CN104130952B - One strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof - Google Patents
One strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof Download PDFInfo
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Abstract
The present invention discloses a strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof. Does is the drip irrigation device of the present invention: a Rhodotorula mucilaginose strain Rhodotorula? does is mucilaginosa, preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, CGMCC? No.8926. Rhodotorula mucilaginosa provided by the invention can be used for fermentative production carotenoid and grease. The present invention utilizes rhodotorula to produce carotenoid and produce grease simultaneously, and production bacterial strain is carried out many wheel ARTP(atmospheric pressure at room plasma bodys) mutagenesis, obtain the mutagenic strain that output is higher, the suitability for industrialized production of carotenoid and grease is provided good application prospect and economic benefit.
Description
Technical field
The invention belongs to yeast technical field, it is specifically related to a strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof.
Background technology
Carotenoid is the general name of the fat-soluble pigment that a group has oxidation-resistance, it is as the class important biomolecule active substance in humans and animals body, there is enhancing body immunizing power, radioprotective, the antitumor and treatment vital role such as photosensitive diseases, have a wide range of applications in industries such as food, makeup, medicine and feeds. At present, carotenoid is regarded as A class nutrition pigment by international organizations such as FAO, the European Community and WHO, and in recent years, the industrialization development around carotenoid is also progressively formed.
The industrialized preparing process of carotenoid can adopt chemical synthesis or plant extraction method. Not only technology is complicated for chemosynthesis carotenoid, and it can affect the taste of food as foodstuff additive use, reduces the ratio of absorption of human body, also there is certain side effect simultaneously. Therefore, the psychology advocating green food with people strengthens day by day, and research direction is turned to the development and utilization of natural pigment by scholars more and more in recent years. Having higher cost and complicated operation owing to extracting traditional preparation technology of carotenoid from plant, therefore, the suitability for industrialized production of carotenoid receives huge restriction. Utilizing fermentable to produce carotenoid, not only with low cost, technique is simple, and can ensure high-quality and the stable yields of product, therefore, utilizes microorganisms producing carotenoid to be the main approach that Biological resources type carotenoid is originated.
At present, the microorganism strains that can produce carotenoid reported mainly trispore Bruce mould and rhodotorula, the former has been in pilot scale and commercial application stage, and the latter Ze Shang is in the laboratory lab scale stage. Although trispore Bruce mould pigment production height, but culture process is complicated, and the production cycle is long, it is simple that rhodotorula production carotenoid then has nutritional requirement, culture cycle is short, and thalline contains the advantage that abundant protein, amino acid and VITAMIN etc. can fully utilize.
Microbial oil (MicrobialOils) is that microorganism utilizes the raw material such as carbohydrate or hydrocarbon polymer, and grease and its content of synthesis in body reach more than the 20% of dry cell weight, usually also known as Unicell Oils and Fats.Along with the worsening shortages of natural resources and the continuous growth of grease demand, the development of conventional grease industry is restricted, imbalance between supply and demand is day by day outstanding, therefore, take renewable resources as raw material, open up a kind of new oil resource-microbial oil, the grease demand meeting people growing is had important meaning. Compared with traditional grease production method, fermentable produce grease because of cost low, cell speedup is fast, and growth cycle is short, and the concern not got more and more by the impact of season, climate change. The product grease microorganism of report mainly contains at present: bacterium, mould, yeast and algae, wherein in the majority with the eukaryotic microorganisms of yeast and mold. Open up the approach of this new production grease of microbial oil, it is a new direction of exploitation oil resource, be possible not only to alleviate traditional mode of production grease situation in short supply, it is also possible to for suitability for industrialized production grease increases a new important channel.
Summary of the invention
The technical problem that the present invention solves there is provided a strain rhodotorula mucilaginosa and the application in fermentative production carotenoid and grease thereof.
Rhodotorula mucilaginose provided by the invention (Rhodotorulamucilaginosa), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCCNo.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, is realized by following step: described rhodotorula mucilaginosa carries out slant culture and seed culture, obtains activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in product fermentative carotenoid substratum, in 27-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, namely obtaining the fermented liquid containing 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 preferred technical scheme further, the pH6.5-7.5 in fermenting process.
