CN104130952A - Rhodotorula mucilaginosa and application in fermentation production of carotenoid and oils - Google Patents
Rhodotorula mucilaginosa and application in fermentation production of carotenoid and oils Download PDFInfo
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- CN104130952A CN104130952A CN201410131054.1A CN201410131054A CN104130952A CN 104130952 A CN104130952 A CN 104130952A CN 201410131054 A CN201410131054 A CN 201410131054A CN 104130952 A CN104130952 A CN 104130952A
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Abstract
The invention discloses a Rhodotorula mucilaginosa strain and application in fermentation production of carotenoid and oils. The main point of the technical scheme involved in the invention lies in that: the Rhodotorula mucilaginosa strain is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No.8926. The Rhodotorula mucilaginosa provided by the invention can be used for fermentation production of carotenoid and oils. According to the invention, Rhodotorula mucilaginosa is utilized to produce carotenoid and oils at the same time, multi-round ARTP (atmospheric and room temperature plasma) mutagenesis is carried out on the production strain so as to obtain a high yield mutant strain, thus providing good application prospects and economic benefits for industrial production of carotenoid and oils.
Description
Technical field
The invention belongs to yeast technical field, be specifically related to a strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof.
Background technology
Carotenoid is one group of general name with the fat-soluble pigment of oxidation-resistance, it is as the class important biomolecule active substance in humans and animals body, there is the vital role such as enhancing body immunizing power, radioprotective, antitumor and treatment photosensitive diseases, in industries such as food, makeup, medicine and feeds, have a wide range of applications.At present, carotenoid is regarded as category-A nutrition pigment by FAO, JiWHODeng international organization of the European Community, and in recent years, around the also progressively formation of industrialization development of carotenoid.
The industrialized preparing process of carotenoid can adopt chemical synthesis or plant extraction method.Chemosynthesis carotenoid is technical sophistication not only, and it uses the taste that can affect food as foodstuff additive, and the ratio of reduction absorption of human body also exists certain side effect simultaneously.Therefore, be accompanied by the psychology that people advocate green food and day by day strengthen, increasing scholar turns to research direction the development and utilization of natural pigment in recent years.Owing to extracting traditional preparation technology of carotenoid from plant, have higher cost and Operating Complexity, therefore, the suitability for industrialized production of carotenoid has been subject to huge restriction.Utilize microorganism fermentative production carotenoid, not only with low cost, technique is simple, and can guarantee high-quality and the stable yields of product, and therefore, utilizing microorganisms producing carotenoid is the main path in Biological resources type carotenoid source.
At present, the microorganism strains that can produce carotenoid of having reported is mainly trispore Bruce mould and rhodotorula, and the former is in pilot scale and commercial application stage, and latter is still in the laboratory lab scale stage.Although trispore Bruce mould pigment production is high, but culture process is complicated, and the production cycle is long, it is simple that rhodotorula production carotenoid has nutritional requirement, culture cycle is short, and thalline contains the advantage that rich in protein, amino acid and VITAMIN etc. can fully utilize.
Microbial oil (Microbial Oils) is that microorganism utilizes the raw materials such as carbohydrate or hydrocarbon polymer, and synthetic grease and its content reach the more than 20% of dry cell weight in vivo, conventionally claim again Unicell Oils and Fats.Along with the worsening shortages of natural resources and the continuous growth of grease demand, conventional grease industrial expansion is restricted, imbalance between supply and demand becomes increasingly conspicuous, therefore, take renewable resources as raw material, open up a kind of new oil resource-microbial oil, to meeting the growing grease demand of people, have great importance.Compare with traditional grease production method, microorganism fermentative production grease because of cost low, cell speedup is fast, growth cycle is short, and is not subject to the impact of season, climate change and receives increasing concern.The Lipid-producing microorganism of report mainly has at present: bacterium, mould, yeast and algae, and wherein in the majority with the eukaryotic microorganisms of yeast and mold.Opening up the approach of this new production grease of microbial oil, is a new direction of exploitation oil resource, not only can alleviate the situation that traditional mode of production grease is in short supply, can also increase a new important channel for suitability for industrialized production grease.
