CN104286415A - Saccharomyces cerevisiae microbial preparation with high yield of glutathione and preparation method thereof - Google Patents
Saccharomyces cerevisiae microbial preparation with high yield of glutathione and preparation method thereof Download PDFInfo
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- CN104286415A CN104286415A CN201410519741.0A CN201410519741A CN104286415A CN 104286415 A CN104286415 A CN 104286415A CN 201410519741 A CN201410519741 A CN 201410519741A CN 104286415 A CN104286415 A CN 104286415A
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Abstract
The invention discloses a saccharomyces cerevisiae microbial preparation with high yield of glutathione and a preparation method thereof. The saccharomyces cerevisiae microbial preparation product with high yield of glutathione is produced by such production processes easy to achieve industrial production as high-density liquid fermentation, solid fermentation and low temperature drying. Each gram of saccharomyces cerevisiae microbial preparation prepared by the method contains 2-4 billions of live bacteria, the percentage of contaminating microorganism is low, in particular, the yield of glutathione of saccharomyces cerevisiae in the saccharomyces cerevisiae microbial preparation is high and can be up to 400 mg/kg above, the conversion rate is high and the production rate is high. The strain prepared by the method is easy to culture, raw materials are readily available and are cheap, reaction conditions are moderate, reaction steps are simple, and the cost is low. The prepared saccharomyces cerevisiae microbial preparation is used as a microbial forage additive to promote the growth of animals.
Description
Technical field:
The invention belongs to field of fodder, be specifically related to a kind of high-yield glutathione saccharomyces cerevisiae microorganism formulation and preparation method thereof.
Background technology:
Saccharomyces cerevisiae is one of the bacterial classification that the Ministry of Agriculture of China allows as feed addictive, and it is developed into feeding micro-ecological preparation more and more.Its metabolite of saccharomyces cerevisiae is rich in the multiple physiologically active ingredients such as amino acid, Cobastab, protein and multiple enzyme, especially containing abundant glutathione (glutathione, GSH), it is a kind of tripeptides containing γ-amido link and sulfydryl, is made up of glutamic acid, cysteine and glycine.Saccharomycete and metabolite thereof can improve animal oxidation resistance; strengthen Immune Function In Animals; improve animal disease resistant ability etc.; these characteristics absorb the intestinal mucosa that watches for animals, immunity moderation system, nutrient digestion such as promotion amino acid, glucose etc.; strengthen animal oxidation resistance, the feed conversion rate and the diseases prevention growth promoting effects that improve animal play an important role.
Therefore the saccharomycete adding high-yield glutathione in feed is the selection of a very green health.
The method of producing glutathione at present both at home and abroad mainly contains extraction, chemical synthesis, fermentation method, enzyme process, solution fermentation is the main method of current production glutathione, the bacterial classification generally used is saccharomycete, the method of using microbe liquid fermentation is produced glutathione and is compared and be easy to industrialization, but in its saccharomyces cerevisiae microorganism formulation prepared, have lost all metabolites that saccharomycete produces, only retain saccharomycete thalline, and yield poorly, price is higher, add in feed simultaneously and can improve feed cost, restrict it and use on a large scale.
Summary of the invention:
The present invention aims to provide a kind of high-yield glutathione saccharomyces cerevisiae microorganism formulation and preparation method thereof, the saccharomyces cerevisiae microorganism formulation utilizing the method to prepare, its glutathione content is high, conversion ratio is high, throughput rate is fast, and the method reaction condition is gentle, reactions steps is simple, with low cost.
