CN102251008A - Method for improving yield of lycopene produced by utilizing Blakeslea trispora - Google Patents
Method for improving yield of lycopene produced by utilizing Blakeslea trispora Download PDFInfo
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Abstract
The invention provides a method for improving yield of lycopene produced by utilizing Blakeslea trispora, and the method is characterized by comprising the following steps: (1) carrying out plate culture on Blakeslea trispora (+)(-) strain spore suspensions and dissolving one of prenol, 3-methyl-3-butene-1-ol, geraniol and mevalonolactone in ethanol to obtain a solution; (2) preparing a seed culture medium; (3) separately adding plate-cultured Blakeslea trispora (+)(-) strains to a triangular flask containing the seed culture medium; and (4) preparing a fermentation medium, mixing 1 bottle of cultured (+) strain seed liquid and 4 bottles of cultured (-) strain seed liquid, adding the fermentation culture medium to the seed liquid for starting fermentation, then adding the solution of one of prenol, 3-methyl-3-butene-1-ol, geraniol and mevalonolactone 0-36 hours after the start of the fermentation and then carrying out constant-temperature culture. The method provided by the invention is simple and easy to operate and control, substantially improves the yield of lycopene and lowers production cost.
Description
Technical field
The present invention relates to a kind of raising and produce the method for yield of lycopene by trispore Bruce mould, belong to by adding a series of precursor substances and improve the research field of microbial liquid fermentative production secondary metabolite, specially refer to and a kind ofly improve the method for the biosynthesizing amount of Lyeopene by adding prenol, Geraniol, 3-methyl-3-butene-1-pure and mild mevalonolactone.
Background technology
Lyeopene is a kind of fat-soluble unsaturated hydrocarbon, belongs to the isoprene compounds, has 11 conjugated double bonds and 2 unconjugated double bonds, and molecular formula is C40H56, and molecular weight is 536.85Da, and molecular structure is as follows:
Lyeopene is a kind of important fat-soluble carotenoid, having very strong anti-oxidation, remove free radical, anticancerly press down cancer, enhancing body immunity, delay senility and important physical function such as preventing cardiovascular disease, is a kind of rising new type functional natural pigment.For production methods of lycopene, except natural extract or chemosynthesis, can also adopt algae and fungi and yeast fermentation to produce carotenoid, it is the most important approach that obtains Biological resources type carotenoid, not limited by envrionment conditions, have advantages such as security, low cost and strong tinting strength and be subjected to especially favor at home and abroad.From analyses such as quality, technology, production, resources costss, utilizing microbial technique to produce natural carotenoid such as Lyeopene is the main direction of development.At present, utilize the mould fermentative production Lyeopene of three spore cloth Laplaces to reach half industrialization degree.Can not high-caliber accumulation Lyeopene but the principal element that restricts fermentation method suitability for industrialized production Lyeopene at present is a microorganism, cause that fermentation production rate is low, production cost is higher.Lyeopene route of synthesis complexity makes up Lyeopene genetically engineered high yield bacterium and does not make a breakthrough as yet so far.Therefore, add precursor substance and fermentation accelerant, and fermentation condition is optimized, be still the main means that improve yield of lycopene.
In the process with the mould production Lyeopene of three spore cloth Laplaces, the analog (prenol, Geraniol, 3-methyl-3-butene-1-pure and mild mevalonolactone) that we find to add a series of mesostate can obviously improve the output of biomass and the Lyeopene of microorganism.
Lyeopene can be synthetic by the isoprenoid pathways metabolism by the mould fermentation of microorganism three spore cloth Laplaces, synthetic precursor is derived by acetyl-CoA, acetyl-CoA generates mevalonic acid through series reaction, further in plastid, generate isopentenylpyrophosphate (IPP), isopentenylpyrophosphate generates dimethyl allene tetra-sodium (DMPP) through isomerization, two molecule dimethyl allene tetra-sodium condensations generate geranylpyrophosphate (GPP), generate yak base geranylpyrophosphate (GGPP) through farnesyl tetra-sodium (FPP) then, through phytoene, sigma carotene generates Lyeopene.Lyeopene can be transformed into carotenoid such as β-Hu Luobusu, gamma carotene, alpha-carotene, β-Hu Luobusu, xenthophylls, zeaxanthin, zeaxanthin diepoxide, neoxanthin under the effect of cyclase.During the fermentation, add blocker between in due course, make Lyeopene only further be converted into other carotenoid, Lyeopene is accumulated, after fermentation, carry out cytoclasis and just can obtain product.