Summary of the invention
The technical problem that the present invention solves has been to provide a strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof.
Rhodotorula mucilaginose provided by the invention (Rhodotorula mucilaginosa), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCC No.8926, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, preservation date: on March 17th, 2014.
Rhodotorula mucilaginosa provided by the invention can be used for fermentative production carotenoid, by following steps, realizes: described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated into and is produced in fermentative carotenoid substratum, in 27-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, obtain the fermented liquid that contains carotenoid, wherein carbon source is glucose or sucrose, and described product fermentative carotenoid medium component is counted with g/L: glucose 31.8, yeast extract paste 1, ammonium sulfate 2, potassium primary phosphate 1, Calcium Chloride Powder Anhydrous 0.1, magnesium sulfate heptahydrate 0.5, sodium-chlor 0.1, all the other compositions are water, pH7.0.
As further preferred technical scheme, the pH6.5-7.5 in fermenting process.
Rhodotorula mucilaginosa provided by the invention can be used for fermentative production grease simultaneously, by following steps, realizes: described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in Lipid-producing fermention medium, in 26-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 30-130:1,190rpm shaking culture 96h, obtains the fermented liquid that contains grease, wherein carbon source is glucose or sucrose, described Lipid-producing fermentation culture based component is counted with g/L: glucose 85.6, and yeast soaks powder 0.5, potassium primary phosphate 1, ammonium sulfate 2, magnesium sulfate heptahydrate 0.5, all the other compositions are water, pH6.0.
28 ℃ of slant culture temperature of the present invention, incubation time 72h, slant culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, agar powder 2, all the other compositions are water.
28 ℃ of seed culture temperature of the present invention, 160rpm cultivates 24h, and seed culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, all the other compositions are water.
The present invention utilizes rhodotorula mucilaginosa produce carotenoid and produce grease simultaneously, and carry out many wheel ARTP(atmospheric pressure at room plasma bodys to producing bacterial strain) mutagenesis, obtain the higher mutagenic strain of output, to the suitability for industrialized production of carotenoid and grease, provide good application prospect and economic benefit.
Accompanying drawing explanation
Fig. 1 is the systematic evolution tree that the present invention is based on yeast 26S rDNA sequence construct, Fig. 2 is the HPLC collection of illustrative plates of β-carotene standard substance, Fig. 3 is the HPLC collection of illustrative plates of the fermented liquid that contains carotenoid of the embodiment of the present invention 1 production, and Fig. 4 is the GC-MS collection of illustrative plates of the fermented liquid that contains grease of embodiment 2 productions.
Drawing explanation: 1, plam oil, 2, oleic acid.
Embodiment
By the following examples foregoing of the present invention is described in further details, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
The screening of bacterial strain and mutagenesis
From the active sludge of antibiotic fermentation waste water, screen and obtain.Adopt YPD substratum separated, YPD culture medium prescription: glucose 20g, peptone 10g, yeast extract paste 5g, agar 15-20g, distilled water 1000mL, pH nature, 115 ℃ of sterilizing 30min, add paraxin before use, and making its final concentration is 100mg/L.Screening process: get 50mL active sludge, shaking table concussion 30min at 150rpm, 28 ℃, after cell is disperseed, dilutes respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, then get each weaker concn 0.1ml liquid spreading to the YPD substratum that contains paraxin, each gradient do two parallel, cultivate for 28 ℃ and observe.To being with coloured bacterial strain to carry out purifying cultivation, obtain single bacterium colony, be then inoculated in YPD liquid fermentation medium, it is higher that final screening obtains bacterial strain KC8 output.
