and every gram stem cell contains the organic zinc amount up to 15 milligrams.
The method that the present invention produces the Zinc-rich saccharomyces cerevisiae product is a fermentation substrate with common wort, amylum hydrolysate of the sugar, molasses or their arbitrary combination, only need to regulate its total sugar content, interpolation zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) etc. can be produced, method is simple, and is with low cost.Production method of the present invention is practical, and is easy and simple to handle, and fermentation equipment and working condition are not had particular requirement, utilizes the equipment of general fermentation plant and working condition to produce, and not only is suitable for the production that large-scale production also is suitable for short run.
Embodiment
The seed selection of embodiment 1, high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896
The seed selection of high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 is divided into following step:
1) according to yeast the resistant determination of zinc sulfate is carried out primary dcreening operation, the yeast of having measured 435 strain different generas is containing 200 mcg/ml, 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 zinc ion concentration of 2000 mcg/ml, peptone 1%, glucose 4%, 1% agar powder) growing state on is therefrom selected the bacterial strain that it is good that 32 strains are grown on the YEPD solid medium that contains 1400 mcg/ml zine ions.
2) measure 32 strain bacterial strains cultured cells biomass and the cell zinc content in the YEPD liquid nutrient medium that contains 400 mcg/ml zine ions 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 zinc content of every gram stem cell is below 5 milligrams) and the higher bacterial strain (zinc content of every gram stem cell is more than 10 milligrams, but the dry cell weight of every liter of nutrient solution is below 8 grams) of 8 strain cell zinc 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 Y-78 (saccharomyces cerevisiae) of spore and a strain cell zinc content is higher, give birth to the good diploid Y-389 (wine yeast) 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 zinc content of haploid strains respectively, therefrom select higher monoploid Y-78-8 (a) of biomass and the higher monoploid Y-389-15 (a) of cell zinc content.
4) respectively monoploid Y-78-8 (a) and monoploid Y-389-15 (a) are carried out mutagenic obtained nutrient defect mutation strain with nitrosoguanidine and ethyl sulfate, from mutant strain, select the Y-78-8-21 (a that has the different aminoacids flaw labeling, his) and Y-389-15-16 (a is met) as merging parental plant.
5) prepare Y-78-8-21 (a according to a conventional method, his) and Y-389-15-16 (a, protoplastis met), with the protoplastis of two bacterial strains by 1: 1 mixed, merge as fusion-inducing agent with polyoxyethylene glycol, go up the screening fusant at minimum medium (YNB substratum).
6) measure fusant cultured cells biomass and cell zinc content in the YEPD substratum that contains 400 mcg/ml zine ions.Therefrom select cellular biomass and cell zinc content and obviously be better than merging the fusant bacterial strain of parental plant, i.e. high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896.High-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 has the good character of parents' strain.The cellular biomass of high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 and cell zinc content are apparently higher than parental plant Y-78 and Y-389, and the dry cell weight of every liter of nutrient solution reaches more than 24 grams; The zinc content of every gram stem cell is more than 12 milligrams.
7) the genetic stability analysis of high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896: high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 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 YEPD substratum, YNB substratum, YNB+HIS substratum and the YNB+MET substratum that contains 1400 mcg/ml zine 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 at random and singly drop on cultivation back mensuration cellular biomass and cell zinc content in the YEPD substratum that contains 600 mcg/ml zine ions, the result shows that cellular biomass and cell zinc content do not have considerable change.The good Zinc-rich saccharomyces cerevisiae bacterium that The above results proof high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94CGMCC № 0896 is a strain inheritance stability, cellular biomass height, cell zinc content is high.
The optimization culture condition of embodiment 2, high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896
1) culture condition that adopts single-factor fermenting experiment and multiplefactor orthogonal experiment to carry out high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 (comprises nutrient media components, wustite concentration, the pH value of substratum, air flow, inoculum size and fermentation time etc.) experiment, the culture condition experiment of passing through, determined the optimization culture condition: molasses culture medium (or malt extract medium or amylum hydrolysate of the sugar substratum), total sugar content is 8%-20%, zinc salt is (as zinc sulfate in the substratum, acetify zinc, zinc chloride, zinc oxide etc.) concentration is 200 micrograms-1000 mcg/ml, the pH of substratum is 4.0-7.0, inoculum size is 5%-30%, 25 ℃-35 ℃ stir culture 16-30 hour.
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 28 grams, and the zinc content of every gram stem cell reaches more than 15 milligrams.
The production of embodiment 3, Zinc-rich saccharomyces cerevisiae
The production technique of the Zinc-rich saccharomyces cerevisiae product of high-biomass and high zinc content may further comprise the steps successively.