Existing studies show that, elementary metabolic intermediate citric acid, equisetic acid, oxysuccinic acid and succsinic acid all have certain promoter action to synthesizing of Lyeopene.By fermenting relevant theory as can be known, suitably add mesostate during the fermentation, as GPP, IPP, DMAPP, GGPP, mevalonic acid (water-solublely easily lactonizes, so the mevalonolactone of buying), can reach the direction that stimulates metabolism to flow to the Lyeopene accumulation and flow, promote the raising of Lyeopene.In experimenting, we find out that, and prenol is the analog of IPP, 3-methyl-3-butene-1-alcohol is the structure group food of DMAPP, Geraniol is the analog of GPP and GGPP, adds these analogs and can play the purpose that improves yield of lycopene equally.GPP, IPP, these intermediate metabolites instabilities of DMAPP, GGPP, and be difficult on the market buy, cannot directly be used for adding, just do not reach the purpose that improves yield of lycopene yet.And prenol, 3-methyl-3-butene-1-alcohol, Geraniol stable in properties can conveniently buy, and cost is very low, can be applied in the production of suitability for industrialized production Lyeopene.
Summary of the invention
The object of the present invention is to provide the submerged fermentation of trispore Bruce mould liquid fermenting to produce the output of Lyeopene.Specific implementation method is to be fermented bacterium with Blakeslea trispora (+) (-), behind the seed single culture 40-44h, positive and negative bacterium is mixed in proportion the access fermention medium and carries out fermentation culture, the analog that adds certain density mesostate in the suitable time respectively, add the nicotine blocker then, finish behind the fermentation culture 120h, carry out the technological processs such as extraction and purification of Lyeopene in the mycelium then.The invention provides the method for a kind of raising by the mould production yield of lycopene of three spore cloth Laplaces, it is characterized in that: concrete technological line of the present invention comprises the steps:
The bacterial classification source
The bacterial classification that produces Lyeopene is trispore Bruce mould (Blakeslea trispora), a kind of filamentous fungus, be under the jurisdiction of mucorales (Muco-rales), the mould section of hairpin, the mould Pseudomonas of Bradley (Blakeslea), divide "+" "-" two kinds of bacterial strains, buy in US mode culture collection warehousing (ATCC), bacterium number is 14271 (+) and 14272 (-).
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, and keeps 20 minutes postcooling of boiling, the elimination potato, add 2g glucose in the clear liquid, 2g agar is in the triangular flask of packing into after the dissolving, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling; Get the three spore cloth mould Blakeslea trispora of Laplace (+) (-) bacterial strain spore suspensions with the glass stick separate application in the flat board that contains the PDA substratum; The flat board of coating is placed 24h under the room temperature behind 26-28 in the incubator ℃ of cultivation 36-40h;
(2) The pretreatment
One of prenol, 3-methyl-3-butene-1-alcohol, Geraniol, mevalonolactone are through the dissolve with ethanol wiring solution-forming;
(3) seed culture
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, pH is adjusted to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with gauze, and 30min sterilizes in 115 ℃ of Autoclaves;
(4) connect bacterium
On the aseptic technique platform, with the cultured three spore cloth mould Blakeslea trispora of Laplace (+) (-) bacterium of flat board with the positive bacterium of each picking of inoculating needle one ring, negative bacterium, join respectively in the triangular flask that the 100mL seed culture medium is housed, positive bacterium connects one bottle, negative bacterium connects four bottles, seals with gauze, is put in the constant temperature shaking table, at 26-28 ℃, cultivate 36-40h under the condition of 180-200r/min respectively and obtain 1 bottle of positive bacterium, 4 bottles of negative bacterium seed liquor;
(4) fermentation culture
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, pH is adjusted to 6.5 with the sodium hydroxide solution of 1mol/L, install in the triangular flask, add the fermention medium of 80mL, seal with gauze, 30min sterilizes in 121 ℃ of Autoclaves;
(6) inoculation and fermentation
In the aseptic processing room, with 1 bottle of positive bacterium of above-mentioned cultivation, 4 bottles of negative bacterium seed liquor, all thorough mixing obtains fermentation seed liquid, and the fermentation seed liquid that mixes that the above-mentioned fermention medium of every 80mL adds 9mL obtains starting fermentation liquid; Add one of the prenol prepare, 3-methyl-3-butene-1-alcohol, Geraniol, mevalonolactone solution again, be put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the volumetric concentration of going into 800uL is 10% nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product;
The interpolation concentration of above-mentioned prenol is that 10-60mg/L starting fermentation liquid is long-pending, the interpolation concentration of Geraniol is that 10-60mg/L starting fermentation liquid is long-pending, the interpolation concentration of 3-methyl-3-butene-1-alcohol is that 10-170mg/L starting fermentation liquid is long-pending, the interpolation concentration of mevalonolactone is that 10-30mg/L starting fermentation liquid is long-pending, and the interpolation time added in fermentation beginning back in 0-36 hour.