The preparation method of high-yield glutathione saccharomyces cerevisiae microorganism formulation of the present invention, is characterized in that, comprise the following steps:
A, actication of culture: be transferred in YEPD fluid nutrient medium after being activated by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139,28 ± 1 DEG C are cultured to bacteria concentration is 1.0 × 10
7cfu/mL ~ 2.0 × 10
8cfu/mL, forms seed culture fluid;
B, seed culture fluid is transferred in the fermentation tank containing one grade fermemtation culture medium with the inoculum concentration of mass percent 3% ~ 5%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 200 ~ 350rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 80 ~ 90%, fermentation to saccharomyces cerevisiae grows into exponential phase, using the bacterium liquid of this exponential phase as primary seed solution;
C, primary seed solution is transferred to containing second order fermentation culture medium fermentation tank with the inoculum concentration of mass percent 2 ~ 10% in carry out second order fermentation, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 150 ~ 250rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 70 ~ 80%, fermentation to saccharomyces cerevisiae grows into exponential phase, using the bacterium liquid of this exponential phase as secondary seed solution;
D, in gnotobasis, by secondary seed solution with in the inoculum concentration of mass percent 2 ~ 10% access wheat bran solid medium, mix, controlling ventilation is: 3 ~ 10 rates of ventilation/hour, temperature 25 ~ 32 DEG C, cultivate 60 ~ 72 hours under humidity 80% ~ 90% condition, stir wheat bran solid medium between culture period, through the inspection of semifinished product, every gram of wet thallus is containing viable count 2,000,000,000 ~ 4,000,000,000 viable bacterias, after 30-45 DEG C of dry process, obtain high-yield glutathione saccharomyces cerevisiae microorganism formulation;
Described firsts and seconds fermentation medium is all: often liter contains glucose 5 ~ 30g, sucrose 2 ~ 20g, molasses 2 ~ 30g, NaCl3 ~ 10g, peptone 5 ~ 20g, dipotassium hydrogen phosphate 1-15g, and surplus is water, pH6.0 ± 0.2;
Described wheat bran solid medium is: by gross mass mark 100%, is made up of wheat bran 38% ~ 50%, dregs of beans 10% ~ 23%, urea 0.1% ~ 0.5%, ammonium chloride 0.1% ~ 0.5% and water 38% ~ 51%.
This high-yield glutathione saccharomyces cerevisiae microorganism formulation is through packaging, and finished product detection, stores, can be used as commodity selling.
Described being activated by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 is preferably by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 streak inoculation to wort agar culture medium test tube slant, cultivates 24 hours for 28 DEG C.
Stir wheat bran solid medium between the culture period of described steps d preferably to stir once every 3 ~ 5 hours.
Described wort agar culture medium belongs to conventional medium of the prior art, and its formula is: often liter contains malt extract powder 130g, agar 20g, chloramphenicol 0.1g, and surplus is water, pH6.0 ~ 6.2.
Described YEPD fluid nutrient medium belongs to conventional medium of the prior art, and its formula is: often liter contains dusty yeast 10g, peptone 20g, glucose 20g, distilled water 1000mL, pH nature, 115 DEG C of moist heat sterilization 20min.
The present invention, compared to prior art, has advantage:
The saccharomyces cerevisiae microorganism formulation prepared according to method of the present invention, its every gram contains viable count 20 ~ 4,000,000,000, and miscellaneous bacteria rate is low, and the glutathione output of the saccharomyces cerevisiae especially in this saccharomyces cerevisiae microorganism formulation is high, can reach more than 400mg/kg, conversion ratio is high, throughput rate is fast.The bacterial classification of the inventive method is easy to cultivate, and raw material sources are convenient cheap, and reaction condition is gentle, reactions steps is simple, with low cost.The saccharomyces cerevisiae preparation prepared is used as additive for microbe feedstuff, can promote the growth of animal.
Accompanying drawing illustrates:
Fig. 1 is the saccharomyces cerevisiae growth curve chart in embodiment 1 in one grade fermemtation;
Fig. 2 is the saccharomyces cerevisiae growth curve chart in embodiment 1 in second order fermentation.