Rhodotorula mucilaginosa provided by the invention can be used for fermentative production grease simultaneously, is realized by following step: described rhodotorula mucilaginosa carries out slant culture and seed culture, obtains activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in product oil fermentation substratum, in 26-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 30-130:1,190rpm shaking culture 96h, namely obtains the fermented liquid containing grease, wherein carbon source is glucose or sucrose, described product oil fermentation medium component is counted with g/L: glucose 85.6, yeast leaching powder 0.5, potassium primary phosphate 1, ammonium sulfate 2, magnesium sulfate heptahydrate 0.5, all the other compositions are water, pH6.0.
Slant culture temperature of the present invention 28 DEG C, incubation time 72h, slant culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, agar powder 2, and all the other compositions are water.
Seed culture temperature of the present invention 28 DEG C, 160rpm cultivates 24h, and seed culture based component is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, and all the other compositions are water.
The present invention utilizes rhodotorula mucilaginosa to produce carotenoid and produce grease simultaneously, and production bacterial strain is carried out many wheel ARTP(atmospheric pressure at room plasma bodys) mutagenesis, obtain the mutagenic strain that output is higher, the suitability for industrialized production of carotenoid and grease is provided good application prospect and economic benefit.
Accompanying drawing explanation
Fig. 1 is the systematic evolution tree of the present invention based on yeast 26SrDNA sequence construct, Fig. 2 is the HPLC collection of illustrative plates of ��-carotene standard substance, Fig. 3 is the HPLC collection of illustrative plates of the fermented liquid containing carotenoid that the embodiment of the present invention 1 is produced, and Fig. 4 is the GC-MS collection of illustrative plates of the fermented liquid containing grease that embodiment 2 is produced.
Figure face illustrates: 1, plam oil, 2, oleic acid.
Embodiment
By the following examples the foregoing of the present invention is described in further details, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to following embodiment. All technology realized based on foregoing of the present invention all belong to the scope of the present invention.
The screening of bacterial strain and mutagenesis
Screen from the active sludge of antibiotic fermentation waste water and obtain. Adopting the separation of YPD substratum, YPD culture medium prescription: glucose 20g, peptone 10g, yeast extract paste 5g, agar 15-20g, distilled water 1000mL, pH nature, 115 DEG C of sterilizing 30min, add paraxin before use so that it is final concentration is 100mg/L. Screening process: get 50mL active sludge, 150rpm, shaking table concussion 30min at 28 DEG C, after cell dispersal, dilute 10 respectively-1��10-2��10-3��10-4��10-5��10-6, then get each weaker concn 0.1ml liquid spreading to, on the YPD substratum containing paraxin, it is parallel that each gradient does two, cultivate for 28 DEG C and observe. Bacterial strain with color being carried out purifying cultivation, obtains 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 distance 2mm; Processing power 120W; Airshed 10SLM, some sample amount 10 �� L, the treatment time is respectively 0s, 15s, 30s, 45s, 60s, 90s, 120s, 180s. Process and in latter 6 hours, carry out coating inoculation, cultivate 3 days for 28 DEG C. Choose and get single bacterium colony and inoculate liquid fermentation medium respectively, after multi-turns screen, obtain carotenoid and grease yield all improves maximum bacterial strain KC8-AR62.
The qualification of bacterial strain
(1) morphologic observation
Adopting the method for line by inoculation to, on solid YPD substratum, the composition of solid YPD substratum is counted with g/L: yeast extract paste 1, peptone 2, glucose 2, agar powder 2, all the other compositions are water. Cultivate 72h, can be observed rounded, red, rat, moistening single bacterium colony for 28 DEG C. Thalline dyes at 1000 times of oily Microscopic observations through sudan black, it can be seen that the yeast form of oval tool gemma, can also see the existence of red pigments simultaneously, and is contaminated the existence for blue product oil cells. After ARTP mutagenesis, the colonial morphology of bacterial strain obviously becomes big.