(7) assay determination
Pulverize dry good mycelium, accurately take by weighing the dry bacterial powder of 0.05g, use petroleum ether extraction, with the content of high effective liquid chromatography for measuring Lyeopene.
Further, the interpolation time of prenol, Geraniol, 3-methyl-3-butene-1-pure and mild mevalonolactone begins to add in fermentation.
Method of the present invention is simple, is easy to operate and control, and increases substantially the productive rate of Lyeopene, has reduced production cost.
Embodiment
Embodiment 1:
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, and keeps 20 minutes postcooling of boiling, the elimination potato, add 2g glucose in the clear liquid, 2g agar is in the 500mL triangular flask of packing into after the dissolving, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling.Get three spore cloth Laplace mould (+) (-) bacterial strain spore suspensions, with the glass stick separate application in the flat board that contains the PDA substratum.The flat board of coating in the incubator 28 ℃ cultivate 40h after, place 24h under the room temperature.
(2) pre-treatment of prenol
Prenol is through dilution process, and 400uL prenol (purity 97%) is dissolved in 75% ethanol of 10ml, is made into the solution that concentration is 32mg/mL.
(3) preparation of seed culture medium
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with 8 layers of gauze, and 30min sterilizes in 115 ℃ of Autoclaves.
(4) connect bacterium
On the aseptic technique platform,, join respectively in the 500mL triangular flask that the 100mL seed culture medium is housed with the positive bacterium of each picking one ring of inoculating needle, negative bacterium, positive bacterium connects one bottle, and negative bacterium connects four bottles, seals with eight layers of gauze, be put in 28 ℃, in the 200r/min constant temperature shaking table, difference single culture 40h.
(5) preparation of fermention medium
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH6.5 with the sodium hydroxide solution of 1mol/L, divide to install in the triangular flask of 6 bottles of 500mL, every bottle of fermention medium that adds as 80mL seals with 8 layers of gauze, and 30min sterilizes in 121 ℃ of Autoclaves.
(6) inoculation and fermentation
In the aseptic processing room, with just (1 bottle), negative bacterium (4 bottles) seed liquor of single culture, whole thorough mixing, six bottles of fermention mediums add the fermentation seed liquid that mixes of 9mL respectively.Three bottles of prenol solution that prepared that add 54uL more respectively wherein, six bottles of fermention mediums are put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the concentration that adds 800uL is 10% (v/v) nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product.
(7) assay determination
Pulverize dry good mycelium, accurately take by weighing the dry bacterial powder of 0.05g, use petroleum ether extraction, with the content of high effective liquid chromatography for measuring Lyeopene.
The gained result is as follows:
The prenol addition is that 40mg/L starting fermentation liquid is long-pending, and the output of Lyeopene is 560mg/L, and blank output is 346mg/L, has improved 62%.
Embodiment 2:
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, keep 20 minutes postcooling of boiling, the elimination potato adds 2g glucose, 2g agar in the clear liquid, pack into after the dissolving in the 500mL triangular flask, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling.Get three spore cloth Laplace mould (+) (-) bacterial strain spore suspensions.The flat board of coating in the incubator 28 ℃ cultivate 40h after, place 24h under the room temperature.
(2) pre-treatment of Geraniol
Geraniol is through dissolving, dilution process, and 40uL Geraniol (purity 98%) is dissolved in 75% ethanol of 1ml, is made into the solution that concentration is 32mg/mL.