Detailed description of the invention:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
Saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 (is preserved in Guangdong Province's Culture Collection, preserving number GIM2.139, this bacterial classification is for sale, can buy) freeze-drying kind on picking one ring thalline be seeded in wort agar culture medium test tube slant culture medium, 28 DEG C cultivate 24 hours; Be transferred in YEPD fluid nutrient medium by test tube slant seed again, cultivate 24h for 28 DEG C, form seed culture fluid, its bacteria concentration is 1.0 × 10
8cfu/mL.The formula of described wort agar culture medium is: often liter contains malt extract powder 130g, agar 20g, chloramphenicol 0.1g, and surplus is water, pH6.0 ~ 6.2,115 DEG C of autoclaving 20min.YEPD Liquid Culture based formulas is: often liter contains dusty yeast 10g, peptone 20g, glucose 20g, distilled water 1000mL, pH nature, 115 DEG C of moist heat sterilization 20min.
Seed culture fluid is proceeded to the inoculum concentration of mass percent 5% in the 30L seeding tank that one grade fermemtation culture medium is housed and carries out fermented and cultured, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 200rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 85 ~ 90%, after inoculation, the light absorption value of 560nm is once surveyed in every 2h sampling, with nonvaccinated culture medium as blank, make saccharomyces cerevisiae growth curve chart (Fig. 1), sharply increased at 8 ~ 12h bacterium number by the known saccharomyces cerevisiae of curve, show that thalline arrives exponential phase, taking fermentation time is that the bacterium liquid of 8 hours makes primary seed solution, by primary seed solution with 10% inoculum concentration, transfer second order fermentation culture medium is housed 300L fermentation tank in expand cultivation further, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 250rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 70 ~ 80%, after inoculation, the light absorption value of 560nm is once surveyed in every 2h sampling, with nonvaccinated culture medium as blank, make saccharomyces cerevisiae growth curve chart (Fig. 2), sharply increased at 12 ~ 25h bacterium number by the known saccharomyces cerevisiae of curve, show that thalline reaches exponential phase, the bacterium liquid of therefore taking this period obtains secondary seed solution, what choose in the present embodiment be fermentation time is that the bacterium liquid of 16 hours is as secondary seed solution.Described firsts and seconds fermentation medium is, often liter contains glucose 10g, sucrose 20g, molasses 15g, NaCl3g, peptone 5g, dipotassium hydrogen phosphate 5g, and surplus is water, pH6.0 ± 0.2, after above-mentioned component is mixed, 121 DEG C of autoclaving 30min.
Before inoculation, first open the ozone generator and ultraviolet violet light switch of cultivating room, cultivate room area: 25m
2sterilization 30min, preparation gnotobasis, secondary seed solution be inoculated in wheat bran solid medium with the inoculum concentration of mass percent 3% in this gnotobasis, mix, the ventilation controlled in wheat bran solid medium is: 10 rates of ventilation/hour, temperature 28 DEG C, humidity is cultivate 60 hours under the condition of 80 ~ 90%, stirs wheat bran solid culture base-material once between culture period every 3 hours, therefrom obtained high-yield glutathione saccharomyces cerevisiae microorganism formulation.Described wheat bran solid medium, by gross mass mark 100%, is made up of wheat bran 38%, dregs of beans 23%, urea 0.5%, ammonium chloride 0.5% and water 38%, and raw material mixes, and stirs.Then at rotary digester or meal braizing machine, pressure, temperature is respectively: 1.05 ~ 1.3Kg/cm
3, under 121 ~ 125 DEG C of conditions, sterilizing 30 ~ 40min, is cooled to 40 ~ 50 DEG C of shallow basins of packing, every basin 5kg, for subsequent use.
One, the detection of viable count and miscellaneous bacteria rate:
1, the method for plate culture count
1.1, the high-yield glutathione saccharomyces cerevisiae microorganism formulation 10g taking the present embodiment, as sample, puts into and fills 90ml sterilized water and with the conical flask of bead, and shaking table 200rpm vibrates 20min, leaves standstill 20 ~ 30S, makes sample suspension.
1.2, draw 1mL sample suspension with 1ml aseptic straw, put into the test tube filling 9mL aqua sterilisa, shake well makes to mix, and namely makes the even dilution of 1:100.
