CN103484520B - A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts - Google Patents
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts Download PDFInfo
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Abstract
The present invention relates to a kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, comprise inclined-plane cultivation, seed culture and fermentation procedure, in fermentation procedure, add any one or two or more combinations in salicylic acid and its esters and nitrous acid and its esters, thereby Blakeslea trispora producing beta-carotene by fermentation has been had to good facilitation. Compared with prior art, it is few that the present invention has chemical reagent use amount, combination by number of chemical reagent is used, can reach good facilitation to beta carotene fermentation level, beta carotene fermentation level improves 55.0 ± 31.6～190.5 ± 27.4% than original process fermentation level, more than can reach 2.25 grams per liters.
Description
Technical field
The present invention relates to carotenoid produces and studying technological domain, specifically, that one utilizes the positive and negative bacterial strain of Blakeslea trispora to mix the fermentation of pairing liquid submersion, and add during the fermentation the combination of a kind of fermentation accelerant or two or more fermentation accelerants, thereby promote the method for the beta carotene level of production.
Background technology
Carrotene (Carotene) is isolated in 1831 by Wackenroder the earliest from carrot, and the molecular formula of beta carotene is measured in 1906 by Willstather, and that structural formula in nineteen twenty-eight-1932 year be Zechmeister etc. is definite. Carrotene is a kind of orange photosynthetic pigments, is extensively present in green and yellow vegetable and fruit, makes it with orange, and in photosynthesis, plays the part of the role of transferring energy. Carrotene belongs to tetraterpenes compound. Respectively there is a β-purple trailing plants ketone ring at the two ends of its molecule, center burst can produce two vitamin As, be the dimer of retinol (vitamin A) and have various structures, topmost two kinds is alpha-carotene and beta carotene, also has in addition three kinds of gamma carotene, δ-carrotene and ε-carrotene.
When the international conference of the effect of the Second Committee antioxidant vitamin of holding Berlin in 1994 in prevent disease, Blumberg proposition beta carotene has the function of following several respects at least: (1) beta carotene is the precursor of vitamin A, and vitamin A is the micro-nutrient composition of needed by human; (2) there is immunologic function, the ability that can improve human immune system and resist pathogen; (3) there is the function of antioxidant, can singlet-oxygen quenching, the impact of removing interior free yl; (4) the pre-anti-cancer of energy, delays the development of cancer; (5) promote to connect and exchange between cell seam. At present; beta carotene is also confirmed more and more in anti-inflammatory, removing toxic substances, anticancer, angiocardiopathy preventing, the physiological action of preventing and treating aspect cataract and protection liver; and be applied to prevention and treatment of diseases (AmJClinNutr, 1995,62:1521S-1526S).
Beta carotene is divided into chemical synthesis beta carotene and the large class of natural beta-carotin two according to mode of production difference. Extensively be present in living nature at interior carotenoid comprising beta carotene, comprise plant, animal, algae and microorganism etc., they are abundant sources of natural carotenoid. Although natural beta-carotin production cost is higher, but owing to there not being carcinogen, water-soluble better, there is good physiologically active, and be usually combined with each other with anti-oxidant natural activity products such as other carotenoid, therefore there is the pharmacologically actives such as good anti-asthma, anti-chromosome aberration and anti-cancer. Natural beta-carotin is approved by IARC and National Cancer Institute at present, and is approved as category-A nutraceutical additive through FAO (Food and Agriculture Organization of the United Nation) (FAO) and the World Health Organization (WHO). Along with people's improving constantly natural nutrition and health product understanding, and the pay attention to day by day of food-safety problem (exposure of the artificial color such as illegal use clenbuterol hydrochloride, sodium formaldehyde sulfoxylate, tonyred, melamine, lemon yellow and phthalic acid ester and food additives phenomenon especially in recent years), the progressively dominate in market of food and feed additive (for example, natural colouring matter beta carotene etc.) of natural colouring matter and safety.