The Zinc-rich saccharomyces cerevisiae that slant strains, liquid spawn, level liquid seed culture, secondary liquid seeds are cultivated, three grades of liquid seeds cultivation, ferment tank cultivation, collection yeast cell and drying, pulverizing obtain high-biomass and high zinc content.Further specify as follows to each production technique below:
(1) slant strains is cultivated
High-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 is inoculated in and contains that total sugar content is 16%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is on the wort solid inclined-plane of 800 mcg/ml,, put into 4 ℃ of refrigerators and preserve after 48 hours 25-35 ℃ of cultivation;
(2) strain cultivation
Behind high-biomass Zinc-rich saccharomyces cerevisiae (Saccharomyces cerevisiae) ZGFH-94 CGMCC № 0896 actication of culture of preserving, connect a garland cells in the triangular flask that the malt extract medium that 150 milliliters of total sugar contents are 10%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is 400 mcg/ml is housed, be liquid spawn in 16 hours in 25-35 ℃ of stir culture;
(3) level liquid seed culture
Inoculum size by 5% inserts liquid spawn in 30 triangular flasks that the malt extract medium that 150 milliliters of total sugar contents are 12%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is 600 mcg/ml is housed, 25-35 ℃ of stir culture 18 hours, be the level liquid inoculum;
(4) the secondary liquid seeds is cultivated
Inoculum size by 15% inserts the first order seed nutrient solution and is equipped with that 45 liters of total sugar contents are 12%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is the Xiao Kashi fermentor tank of the malt extract medium of 800 mcg/ml, 25-35 ℃ of stir culture 17 hours, be secondary liquid seeds culture;
(5) three grades of liquid seeds are cultivated
Inoculum size by 20% inserts the secondary seed nutrient solution and is equipped with that 450 liters of total sugar contents are 16%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is the Da Kashi fermentor tank of the malt extract medium of 700 mcg/ml, 25-35 ℃ of stir culture 19 hours, be three grades of liquid seeds cultures;
(6) ferment tank is cultivated
Inoculum size by 30% inserted three grades of seed culture fluids and is equipped with that 2250 liters of total sugar contents are 14%, zinc salt (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.) concentration is the fermentor tank of the malt extract medium of 500 mcg/ml, 25-35 ℃ of stir culture 18 hours;
(7) collect yeast cell and drying
Adopt sheet frame squeezing or centrifugal collection Zinc-rich saccharomyces cerevisiae cell, 45~85 ℃ of air-dry its water content that make less than 5%;
(8) pulverize
With pulverizer with air-dry Zinc-rich saccharomyces cerevisiae cell pulverization;
(9) packing
Use air-locked packaging bags, be the Zinc-rich saccharomyces cerevisiae (the dried Zinc-rich saccharomyces cerevisiae cell of every gram contains organic zinc up to more than 15 milligrams) of high-biomass, high rich zinc content.
The production of embodiment 4, Zinc-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 8% amylum hydrolysate of the sugar, 0.5% corn hydrolyzed solution, 0.4% ammonium sulfate, the zinc salt of 200 mcg/ml (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.), the pH of substratum is 6, inoculum size is 15%, under 25-35 ℃ condition, stir culture 22 hours.
Its production technique may further comprise the steps successively: slant strains, liquid spawn, level liquid seed culture, secondary liquid seeds cultivate, three grades of liquid seeds are cultivated, the level Four liquid seeds is cultivated, ferment tank is cultivated, collect yeast cell and drying, pulverizing, obtains the Zinc-rich saccharomyces cerevisiae of high-biomass high zinc content.
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, Zinc-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: 3,0.5% corn hydrolyzed solution, 0.8% ammonium sulfate, 0.10% phosphoric acid, the zinc salt of 1000 mcg/ml (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.), the pH of substratum was 7.0, and inoculum size is 20%, 25-35 ℃ of stir culture 20 hours.
Its production technique may further comprise the steps successively: liquid spawn, level liquid seed culture, secondary liquid seeds cultivate, three grades of liquid seeds are cultivated, the level Four liquid seeds is cultivated, the Pyatyi liquid seeds is cultivated, ferment tank is cultivated, collect yeast cell and drying, pulverizing, obtains the Zinc-rich saccharomyces cerevisiae of high-biomass high zinc 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.
The production of embodiment 6, Zinc-rich saccharomyces cerevisiae
With sucrose molasses and wort is substratum, its fermentative medium formula and culture condition are: the sucrose molasses and the wort of total reducing sugar amount 16%, its ratio of weight and number is 4: 1,1.0% corn hydrolyzed solution, 0.6% ammonium sulfate, 0.05% phosphoric acid, the zinc salt of 400 mcg/ml (as zinc sulfate, acetify zinc, zinc chloride, zinc oxide etc.), the pH of substratum was 7.5, and inoculum size is 30%, 25-35 ℃ of stir culture 18 hours.
Its production technique may further comprise the steps successively: liquid spawn, level liquid seed culture, secondary liquid seeds cultivate, three grades of liquid seeds are cultivated, the level Four liquid seeds is cultivated, the Pyatyi liquid seeds is cultivated, ferment tank is cultivated, collect yeast cell and drying, pulverizing, obtains the Zinc-rich saccharomyces cerevisiae of high-biomass high zinc 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.