(3) preparation of seed culture medium
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with 8 layers of gauze, and 30min sterilizes in 115 ℃ of Autoclaves.
(4) connect bacterium
On the aseptic technique platform,, join respectively in the 500mL triangular flask that the 100mL seed culture medium is housed with the positive bacterium of each picking one ring of inoculating needle, negative bacterium, positive bacterium connects one bottle, and negative bacterium connects four bottles, seals with eight layers of gauze, be put in 28 ℃, in the 200r/min constant temperature shaking table, difference single culture 40h.
(5) preparation of fermention medium
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH6.5 with the sodium hydroxide solution of 1mol/L, divide to install in the triangular flask of 6 bottles of 500mL, every bottle of fermention medium that adds 80mL seals with 8 layers of gauze, and 30min sterilizes in 121 ℃ of Autoclaves.
(6) inoculation and fermentation
In the aseptic processing room, with just (1 bottle), negative bacterium (4 bottles) seed liquor of single culture, whole thorough mixing, six bottles of fermention mediums add the fermentation seed liquid that mixes of 9mL respectively.Three bottles of Geraniol solution that prepared that add 112uL more respectively wherein, six bottles of fermention mediums are put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the concentration of going into 800uL is 10% (v/v) nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product.
(7) assay determination
Pulverize dry good mycelium, accurately take by weighing the dry bacterial powder of 0.05g, use petroleum ether extraction, with the content of high effective liquid chromatography for measuring Lyeopene.
The gained result is as follows:
The Geraniol addition is 42mg/L starting fermentation liquid when long-pending, and the output of Lyeopene is 549mg/L, and blank output is 297mg/L, has improved 85%.
Embodiment 3:
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, and keeps 20 minutes postcooling of boiling, the elimination potato, add 2g glucose in the clear liquid, 2g agar is in the 500mL triangular flask of packing into after the dissolving, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling.Get three spore cloth Laplace mould (+) (-) bacterial strain spore suspensions with the glass stick separate application in the flat board that contains the PDA substratum.The flat board of coating in the incubator 28 ℃ cultivate 40h after, place 24h under the room temperature.
(2) pre-treatment of 3-methyl-3-butene-1-alcohol
3-methyl-3-butene-1-alcohol is through dilution process, and 40uL prenol (purity 97%) is dissolved in 75% ethanol of 1ml, is made into the solution that concentration is 30mg/mL.
(3) preparation of seed culture medium
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with 8 layers of gauze, and 30min sterilizes in 115 ℃ of Autoclaves.
(4) connect bacterium
On the aseptic technique platform,, join respectively in the 500mL triangular flask that the 100mL seed culture medium is housed with the positive bacterium of each picking one ring of inoculating needle, negative bacterium, positive bacterium connects one bottle, and negative bacterium connects four bottles, seals with eight layers of gauze, be put in 28 ℃, in the 200r/min constant temperature shaking table, difference single culture 40h.
(5) preparation of fermention medium
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH6.5 with the sodium hydroxide solution of 1mol/L, divide to install in the triangular flask of 6 bottles of 500mL, every bottle of fermention medium that adds 80mL seals with 8 layers of gauze, and 30min sterilizes in 121 ℃ of Autoclaves.
(6) inoculation and fermentation
In the aseptic processing room, with just (1 bottle), negative bacterium (4 bottles) seed liquor of single culture, whole thorough mixing, six bottles of fermention mediums add the fermentation seed liquid that mixes of 9mL respectively.The three bottles of 3-that has prepared methyl-3-butene-1-alcoholic solutions that add 400uL more respectively wherein, six bottles of fermention mediums are put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the concentration of going into 800uL is 10% (v/v) nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product.
(7) assay determination
Pulverize dry good mycelium, accurately take by weighing the dry bacterial powder of 0.05g, use petroleum ether extraction, with the content of high effective liquid chromatography for measuring Lyeopene.
The gained result is as follows:
3-methyl-3-butene-1-pure addition is 150mg/L starting fermentation liquid when long-pending, and the output of Lyeopene is 460mg/L, and blank output is 311mg/L, has improved 47%.