1.3, draw 1:100 dilution 1mL with 1mL aseptic straw, slowly inject containing 9mL aqua sterilisa in vitro along tube wall, jolting test tube, mixes, and makes the dilution of 1:1000.Separately get 1mL sterilizing suction pipe, by aforesaid operations order, do 10 times and increase progressively dilution, make 10 respectively
-4, 10
-5, 10
-610
-9gradient dilution liquid, so often increases progressively dilution once, namely uses 1 1mL sterilizing suction pipe instead.
1.4,2-3 acceptable diluent degree is selected (generally to get 10
-6, 10
-7, 10
-8), with sterilizing 100 μ L micropipettor, respectively get 0.1mL and be inoculated in the beef extract-peptone agar culture plate that prepared, each dilution factor makees 3 flat boards, is evenly applied to whole surface with aseptic push rod.
1.5, plate is inverted in after cultivating 48h ± 2h in 28 DEG C ± 1 DEG C constant incubator, chooses the plate count of clump count between 30-300.
2, S. cervisiae colonial morphology
Morphological feature: milky, circular, protuberance, smooth-shaped, opaque, neat in edge.
3, colony counting
3.1, dilution selection
3.1.1, the dilution factor of average colony between 30-300 should be selected, be multiplied by extension rate and report it.
If 3.1.2 there are two dilution factors.Its growth clump count all between 30-300, if then depending on how deciding it between the two
Than being less than or equal to 2, its average should be reported; If be greater than 2, then report wherein less numeral.
If 3.1.3 all dilution average colony numbers are all greater than 300.Then should be multiplied by extension rate by the average colony number that dilution factor is the highest and report it.
If 3.1.4 all dilution factor average colony numbers are all less than 30, then should be multiplied by extension rate by the minimum average colony number of dilution factor and report it.
3.2, suspicious colony identification
3.2.1 random choose 5 bacterium colonies are identified, comprise colonial morphology, thalli morphology and the microscopic examination of ascospore form, and Physiology and biochemistry qualification.Its method of operating see " Yeast Identification and classification manual " (publishing) for 1991.
3.2.2 qualification result
Colonial morphology: bacterium colony is large and moistening, protuberance, milky, smooth surface is non-wrinkled, edge clear.
Thalli morphology: thalline is in basis of microscopic observation, and cell is oval, ellipse or circular.
Ascospore form: ascospore in basis of microscopic observation, in smooth circle or oval.
Biochemical identification: saccharomyces cerevisiae energy glucose fermentation, maltose, unfermentable lactose, can not assimilate nitrate.
3.2.3 the report of clump count
The plate count of picking colony sum between 30-300, after random picking 5 suspicious colony identification, draw S. cervisiae number in every gram of sample according to following formulae discovery, formula is:
A＝B×C/5×f×10
In formula:
The saccharomyces cerevisiae bacterium colony number of A-mensuration, unit is cfu/g;
The suspicious saccharomyces cerevisiae average colony number that B-same dilution factor three times repeats;
The clump count of saccharomyces cerevisiae is confirmed as in the bacterium colony of C-5 qualification;
F-extension rate;
10-conversion factor.
Miscellaneous bacteria rate (%)=miscellaneous bacteria sum/(saccharomyces cerevisiae viable count+miscellaneous bacteria sum)
The high-yield glutathione saccharomyces cerevisiae microorganism formulation of the present embodiment to be tested discovery, its every gram containing viable count 3,000,000,000 viable bacterias, miscellaneous bacteria rate 0.05%.
Saccharomyces cerevisiae produces glutathione: by the saccharomyces cerevisiae access wheat bran solid medium in high-yield glutathione saccharomyces cerevisiae microorganism formulation, inoculum concentration is weight percentage: 2%, temperature 28 DEG C, cultivates 60 hours.Will be soluble in water through above-mentioned fermentation solid content, 85 DEG C of water-bath 15min, the centrifugal 10 ~ 15min of 5000rpm in centrifuge.Get supernatant DTNB (5-sulfo--2-nitrobenzoic acid) method and measure glutathione content, record glutathione content and reach more than 500mg/kg.
By this high-yield glutathione saccharomyces cerevisiae microorganism formulation through 30-45 DEG C of vacuum drying treatment, be packaged into finished product, detect qualified after, as commodity selling.