Microbe fermentation method has to produce to be stablized, and affected by season, weather, time and region factor little, and the production cycle is shorter, and quality control is relatively easy, and the advantages such as product free from extraneous odour that obtain. In addition, by the amplification of fermentation scale, also comparatively easily meet the market demand growing to natural carotenoid at present. In view of above feature, the product beta carotene microorganism that researcher obtains natural separation has carried out comparison and the research of system, and by mutagenesis screening and fermentation research, has found that some utmost points have the industrial microorganism bacterial strain of using value. Wherein, Blakeslea trispora is owing to producing and the beta carotene application safety that obtains, and the advantage such as biomass is large, beta carotene output height, therefore becomes the key industry application bacterial strain that biological fermentation process is prepared natural beta-carotin.
For strengthening the process of Blakeslea trispora preparing natural beta-carotin by fermentation, research worker is except obtaining superior strain by mutagenic and breeding, screening and optimizing fermentation medium carbon nitrogen source and derivant, improving fermentation stirs, outside oxygen supply and pH and temperature regulate, promoting the factor to improve the fermenting property of Blakeslea trispora by add a small amount of fermentation at sweat, is also one of important way of strengthening beta carotene fermenting and producing.
Find by literature search, research worker all can promote the biosynthesis of beta carotene and final fermentation level in various degree by utilize in the middle of Blakeslea trispora and the inhibitor of cometabolism and analogue, metal ion, surfactant, alkane, each quasi-grease, oxidant and antioxidant, the oxygen stress-inducing factor and branched metabolic pathway etc. both at home and abroad. For example: K.Nanou and T.Roukas (ApplBiochemBiotechnol, 2010,160:2415-2423) once utilized BHT induction Blakeslea trispora to produce oxygen stress response, and then caused that carrotene was in intramycelial a large amount of synthetic and accumulation. In the time that BHT working concentration is 20mM, in Blakeslea trispora mycetocyte, carrotene accumulation reaches peak (content is 125mg/g stem cell), improves 5 times than control group (content is 25mg/g stem cell).
Nanou, K and Roukas, T by adding 20mM butylated hydroxytoluene (Butylatedhydroxytoluene in Blakeslea trispora bacteria fermentation culture medium, BHT), thereby make the content beta-carotene in somatic cells improve 5 times, reach 125.0mg/g stem cell heavy (ApplBiochemBiotechnol, 2010,160:2415-2423).
The people such as Zhang Tingting, by add the mesostate (natrium citricum and sodium succinate) of tricarboxylic acid cycle circulation in fermentation medium, thereby make Blakeslea trispora produce beta carotene level and have raising (food science and technology in various degree, 2009, 4:2-7) Tang, the people such as Q are by using 30mM ergosterol synthetic inhibitor ketoconazole (Ketoconazole, KCZ), thereby hmgR and the isogenic transcriptional level of carRA on Blakeslea trispora beta carotene route of synthesis are raised, and further promote the synthetic of beta carotene. when fermentation 24h, the content of beta carotene in cell (0.41 ± 0.05mg/g stem cell is heavy) improves approximately 2.7 times (CurrMicrobiol, 2008,57:527-531) than the control group that does not add KCZ.
The research of the robot system such as SheetalM.Choudhari and RekhaS.Singhal comprise surfactant, tricarboxylic acid cycle mesostate and vitamin A and antibiotic produce the impact (BioresourTechnol of beta carotene at interior all kinds of promotion factor pair Blakeslea trisporas, 2008,99:3166-3173). By adding 0.2% Span20, thereby make beta carotene output be increased to 0.318g/L from 0.139g/L. Subsequently, by add the retinyl acetate of 1000ppm in culture medium, thereby make carrotene fermentation level further be increased to 830 ± 6mg/L.
Bang beautiful and remaining dawn of refined experimental study the impacts (food and biotechnology journal, 2007,26:97-101) of 3 kinds of nonionic surface active agent such as polyoxyethylene nonylphenol ether, Span20, Tween80 on the synthetic beta carotene of Blakeslea trispora. By adding 0.075% OP, 0.05% Span20 and 1% Tween80, beta carotene output increases production respectively 15%, 10% and 50%. Wherein add 0.025% antioxidant ethoxyquin (Ethoxyquin), can make the output of beta carotene improve 70%.