Utilize ARTP(atmospheric pressure at room plasma body) mutagenic condition: process apart from 2mm; Processing power 120W; Airshed 10SLM, point sample amount 10 μ L, the treatment time is respectively 0s, 15s, 30s, 45s, 60s, 90s, 120s, 180s.Process in latter 6 hours and be coated with inoculation, cultivate 3 days for 28 ℃.Picking list bacterium colony is inoculated respectively liquid fermentation medium, obtains carotenoid and grease yield and all improve maximum bacterial strain KC8-AR62 after multi-turns screen.
The evaluation of bacterial strain
(1) morphologic observation
The method of employing line is by inoculation to solid YPD substratum, and the composition of solid YPD substratum is counted with g/L: yeast extract paste 1, and peptone 2, glucose 2, agar powder 2, all the other compositions are water.Cultivate 72h for 28 ℃, can be observed rounded, red, rat, moistening single bacterium colony.Thalline at 1000 times of oily Microscopic observations, can be seen the yeast form of oval tool gemma through sudan black dyeing, can also see the existence of red pigments, and be dyed the existence for blue Lipid-producing cell simultaneously.After ARTP mutagenesis, it is large that the colonial morphology of bacterial strain obviously becomes.
(2) using the method of CTAB extraction strains of DNA, with 26 s?RDNA?D1 / D2 area augmentation, sequencing sequence is:TAAAAGGATTGCCCTAGTAGCGGCGAGCGAAGCGGGAAGAGCTCAAATTTATAATCTGGCACCTTCGGTGTCCGAGTTGTAATCTCTAGAAATGTTTTCCGCGTTGGACCGCACACAAGTCTGTTGGAATACAGCGGCATAGTGGTGAGACCCCCGTATATGGTGCGGACGCCCAGCGCTTTGTGATACATTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAATTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAACAGTACGTGAAATTGTTGGAAGGGAAACGCTTGAAGTCAGACTTGCTTGCCGAGCAATCGGTTTGCAGGCCAGCATCAGTTTTCCGGGATGGATAATGGTAGAGAGAAGGTAGCAGTTTCGGCTGTGTTATAGCTCTCTGCTGGATACATCTTGGGGGACTGAGGAACGCAGTGTGCCTTTGGCGGGGGTTTCGACCTCTTCACACTTAGGATGCTGGTGGAATGGCTTTAAACGACCCGTCTTAA。Through Blast sequence alignment phylogenetic tree construction, be illustrated in fig. 1 shown below.
Embodiment 1
Described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid, according to the inoculum size of volume ratio 10%, seed liquor is inoculated into and is produced in fermentative carotenoid substratum, in 27-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 25-35:1, 190rpm shaking culture 96h, obtain the fermented liquid that contains carotenoid, wherein carbon source is glucose or sucrose, take glucose as best, the production peak of carotenoid can reach 22.93mg/L, described product fermentative carotenoid medium component is counted with g/L: glucose 31.8, yeast extract paste 1, ammonium sulfate 2, potassium primary phosphate 1, Calcium Chloride Powder Anhydrous 0.1, magnesium sulfate heptahydrate 0.5, sodium-chlor 0.1, all the other compositions are water, pH7.0.The pigment of producing is through high-performance liquid chromatogram determination, and main component is carotenoid, as shown in Figures 2 and 3.
This bacterial strain all can produce higher carotenoid at pH4.5-8.0, but High Yield of Carotenoid scope is between pH6.5-7.5, dextrose plus saccharose can be as the carbon source that produces carotenoid, but best with glucose, carbon-nitrogen ratio is 25-35:1, temperature is between 27-30 ℃, and fermentation 96h carotenoid output is the highest, and production peak can reach 22.93mg/L.