Embodiment 2:
Saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 (is preserved in Guangdong Province's Culture Collection, preserving number GIM2.139, this bacterial classification is for sale, can buy) freeze-drying kind on picking one ring thalline be seeded in wort agar culture medium test tube slant culture medium, 28 DEG C cultivate 24 hours; Be transferred in YEPD fluid nutrient medium by test tube slant seed again, cultivate 24h for 28 DEG C, form seed culture fluid, its bacteria concentration is 8.0 × 10
7cfu/mL.The formula of described brewer's wort fine jade culture medium is: often liter contains malt extract powder 130g, agar 20g, chloramphenicol 0.1g, and surplus is water, pH6.0 ~ 6.2,115 DEG C of autoclaving 20min.YEPD Liquid Culture based formulas is: often liter contains dusty yeast 10g, peptone 20g, glucose 20g, distilled water 1000mL, pH nature, 115 DEG C of moist heat sterilization 20min.
Seed culture fluid is proceeded to the inoculum concentration of mass percent 3% in the 30L seeding tank that one grade fermemtation culture medium is housed and carries out fermented and cultured, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 350rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 85 ~ 90%, fermentation time is that the bacterium liquid (entering exponential phase) of 10 hours makes primary seed solution; By primary seed solution with the inoculum concentration of mass percent 10%, transfer second order fermentation culture medium is housed 300L fermentation tank in expand cultivation further, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 150rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 70 ~ 80%, fermentation time is that the bacterium liquid (entering exponential phase) of 22 hours is as secondary seed solution.Described firsts and seconds fermentation medium is, often liter contains glucose 30g, sucrose 10g, molasses 30g, NaCl5g, peptone 10g, dipotassium hydrogen phosphate 1g, and surplus is water, pH6.0 ± 0.2, after above-mentioned component is mixed, 121 DEG C of autoclaving 30min.
Before inoculation, first open the ozone generator and ultraviolet violet light switch of cultivating room, cultivate room area: 25m
2sterilization 30min, preparation gnotobasis, secondary seed solution be inoculated in wheat bran solid medium with the inoculum concentration of 10% (mass percent) in this gnotobasis, mix, the ventilation controlled in wheat bran solid medium is: 10 rates of ventilation/hour, temperature 25 DEG C, humidity is cultivate 72 hours under the condition of 80 ~ 90%, stirs wheat bran solid culture base-material once between culture period every 3 hours, therefrom obtained high-yield glutathione saccharomyces cerevisiae microorganism formulation.Described wheat bran solid medium, by gross mass mark 100%, forms containing wheat bran 38.8%, dregs of beans 10%, urea 0.1%, ammonium chloride 0.1% and water 51%, and raw material mixes, and stirs.Then at rotary digester or meal braizing machine, pressure, temperature is respectively: 1.05 ~ 1.3Kg/cm
3, under 121 ~ 125 DEG C of conditions, sterilizing 30 ~ 40min, is cooled to 40 ~ 50 DEG C of shallow basins of packing, every basin 5kg, for subsequent use.
According to the method for embodiment 1, discovery of being tested by the high-yield glutathione saccharomyces cerevisiae microorganism formulation of the present embodiment, its every gram contains viable count 2,000,000,000 viable bacterias, miscellaneous bacteria rate 0.08%.
Saccharomyces cerevisiae produces glutathione: by the saccharomyces cerevisiae access wheat bran solid medium in high-yield glutathione saccharomyces cerevisiae microorganism formulation, inoculum concentration is weight percentage: 4%, temperature 25 DEG C, cultivates 70 hours.Will be soluble in water through above-mentioned fermentation solid content, 85 DEG C of water-bath 15min, the centrifugal 10 ~ 15min of 5000rpm in centrifuge.Get supernatant DTNB (5-sulfo--2-nitrobenzoic acid) method and measure glutathione content, record glutathione content and reach more than 400mg/kg.
By this high-yield glutathione saccharomyces cerevisiae through 30-45 DEG C of vacuum drying treatment, be packaged into finished product, detect qualified after, as commodity selling.