Xu Zhi waits by force people to study Mg2+On the impact (Wuxi Light Industry Univ.'s journal, 2003,22:105-107) of the synthetic beta carotene of Blakeslea trispora, found that in the time of low concentration Mg2+The output of thalli growth and beta carotene is had to certain facilitation, in addition, add separately and add Mg with alpha, beta-lonone simultaneously2+, its facilitation is mainly all at 72～96h. Fermentation 48h adds 0.3g/LMg2+, beta carotene output increases by 40%. Add Mg simultaneously2+And when β-ionone, beta carotene output improves approximately 11% than the beta carotene output of only adding merely β-ionone control group.
Jeong, the people such as JC confirm (Biotechnologyletters, 1999,21:683-686), by add the H of 10 μ M in the fermentation Blakeslea trispora bacteria culture fluid of 1.5 days2O2, can effectively stimulate the synthetic of beta carotene and accumulate. Under same culture conditions, H2O2Add experimental group and can improve 46% than the output of control group.
Ciegler, people's reports such as A, while utilizing the positive and negative bacterial strain mixed culture fermentation of Blakeslea trispora, be cultured to the kerosene (or adding the kerosene through acidifying deodorization in initial fermentation) that adds 5% on the 2nd day, can improve beta carotene output 100～150%, carrotene output reaches 0.86g/L (ApplMicrobiol, 1962,10:132-136). In addition, Ciegler, the people such as A also study discovery, except β-purple trailing plants ketone, australene (α-pinene), carveol (carveol), pimenta oil (pimentaoil), isoborneol (isoborneol), terpinol (terpineol), geraniol (geraniol), citrene (d-Limonene), oranges and tangerines (citrus) processed side product (tangerine oil, oranges and tangerines meat slurry and oranges and tangerines molasses) all can promote Blakeslea trispora producing beta-carotene by fermentation in varying degrees. Wherein, the oranges and tangerines meat of use 3% slurry (beta carotene output is 1.080g/L) can effectively substitute β-purple trailing plants ketone (beta carotene output is 1.018g/L) of 0.1%. In the time that the working concentration of oranges and tangerines meat slurry is 5%, beta carotene output reaches 1.29g/L, improves 4.1 times than the control group (0.313g/L) that does not add β-purple trailing plants ketone.
Anderson, R.F. wait people to study discovery, in the time of Blakeslea trispora shake flask fermentation to the second day, be β-ionone of 0.1% by add working concentration in the culture medium that contains vegetable oil and surfactant (TritonX-100), can make beta carotene fermentation level double (JAgricFoodChem, 1958,6:543-545).
In the existing patented technology of China:
Zhejiang Medicine Co is permitted the human hairs such as new moral and is understood " methods and applications that a kind of fermentation method is produced beta carotene " (publication number: CN102787158A; Open day: 2012.11.21). The method is Blakeslea trispora spore suspension to be inoculated in to fermentation cylinder for fermentation prepare mycelium. Wherein, fermentation medium composition comprises: cornstarch, corn steep liquor, rapeseed oil, Hectometer cake powder, glucose vegetable oil, thiamine hydrochloride, magnesium sulfate, emulsifying agent. Emulsifying agent is mainly sapn series of products, tween series of products, lecithin or single double glyceride. Before fermentation, culture medium carries out high speed homogenization emulsification with colloid mill to vegetable oil. By in pH6.5～6.8,27～30 DEG C of fermentation 96～100h, beta carotene fermentation level reaches 9.65～10.78g/L. Process mycelium with alkali treatment and hydrophobicity non-polar organic solvent subsequently, and with the beta carotene in ester class organic solvent extraction mycelium. By add monohydric alcohol and dihydroxylic alcohols in extract, crystallization obtains beta carotene crystal (purity is 96.4～97.1%, and yield reaches 81.2～86.1%).