Embodiment 2
Described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid, according to the inoculum size of volume ratio 10%, seed liquor is inoculated in Lipid-producing fermention medium, in 26-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 30-130:1, 190rpm shaking culture 96h, obtain the fermented liquid that contains grease, wherein carbon source is glucose or sucrose, dextrose plus saccharose is can produce grease under the condition of carbon source, the grease of producing is up to 27.8%, described Lipid-producing fermentation culture based component is counted with g/L: glucose 85.6, yeast soaks powder 0.5, potassium primary phosphate 1, ammonium sulfate 2, magnesium sulfate heptahydrate 0.5, all the other compositions are water, pH6.0, utilize Soxhlet extraction method to carry out grease extracting, through GC-MS, the grease producing is identified, the grease of producing is mainly C16 and C18 unsaturated fatty acids, for example palmitinic acid and oleic acid, as described in Figure 4, wherein the content of palmitinic acid is 14.57%, the content of oleic acid is 70.21%, other also has stearic acid and linolic acid etc.
Above embodiment has described ultimate principle of the present invention, principal character and advantage.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; do not departing under the scope of the principle of the invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the scope of protection of the invention.
Claims (10)
1. a Rhodotorula mucilaginose strain Rhodotorula mucilaginosa, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.8926.
2. the application of rhodotorula mucilaginosa claimed in claim 1 in fermentative production carotenoid.
3. the application of rhodotorula mucilaginosa according to claim 2 in fermentative production carotenoid, is characterized in that realizing by following steps: described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated into and is produced in fermentative carotenoid substratum, in 27-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, obtain the fermented liquid that contains carotenoid, wherein carbon source is glucose or sucrose, and described product fermentative carotenoid medium component is counted with g/L: glucose 31.8, yeast extract paste 1, ammonium sulfate 2, potassium primary phosphate 1, Calcium Chloride Powder Anhydrous 0.1, magnesium sulfate heptahydrate 0.5, sodium-chlor 0.1, all the other compositions are water, pH7.0.
4. the application of rhodotorula mucilaginosa according to claim 2 in fermentative production carotenoid, is characterized in that realizing by following steps: described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated into and is produced in fermentative carotenoid substratum, in 27-30 ℃, pH6.5-7.5, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, obtain the fermented liquid that contains carotenoid, wherein carbon source is glucose or sucrose, and described product fermentative carotenoid medium component is counted with g/L: glucose 31.8, yeast extract paste 1, ammonium sulfate 2, potassium primary phosphate 1, Calcium Chloride Powder Anhydrous 0.1, magnesium sulfate heptahydrate 0.5, sodium-chlor 0.1, all the other compositions are water, pH7.0.
5. the application in fermentative production carotenoid according to the rhodotorula mucilaginosa described in claim 3 or 4, is characterized in that: 28 ℃ of described slant culture temperature, incubation time 72h, slant culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, agar powder 2, all the other compositions are water.
6. the application in fermentative production carotenoid according to the rhodotorula mucilaginosa described in claim 3 or 4, is characterized in that: 28 ℃ of described seed culture temperature, and 160rpm cultivates 24h, seed culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, all the other compositions are water.
7. the application of rhodotorula mucilaginosa claimed in claim 1 in fermentative production grease.
8. the application of rhodotorula mucilaginosa according to claim 7 in fermentative production grease, is characterized in that realizing by following steps: described rhodotorula mucilaginosa is carried out to slant culture and seed culture, obtain activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in Lipid-producing fermention medium, in 26-30 ℃, pH4.5-8.0, carbon-nitrogen ratio 30-130:1,190rpm shaking culture 96h, obtains the fermented liquid that contains grease, wherein carbon source is glucose or sucrose, described Lipid-producing fermentation culture based component is counted with g/L: glucose 85.6, and yeast soaks powder 0.5, potassium primary phosphate 1, ammonium sulfate 2, magnesium sulfate heptahydrate 0.5, all the other compositions are water, pH6.0.
9. the application of rhodotorula mucilaginosa according to claim 8 in fermentative production grease, is characterized in that: 28 ℃ of described slant culture temperature, incubation time 72h, slant culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, agar powder 2, all the other compositions are water.
10. the application of rhodotorula mucilaginosa according to claim 8 in fermentative production grease, is characterized in that: 28 ℃ of described seed culture temperature, and 160rpm cultivates 24h, seed culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, all the other compositions are water.