Embodiment 3:
Saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 (is preserved in Guangdong Province's Culture Collection, preserving number GIM2.139, this bacterial classification is for sale, can buy) freeze-drying kind on picking one ring thalline be seeded in wort agar test tube slant culture medium, 28 DEG C cultivate 24 hours; Be transferred in YEPD fluid nutrient medium by test tube slant seed again, cultivate 24h for 28 DEG C, form seed culture fluid, its bacteria concentration is 1.0 × 10
7cfu/mL.The formula of described brewer's wort fine jade culture medium is: often liter contains malt extract powder 130g, agar 20g, chloramphenicol 0.1g, and surplus is water, pH6.0 ~ 6.2,115 DEG C of autoclaving 20min.YEPD Liquid Culture based formulas is: often liter contains dusty yeast 10g, peptone 20g, glucose 20g, distilled water 1000mL, pH nature, 115 DEG C of moist heat sterilization 20min.
Seed culture fluid is proceeded to the inoculum concentration of mass percent 4% in the 30L seeding tank that one grade fermemtation culture medium is housed and carries out fermented and cultured, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 350rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 80 ~ 90%, fermentation time is that the bacterium liquid (entering exponential phase) of 12 hours makes primary seed solution; By primary seed solution with the inoculum concentration of mass percent 2%, transfer second order fermentation culture medium is housed 300L fermentation tank in expand cultivation further, liquid amount 70%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 150rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 70 ~ 80%, fermentation time is that the bacterium liquid (entering exponential phase) of 14 hours is as secondary seed solution.Described firsts and seconds fermentation medium is, often liter contains glucose 5g, sucrose 2g, molasses 2g, NaCl10g, peptone 20g, dipotassium hydrogen phosphate 15g, and surplus is water, pH6.0 ± 0.2, after above-mentioned component is mixed, 121 DEG C of autoclaving 30min.
Before inoculation, first open the ozone generator and ultraviolet violet light switch of cultivating room, cultivate room area: 25m
2sterilization 30min, preparation gnotobasis, secondary seed solution be inoculated in wheat bran solid medium with the inoculum concentration of mass percent 2% in this gnotobasis, mix, the ventilation controlled in wheat bran solid medium is: 3 rates of ventilation/hour, temperature 32 DEG C, humidity is cultivate 65 hours under the condition of 80 ~ 90%, stirs wheat bran solid culture base-material once between culture period every 5 hours, therefrom obtained high-yield glutathione saccharomyces cerevisiae microorganism formulation.Described wheat bran solid medium, by gross mass mark 100%, forms containing wheat bran 50%, dregs of beans 10.4%, urea 0.3%, ammonium chloride 0.3% and water 39%, and raw material mixes, and stirs.Then at rotary digester or meal braizing machine, pressure, temperature is respectively: 1.05 ~ 1.3Kg/cm
3, under 121 ~ 125 DEG C of conditions, sterilizing 30 ~ 40min, is cooled to 40 ~ 50 DEG C of shallow basins of packing, every basin 5kg, for subsequent use.
According to the method for embodiment 1, discovery of being tested by the high-yield glutathione saccharomyces cerevisiae microorganism formulation of the present embodiment, its every gram contains viable count 2,500,000,000 viable bacterias, miscellaneous bacteria rate 0.02%.
Saccharomyces cerevisiae produces glutathione: by the saccharomyces cerevisiae access wheat bran solid medium in high-yield glutathione saccharomyces cerevisiae microorganism formulation, inoculum concentration is weight percentage: 8%, temperature 32 DEG C, cultivates 65 hours.Will be soluble in water through above-mentioned fermentation solid content, 85 DEG C of water-bath 15min, the centrifugal 10 ~ 15min of 5000rpm in centrifuge.Get supernatant DTNB (5-sulfo--2-nitrobenzoic acid) method and measure glutathione content, record glutathione content and reach more than 400mg/kg.