The human hairs such as the Cai Jun of Hubei University Of Technology understand " a kind of method of utilizing Blakeslea trispora fermentation to prepare beta carotene " (publication number: CN102757995A; Open day: 2012.10.31). The method is utilized Blakeslea trispora ATCC14271 (+) and ATCC14272 (-), through fermentation preparation, obtains the higher mycelium of content beta-carotene. Wherein fermentation medium is wheat bran leachate, maltose, magnesium sulfate, soy meal, potassium dihydrogen phosphate, soybean oil, biotin. By at pH6-7,26-28 DEG C of fermentation 144-168h, carrotene fermentation level is 0.103g/L, and mycelium dry weight is 21.5g/L, and content beta-carotene is 4.8% (w/w).
The human hairs such as the virgin Hypon of Shanghai Chemical Research Inst understand a kind of " recycling the new technology of fermented waste fluid production natural beta-carotin " (publication number: CN1163618C; Open day: 2004.8.25). The method is that after utilizing fermentation, the mycelial waste liquid configuration of filtering culture medium carrys out preparing natural beta-carotin by fermentation, described fermented and cultured and consist of starch 5～8% (w/v), Hectometer cake powder 1.2～2.8%, vegetable oil 5.1～7.0%, potassium dihydrogen phosphate 0.04～0.09% (w/v), non-ionic surface active agent APES 0.05～0.20% (w/v), orotic acid～10ppm, manganese sulfate 0.01～0.06% (w/v). By at 26～28 DEG C of fermented and cultured 120～168h, while recycling waste liquid 8 times, beta carotene output reaches 2.436g/L, and control fermentation level is 2.150g/L.
The human hairs such as the virgin Hypon of Shanghai Chemical Research Inst understand " a kind of method of preparing natural beta-carotin by fermentation " (publication number: CN1331343A; Open day: 2002.1.16). This invention is the positive and negative inoculation of filamentous fungi to be cultivated to filamentous fungi fermentation at stirred fermentor prepare beta carotene, on using, middle level is wide leaf push type, lower floor is the Combined stirring paddle leaf of turbine type, the mass-transfer performance and the gas liquid contacting efficiency that have improved zymotic fluid, be more suitable for mycelial growth. At 26～28 DEG C of fermented and cultured 120～168h, more than fermentation level reaches 2.0g/L. The fermentation medium that wherein used and consist of starch 5～8% (w/v), Hectometer cake powder 1.2～2.8%, vegetable oil 5.1～7.0%, potassium dihydrogen phosphate 0.04～0.09% (w/v), non-ionic surface active agent APES 0.05～0.20% (w/v), orotic acid～10ppm, manganese sulfate 0.01～0.06% (w/v).
The human hairs such as Jiangsu Prov. Inst. of Microbiology Jiang Wen time understand " a kind of method of producing beta-carotene by fermentation " (publication number: CN1193048A; Open day: 1998.9.16). This invention by cultivating the positive and negative bacterial strain of Blakeslea trispora and carrying out beta carotene fermentation in airlift fermentor. Its fermentation medium consists of: starch 2～4%, Hectometer cake powder 3～5%, vegetable oil 3～5%, corn steep liquor 3～5%, dipotassium hydrogen phosphate 0.1～0.3%, magnesium sulfate 0.01～0.05%, thiamine hydrochloride 0.001～0.003%. 24～30 DEG C of fermentations 90～120 hours, beta carotene output can reach 1.5g/L.
Known according to above-mentioned technical background information analysis, there is not yet the fermentation accelerant utilizing described in this patent to improve the report of fermentation beta carotene output.
Summary of the invention
Object of the present invention be exactly for provide a kind of can be in the situation that not changing other fermentation condition and fermentation medium composition, stimulate Blakeslea trispora to generate in a large number beta carotene, thereby improve beta carotene fermentation preparation efficiency and reduce production costs.
Object of the present invention can be achieved through the following technical solutions:
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that filamentous fungi Blakeslea trispora (Blakesleatrispora) is seeded on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 26～28 DEG C, cultivates 48～96h, until grow spore;
Seed culture be by the Blakeslea trispora spore inoculating of cultivating through inclined-plane to seed culture medium, regulate pH6.5～7.5, cultivate 16～24h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, and the temperature that controlled fermentation is cultivated is 24～30 DEG C, cultivation 120～168h;
One or more of adding in the time of fermented and cultured in salicylic acid, salicylate, nitrous acid or nitrite are mixed as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation.