Embodiment 4:
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, and keeps 20 minutes postcooling of boiling, the elimination potato, add 2g glucose in the clear liquid, 2g agar is in the 500mL triangular flask of packing into after the dissolving, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling.Get three spore cloth Laplace mould (+) (-) bacterial strain spore suspensions with the glass stick separate application in the flat board that contains the PDA substratum.The flat board of coating in the incubator 28 ℃ cultivate 40h after, place 24h under the room temperature.
(2) pre-treatment of mevalonolactone
Geraniol is through dilution process, and 80uL prenol (purity 97%) is dissolved in 75% ethanol of 4ml, is made into the solution that concentration is 20mg/mL.
(3) preparation of seed culture medium
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with 8 layers of gauze, and 30min sterilizes in 115 ℃ of Autoclaves.
(4) connect bacterium
On the aseptic technique platform,, join respectively in the 500mL triangular flask that the 100mL seed culture medium is housed with the positive bacterium of each picking one ring of inoculating needle, negative bacterium, positive bacterium connects one bottle, and negative bacterium connects four bottles, seals with eight layers of gauze, be put in 28 ℃, in the 200r/min constant temperature shaking table, difference single culture 40h.
(5) preparation of fermention medium
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, fermented liquid is adjusted to pH6.5 with the sodium hydroxide solution of 1mol/L, divide to install in the triangular flask of 6 bottles of 500mL, every bottle of fermention medium that adds 80mL seals with 8 layers of gauze, and 30min sterilizes in 121 ℃ of Autoclaves.
(6) inoculation and fermentation
In the aseptic processing room, with just (1 bottle), negative bacterium (4 bottles) seed liquor of single culture, whole thorough mixing, six bottles of fermention mediums add the fermentation seed liquid that mixes of 9mL respectively.Three bottles of mevalonolactone solution that prepared that add 70uL more respectively wherein, six bottles of fermention mediums are put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the concentration of going into 800uL is 10% (v/v) nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product.
(7) assay determination
Pulverize dry good mycelium, accurately take by weighing the dry bacterial powder of 0.05g, use petroleum ether extraction, with the content of high effective liquid chromatography for measuring Lyeopene.
The gained result is as follows:
The mevalonolactone addition is 17.5mg/L starting fermentation liquid when long-pending, and the output of Lyeopene is 363mg/L, and blank output is 250mg/L, has improved 44%.
Claims (2)
1. the raising method of producing yield of lycopene by trispore Bruce mould is characterized in that step is as follows:
(1) the dull and stereotyped cultivation
Remove skin potato 20g, the deionized water that adds 200mL is boiled, and keeps 20 minutes postcooling of boiling, the elimination potato, add 2g glucose in the clear liquid, 2g agar is in the triangular flask of packing into after the dissolving, 115 ℃ of sterilization 30min pour plate while hot into, can make solid PDA substratum after cooling; Get the three spore cloth mould Blakeslea trispora of Laplace (+) (-) bacterial strain spore suspensions with the glass stick separate application in the flat board that contains the PDA substratum; The flat board of coating is placed 24h under the room temperature behind 26-28 in the incubator ℃ of cultivation 36-40h;
(2) The pretreatment
One of prenol, 3-methyl-3-butene-1-alcohol, Geraniol, mevalonolactone are through the dissolve with ethanol wiring solution-forming;
(3) seed culture
20g starch, 25g corn steep liquor, 12.5g glucose are added in the beaker of 1L, to 500mL, be put on the water-bath with the tap water constant volume, gelatinization 40min under 100 ℃ of conditions, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, pH is adjusted to 6.5 with the sodium hydroxide solution of 3mol/L, divide then to install in the triangular flask of 5 500mL, every bottle of seed culture medium that adds 100mL seals with gauze, and 30min sterilizes in 115 ℃ of Autoclaves;
(4) connect bacterium