By this high-yield glutathione saccharomyces cerevisiae through 30-45 DEG C of vacuum drying treatment, be packaged into finished product, detect qualified after, as commodity selling.
Claims (4)
1. a preparation method for high-yield glutathione saccharomyces cerevisiae microorganism formulation, is characterized in that, comprises the following steps:
A, actication of culture: be transferred in YEPD fluid nutrient medium after being activated by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139,28 ± 1 DEG C are cultured to bacteria concentration is 1.0 × 10
7cfu/mL ~ 2.0 × 10
8cfu/mL, forms seed culture fluid;
B, seed culture fluid is transferred in the fermentation tank containing one grade fermemtation culture medium with the inoculum concentration of mass percent 3% ~ 5%, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 200 ~ 350rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 80 ~ 90%, fermentation to saccharomyces cerevisiae grows into exponential phase, using the bacterium liquid of this exponential phase as primary seed solution;
C, primary seed solution is transferred to containing second order fermentation culture medium fermentation tank with the inoculum concentration of mass percent 2 ~ 10% in carry out second order fermentation, fermentation temperature 28 ± 1 DEG C, stir speed (S.S.) 150 ~ 250rpm, throughput 0.8 ~ 0.9V/Vmin, oxyty 70 ~ 80%, fermentation to saccharomyces cerevisiae grows into exponential phase, using the bacterium liquid of this exponential phase as secondary seed solution;
D, in gnotobasis, by secondary seed solution with in the inoculum concentration of mass percent 2 ~ 10% access wheat bran solid medium, mix, control ventilation is: 3 ~ 10 rates of ventilation/hour, temperature 25 ~ 32 DEG C, cultivates 60 ~ 72 hours under humidity 80% ~ 90% condition, stirs wheat bran solid medium between culture period, after 30-45 DEG C of dry process, obtain high-yield glutathione saccharomyces cerevisiae microorganism formulation;
Described firsts and seconds fermentation medium is all: often liter contains glucose 5 ~ 30g, sucrose 2 ~ 20g, molasses 2 ~ 30g, NaCl 3 ~ 10g, peptone 5 ~ 20g, dipotassium hydrogen phosphate 1-15g, and surplus is water, pH6.0 ± 0.2;
Described wheat bran solid medium is: by gross mass mark 100%, is made up of wheat bran 38% ~ 50%, dregs of beans 10% ~ 23%, urea 0.1% ~ 0.5%, ammonium chloride 0.1% ~ 0.5% and water 38% ~ 51%.
2. preparation method according to claim 1, it is characterized in that, described being activated by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 is by saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.139 streak inoculation to wort agar culture medium test tube slant, cultivates 24 hours for 28 DEG C.
3. preparation method according to claim 1, is characterized in that, stirs wheat bran solid medium for stir once every 3 ~ 5 hours between the culture period of described steps d.
4. the high-yield glutathione saccharomyces cerevisiae microorganism formulation prepared according to the preparation method described in claim 1,2 or 3.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106578528A (en) * | 2016-12-27 | 2017-04-26 | 北京三元禾丰牧业有限公司 | Antibiotic-free fermented compound feed capable of improving pork flavor and preparation method thereof |
| CN109609313A (en) * | 2019-02-14 | 2019-04-12 | 十里香股份公司 | A method of improving ethyl hexanoate content in Luzhou-flavor liquo fermentation fermented grain |
| CN114350731A (en) * | 2021-12-15 | 2022-04-15 | 华南理工大学 | Preparation method of glutathione |
-
2014
- 2014-09-30 CN CN201410519741.0A patent/CN104286415A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106578528A (en) * | 2016-12-27 | 2017-04-26 | 北京三元禾丰牧业有限公司 | Antibiotic-free fermented compound feed capable of improving pork flavor and preparation method thereof |
| CN109609313A (en) * | 2019-02-14 | 2019-04-12 | 十里香股份公司 | A method of improving ethyl hexanoate content in Luzhou-flavor liquo fermentation fermented grain |
| CN114350731A (en) * | 2021-12-15 | 2022-04-15 | 华南理工大学 | Preparation method of glutathione |
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