Filamentous fungi Blakeslea trispora divides positive and negative two kinds of bacterial strains. Positive and negative two kinds of bacterial strains are inoculated into respectively PDA slant medium and cultivate, and the positive and negative bacterial strain spore of filamentous fungi Blakeslea trispora then cultivation being obtained, respectively by every liter of culture medium access 104～109In the inoculum concentration access seed culture medium of individual spore, cultivate.
The concentration of salicylic acid, salicylate, nitrous acid, nitrite is 0.01～200mM, after sterilizing, in fermented and cultured operation, adds.
As preferred embodiment, the concentration of salicylic acid, salicylate, nitrous acid, nitrite is 1～200mM.
Salicylate is selected from one or more in sodium salicylate, potassium salicylate, salicylic acid ammonia, calcium salicylate, salicylic acid ferrous iron, zinc salicylate, magnesium salicylate, gaultherolin, salethyl, propyl salicylate or isopropyl salicylate.
Nitrite is selected from one or more in natrium nitrosum, potassium nitrite, nitrous acid ammonia, calcium nitrite, nitrous acid ferrous iron, zinc nitrite, magnesium nitrite, methyl nitrite, nitrous ether (ethyl nitrite), propyl nitrite or Isopropyl Nitrite.
Fermentation accelerant once or repeatedly adds after sterilizing in fermented and cultured process.
Compared with prior art, the present invention has the following advantages:
(1) the present invention is owing to having added the fermentation accelerant drawing through Experimental Comparison in fermentation medium, by fermentation accelerant by means of signal transduction paths such as the intracellular active oxygens of Blakeslea trispora (ROS), regulate and control this bacterial strain strengthening beta carotene metabolic pathway of synthesizing (comprising the metabolic pathway such as primary metabolite and methylol valeric acid) related gene expression or (with) weaken the expression of the synthetic branched metabolic pathway related gene of beta carotene, thereby cause a large amount of synthetic and accumulation of beta carotene in cell, and and then stimulate Blakeslea trispora produce beta carotene reach 2.25g/L, improve 55～190% than original process fermentation level (1.28g/L).
(2) the present invention owing to having added the fermentation accelerant drawing through Experimental Comparison in fermentation medium, and its consumption is few, in stimulating Blakeslea trispora to produce beta carotene in a large number, can't obviously increase production cost.
(3) the present invention owing to having added the fermentation accelerant drawing through Experimental Comparison in fermentation medium, and its consumption is few, and can utilize the technique such as washing or ethanol cleaning of microorganism collection after fermentation ends to be removed. The residual concentration of fermentation accelerant is lower than 0.1mg/kg thalline (or beta carotene goods), be generally the requirement of 3-5mg/kg far below " pollutants in food limitation national standard " regulation limitation, meet the relevant criterion of the Ministry of Public Health, State Food and Drug Administration.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail, but following examples are not construed as limiting the invention.
Comparative example 1
The bacterial strain that uses be Blakeslea trispora (Blakesleatrispora), fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 120 hours and is obtained zymotic fluid, and analysis result is content beta-carotene 1.28g/L in zymotic fluid.
Embodiment 1
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 51 hours and is added natrium nitrosum 2～20mM, and the comparable control group that does not add natrium nitrosum of its fermentation level improves 21～66%.
Embodiment 2
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 51 hours and is added sodium salicylate 2～200mM, and the comparable control group that does not add sodium salicylate of its fermentation level improves 21.34%～54.99%.
Embodiment 3
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds cultivation adds nitrous acid 2～20mM and regulates pH value to 6.5～6.8 with NaOH after 51 hours, the comparable control group that does not add nitrous acid of its fermentation level improves 11%～67%.
Embodiment 4
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds cultivation adds salicylic acid 2～200mM and regulates pH value to 6.5～6.8 with NaOH after 51 hours, the comparable control group that does not add sodium salicylate of its fermentation level improves 9%～50%.
Embodiment 5
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 51 hours and is added potassium nitrite 2～200mM, and the comparable control group that does not add potassium nitrite of its fermentation level improves 5%～63%.