On the aseptic technique platform, with the cultured three spore cloth mould Blakeslea trispora of Laplace (+) (-) bacterium of flat board with the positive bacterium of each picking of inoculating needle one ring, negative bacterium, join respectively in the triangular flask that the 100mL seed culture medium is housed, positive bacterium connects one bottle, negative bacterium connects four bottles, seals with gauze, is put in the constant temperature shaking table, at 26-28 ℃, cultivate 36-40h under the condition of 180-200r/min respectively and obtain 1 bottle of positive bacterium, 4 bottles of negative bacterium seed liquor;
(4) fermentation culture
Take by weighing 20g starch, 22.5g corn steep liquor, join in the 1L flask, add the deionized water constant volume to 500mL, stir, be put in 100 ℃ the water-bath gelatinization 40min then, the cooling back adds the potassium primary phosphate of 0.5g, the magnesium sulfate heptahydrate of 0.05g, the vitamins B of 0.005g
1, after stirring, pH is adjusted to 6.5 with the sodium hydroxide solution of 1mol/L, install in the triangular flask, add the fermention medium of 80mL, seal with gauze, 30min sterilizes in 121 ℃ of Autoclaves;
(6) inoculation and fermentation
In the aseptic processing room, with 1 bottle of positive bacterium of above-mentioned cultivation, 4 bottles of negative bacterium seed liquor, all thorough mixing obtains fermentation seed liquid, and the fermentation seed liquid that mixes that the above-mentioned fermention medium of every 80mL adds 9mL obtains starting fermentation liquid; Add one of the prenol prepare, 3-methyl-3-butene-1-alcohol, Geraniol, mevalonolactone solution again, be put in the constant incubator then, the constant incubator rotating speed is 220r/min, and temperature is controlled at 28 ℃, during fermentation 48h, the volumetric concentration of going into 800uL is 10% nicotine, behind the fermentation culture 120h following jar, collect thalline, at 45 ℃, 0.08Mpa vacuum drying oven in dry, take out behind the 24h and get final product;
The interpolation concentration of above-mentioned prenol is that 10-60mg/L starting fermentation liquid is long-pending, the interpolation concentration of Geraniol is that 10-60mg/L starting fermentation liquid is long-pending, the interpolation concentration of 3-methyl-3-butene-1-alcohol is that 10-170mg/L starting fermentation liquid is long-pending, the interpolation concentration of mevalonolactone is that 10-30mg/L starting fermentation liquid is long-pending, and the interpolation time added in fermentation beginning back in 0-36 hour.
2. according to the method for claim 1, it is characterized in that: wherein the interpolation time of prenol, Geraniol, 3-methyl-3-butene-1-pure and mild mevalonolactone begins to add in fermentation.
Priority Applications (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757995A (en) * | 2012-06-25 | 2012-10-31 | 湖北工业大学 | Method for preparing beta-carotene through fermentation of Blakeslea trispora |
| CN103276019A (en) * | 2013-05-28 | 2013-09-04 | 北京化工大学 | Method for promoting lycopene synthesis in blakeslea trispora |
| CN103276018A (en) * | 2013-05-28 | 2013-09-04 | 北京化工大学 | Method for producing lycopene by improving fermentation of Blakeslea trispora |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1353765A (en) * | 1999-06-09 | 2002-06-12 | 维塔特内有限公司 | Lycopen production method |
| JP2003304895A (en) * | 2002-04-12 | 2003-10-28 | Kanegafuchi Chem Ind Co Ltd | Method for fermentation production of lycopene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1353765A (en) * | 1999-06-09 | 2002-06-12 | 维塔特内有限公司 | Lycopen production method |
| JP2003304895A (en) * | 2002-04-12 | 2003-10-28 | Kanegafuchi Chem Ind Co Ltd | Method for fermentation production of lycopene |
Non-Patent Citations (1)
| Title |
|---|
| SHEETAL M. CHOUDHARI等: "Use of metabolic stimulators and inhibitors for enhanced production of b-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896", 《BIORESOURCE TECHNOLOGY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757995A (en) * | 2012-06-25 | 2012-10-31 | 湖北工业大学 | Method for preparing beta-carotene through fermentation of Blakeslea trispora |
| CN103276019A (en) * | 2013-05-28 | 2013-09-04 | 北京化工大学 | Method for promoting lycopene synthesis in blakeslea trispora |
| CN103276018A (en) * | 2013-05-28 | 2013-09-04 | 北京化工大学 | Method for producing lycopene by improving fermentation of Blakeslea trispora |
| CN103276018B (en) * | 2013-05-28 | 2015-03-04 | 北京化工大学 | Method for producing lycopene by improving fermentation of Blakeslea trispora |
| CN103276019B (en) * | 2013-05-28 | 2015-07-01 | 北京化工大学 | Method for promoting lycopene synthesis in blakeslea trispora |
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| CN102251008B (en) | 2013-05-29 |
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