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Cited By (11)
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| CN104845893A (en) * | 2015-05-13 | 2015-08-19 | 威海利达生物科技有限公司 | Rhodotorula mucilaginosa and application thereof to fermentation production of malus micromalus astaxanthin |
| CN104846049A (en) * | 2015-05-13 | 2015-08-19 | 威海利达生物科技有限公司 | Method for improving fermentation productivity of carotenoid |
| CN105039484A (en) * | 2015-08-17 | 2015-11-11 | 青岛大学 | High-yield carotinoid and copper fermentation culture method by using Rhodotorula benthica |
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| CN105755086A (en) * | 2016-03-01 | 2016-07-13 | 临沂市康发食品饮料有限公司 | Method for producing carotenoid by utilizing canned yellow peach leftovers through fermentation |
| CN105907731A (en) * | 2016-06-30 | 2016-08-31 | 江南大学 | Method for promoting microbes to produce laccase |
| CN107653195A (en) * | 2017-09-14 | 2018-02-02 | 贵州东智生物科技有限公司 | A kind of method that rhodotorula mucilaginosa is separated from fermenting black garlic |
| CN109548984A (en) * | 2018-12-19 | 2019-04-02 | 江南大学 | A method of oil crops are processed using rhodotorula |
| CN111500475A (en) * | 2019-12-13 | 2020-08-07 | 燕山大学 | Rhodotorula mucilaginosa ZOR1 and method for producing mannan and carotenoid |
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| CN104830703B (en) * | 2015-05-13 | 2018-09-11 | 威海利达生物科技有限公司 | One plant of rhodotorula mucilaginosa and its application in the red astaxanthin in fermenting and producing sea |
| CN104845893A (en) * | 2015-05-13 | 2015-08-19 | 威海利达生物科技有限公司 | Rhodotorula mucilaginosa and application thereof to fermentation production of malus micromalus astaxanthin |
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| CN104830703A (en) * | 2015-05-13 | 2015-08-12 | 威海利达生物科技有限公司 | Rhodotorula mucilaginosa and application thereof in fermentation production of sea red astaxanthin |
| CN105039484A (en) * | 2015-08-17 | 2015-11-11 | 青岛大学 | High-yield carotinoid and copper fermentation culture method by using Rhodotorula benthica |
| CN105039484B (en) * | 2015-08-17 | 2018-11-09 | 青岛大学 | It is a kind of to utilize ocean rhodotorula High Yield of Carotenoid and copper fermentation culture method |
| CN105028954A (en) * | 2015-08-26 | 2015-11-11 | 天津天绿健科技有限公司 | Feed additive product |
| CN105755086A (en) * | 2016-03-01 | 2016-07-13 | 临沂市康发食品饮料有限公司 | Method for producing carotenoid by utilizing canned yellow peach leftovers through fermentation |
| CN105907731A (en) * | 2016-06-30 | 2016-08-31 | 江南大学 | Method for promoting microbes to produce laccase |
| CN107653195A (en) * | 2017-09-14 | 2018-02-02 | 贵州东智生物科技有限公司 | A kind of method that rhodotorula mucilaginosa is separated from fermenting black garlic |
| CN109548984A (en) * | 2018-12-19 | 2019-04-02 | 江南大学 | A method of oil crops are processed using rhodotorula |
| WO2020124632A1 (en) * | 2018-12-19 | 2020-06-25 | 江南大学 | Method for processing oil crops using rhodotorula |
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| CN111500475A (en) * | 2019-12-13 | 2020-08-07 | 燕山大学 | Rhodotorula mucilaginosa ZOR1 and method for producing mannan and carotenoid |
| CN111500475B (en) * | 2019-12-13 | 2022-06-03 | 燕山大学 | Rhodotorula mucilaginosa ZOR1 and method for producing mannan and carotenoid |
| CN114874940A (en) * | 2022-05-10 | 2022-08-09 | 吉林省农业科学院 | Complex microbial inoculant, and fermentation product and application thereof |
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