Embodiment 6
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 51 hours and is added potassium salicylate 2～200mM, and the comparable control group that does not add potassium salicylate of its fermentation level improves 10%～57%.
Embodiment 7
Fermentation medium consists of starch 8% (w/v), dregs of rice cake powder 1.6% (w/v), vegetable oil 6.6% (v/v), KH2PO40.06％(w/v)、MnSO4·H2O0.03% (w/v), regulating pH is 7.0, after the fermentation medium sterilizing being configured to, access positive and negative bacterium seed mixture liquid, the shaking table top fermentation that is 230rpm at 27 DEG C of rotating speeds is cultivated after 51 hours and is added sodium salicylate 0.01～200mM and natrium nitrosum 0.01～200mM, and the comparable control group that does not add sodium salicylate and natrium nitrosum of its fermentation level improves 55～190%.
Embodiment 8
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, this technique comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that positive and negative filamentous fungi Blakeslea trispora bacterial strain is seeded in respectively on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 26 DEG C, cultivates 96h, until grow spore;
Seed culture is by every liter of culture medium access 10 by positive and negative the Blakeslea trispora of cultivating through inclined-plane bacterial strain spore4～109In the inoculum concentration access seed culture medium of individual spore, cultivate, regulating pH is 6.5, cultivates 24h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, in the time of fermented and cultured, adding concentration is that 0.01mM salicylic acid is as fermentation accelerant, after sterilizing in fermented and cultured process disposable adding in fermentation medium, the temperature that controlled fermentation is cultivated is 24 DEG C, cultivation 168h, preparing natural beta-carotin by fermentation.
Embodiment 9
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, this technique comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that positive and negative filamentous fungi Blakeslea trispora bacterial strain is seeded in respectively on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 27 DEG C, cultivates 72h, until grow spore;
Seed culture is by every liter of culture medium access 10 by positive and negative the Blakeslea trispora of cultivating through inclined-plane bacterial strain spore4～109In the inoculum concentration access seed culture medium of individual spore, cultivate, regulating pH is 7, cultivates 18h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, in the time of fermented and cultured, add salicylic acid, calcium salicylate, nitrous ether (ethyl nitrite) as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation, wherein, the concentration of salicylic acid, calcium salicylate, nitrous ether (ethyl nitrite) is 1mM, after sterilizing in fermented and cultured process disposable adding in fermentation medium, the temperature that controlled fermentation is cultivated is 28 DEG C, cultivation 150h, preparing natural beta-carotin by fermentation.
Embodiment 10
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, this technique comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that positive and negative filamentous fungi Blakeslea trispora bacterial strain is seeded in respectively on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 28 DEG C, cultivates 48h, until grow spore;
Seed culture is by every liter of culture medium access 10 by positive and negative the Blakeslea trispora of cultivating through inclined-plane bacterial strain spore4～109In the inoculum concentration access seed culture medium of individual spore, cultivate, regulating pH is 7.5, cultivates 16h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, in the time of fermented and cultured, add salicylic acid, salicylic acid ferrous iron, zinc salicylate as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation, after fermentation accelerant sterilizing, in fermented and cultured operation, added once every 24 hours, concentration after each interpolation is maintained at 1～200mM, the temperature that controlled fermentation is cultivated is 30 DEG C, cultivation 120h, preparing natural beta-carotin by fermentation.
Embodiment 11
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, this technique comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that positive and negative filamentous fungi Blakeslea trispora bacterial strain is seeded in respectively on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 26 DEG C, cultivates 84h, until grow spore;
Seed culture is by every liter of culture medium access 10 by positive and negative the Blakeslea trispora of cultivating through inclined-plane bacterial strain spore4～109In the inoculum concentration access seed culture medium of individual spore, cultivate, regulating pH is 7, cultivates 16h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, in the time of fermented and cultured, add nitrous acid that concentration is 1mM, natrium nitrosum, potassium nitrite as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation, after sterilizing in fermented and cultured process disposable adding in fermentation medium, the temperature that controlled fermentation is cultivated is 24 DEG C, cultivation 120h, preparing natural beta-carotin by fermentation.
Embodiment 12
A kind of fermentation accelerant strengthening carrotene zymotechnique that adopts, this technique comprises inclined-plane cultivation, seed culture, fermented and cultured operation,
It is that positive and negative filamentous fungi Blakeslea trispora bacterial strain is seeded in respectively on PDA slant medium that inclined-plane is cultivated, and controlling temperature is 28 DEG C, cultivates 60h, until grow spore;
Seed culture is by every liter of culture medium access 10 by positive and negative the Blakeslea trispora of cultivating through inclined-plane bacterial strain spore4～109In the inoculum concentration access seed culture medium of individual spore, cultivate, regulating pH is 7.5, cultivates 24h;
Fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, in the time of fermented and cultured, add concentration and be the nitrous ether (ethyl nitrite) of 1mM as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation, after sterilizing in fermented and cultured process disposable adding in fermentation medium, the temperature that controlled fermentation is cultivated is 30 DEG C, cultivation 168h, preparing natural beta-carotin by fermentation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (5)
1. adopt a fermentation accelerant strengthening carrotene zymotechnique, this technique comprises that inclined-plane is cultivated, seedCultivation, fermented and cultured operation,
It is that filamentous fungi Blakeslea trispora (Blakesleatrispora) is seeded in to PDA is oblique that described inclined-plane is cultivatedOn face culture medium, controlling temperature is 26～28 DEG C, cultivates 48～96h, until grow spore;
Described seed culture be by the Blakeslea trispora spore inoculating of cultivating through inclined-plane to seed culture medium, regulatePH6.5～7.5, cultivate 16～24h;
Described fermented and cultured is that the Blakeslea trispora seed liquor through seed culture is inoculated in fermentation medium, controlThe temperature of fermented and cultured processed is 24～30 DEG C, cultivation 120～168h;
It is characterized in that, in the time of fermented and cultured, add one in salicylic acid, salicylate, nitrous acid or nitriteKind or several mixing, as fermentation accelerant, promote the fermentation level of preparing natural beta-carotin by fermentation; DescribedSalicylic acid, salicylate, nitrous acid, the concentration of nitrite be 0.01～200mM, after sterilizing in fermentation trainingSupport in operation and add;
Described salicylate be selected from sodium salicylate, potassium salicylate, salicylic acid ammonia, calcium salicylate, salicylic acid ferrous iron,In zinc salicylate, magnesium salicylate, gaultherolin, salethyl, propyl salicylate or isopropyl salicylateOne or more; Described nitrite is selected from natrium nitrosum, potassium nitrite, nitrous acid ammonia, calcium nitrite, AsiaFerrous nitrate, zinc nitrite, magnesium nitrite, methyl nitrite, nitrous ether (ethyl nitrite), propyl nitrite or nitrous acidOne or more in isopropyl ester.
2. a kind of adopt fermentation accelerant strengthening carrotene zymotechnique, its spy according to claim 1Levy and be, described filamentous fungi Blakeslea trispora divides positive and negative two kinds of bacterial strains.
3. a kind of adopt fermentation accelerant strengthening carrotene zymotechnique, its spy according to claim 2Levy and be, positive and negative two kinds of bacterial strains are inoculated into respectively PDA slant medium and cultivate, the silk then cultivation being obtainedThe positive and negative bacterial strain spore of shape fungi Blakeslea trispora, respectively by every liter of culture medium access 104～109The inoculation of individual sporeIn amount access seed culture medium, cultivate.
4. a kind of adopt fermentation accelerant strengthening carrotene zymotechnique, its spy according to claim 1Levy and be, the concentration of described salicylic acid, salicylate, nitrous acid, nitrite is 1～200mM.
5. a kind of adopt fermentation accelerant strengthening carrotene zymotechnique, its spy according to claim 1Levy and be, described fermentation accelerant once or repeatedly adds after sterilizing in fermented and cultured process.
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| CN102787158A (en) * | 2011-05-20 | 2012-11-21 | 浙江医药股份有限公司新昌制药厂 | Method for producing natural beta-carotene by fermentation and application |
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| CN102757995A (en) * | 2012-06-25 | 2012-10-31 | 湖北工业大学 | Method for preparing beta-carotene through fermentation of Blakeslea trispora |
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