��2������CTAB����ȡ����DNA������26SrDNAD1/D2������,��������Ϊ��TAAAAGGATTGCCCTAGTAGCGGCGAGCGAAGCGGGAAGAGCTCAAATTTATAATCTGGCACCTTCGGTGTCCGAGTTGTAATCTCTAGAAATGTTTTCCGCGTTGGACCGCACACAAGTCTGTTGGAATACAGCGGCATAGTGGTGAGACCCCCGTATATGGTGCGGACGCCCAGCGCTTTGTGATACATTTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAATTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAACAGTACGTGAAATTGTTGGAAGGGAAACGCTTGAAGTCAGACTTGCTTGCCGAGCAATCGGTTTGCAGGCCAGCATCAGTTTTCCGGGATGGATAATGGTAGAGAGAAGGTAGCAGTTTCGGCTGTGTTATAGCTCTCTGCTGGATACATCTTGGGGGACTGAGGAACGCAGTGTGCCTTTGGCGGGGGTTTCGACCTCTTCACACTTAGGATGCTGGTGGAATGGCTTTAAACGACCCGTCTTAA��Through Blast sequence alignment and phylogenetic tree construction, be illustrated in fig. 1 shown below.
Embodiment 1
Described rhodotorula mucilaginosa is carried out slant culture and seed culture, obtains activated seed liquid, according to the inoculum size of volume ratio 10%, seed liquor is inoculated in product fermentative carotenoid substratum, in 27-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 25-35:1, 190rpm shaking culture 96h, namely the fermented liquid containing carotenoid is obtained, wherein carbon source is glucose or sucrose, taking 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 produced 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 most high yield carotenoid scope is between pH6.5-7.5, dextrose plus saccharose can as the carbon source producing carotenoid, but it is best with glucose, carbon-nitrogen ratio is 25-35:1, between temperature is 27-30 DEG C, fermentation 96h carotenoid output is the highest, and production peak can reach 22.93mg/L.
Embodiment 2
Described rhodotorula mucilaginosa is carried out slant culture and seed culture, obtains activated seed liquid, according to the inoculum size of volume ratio 10%, seed liquor is inoculated in product oil fermentation substratum, in 26-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 30-130:1, 190rpm shaking culture 96h, namely the fermented liquid containing grease is obtained, wherein carbon source is glucose or sucrose, grease can be produced when dextrose plus saccharose is carbon source, the grease produced is up to 27.8%, described product oil fermentation medium component is counted with g/L: glucose 85.6, yeast leaching powder 0.5, potassium primary phosphate 1, ammonium sulfate 2, magnesium sulfate heptahydrate 0.5, all the other compositions are water, pH6.0, Soxhlet extraction method is utilized to carry out grease extracting, through GC-MS, the grease produced is identified, the grease produced is mainly C16 and C18 unsaturated fatty acids, such as palmitinic acid and oleic acid, as shown 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.
Embodiment above describes the ultimate principle of the present invention, main feature and advantage. The technician of the industry should understand; the present invention is not restricted to the described embodiments; the principle that the present invention is just described described in above-described embodiment and specification sheets; under the scope not departing from 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 Rhodotorulamucilaginosa, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCCNo.8926.
2. the application of Rhodotorula mucilaginose Rhodotorulamucilaginosa according to claim 1 in fermentative production carotenoid.
3. the application of Rhodotorula mucilaginose Rhodotorulamucilaginosa according to claim 2 in fermentative production carotenoid, it is characterized in that being realized by following step: described Rhodotorula mucilaginose Rhodotorulamucilaginosa is carried out slant culture and seed culture, obtains activated seed liquid;According to the inoculum size of volume percent 10%, seed liquor is inoculated in product fermentative carotenoid substratum, in 27-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, namely obtaining the fermented liquid containing carotenoid, wherein carbon source is glucose, 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 mucilaginose Rhodotorulamucilaginosa according to claim 2 in fermentative production carotenoid, it is characterized in that being realized by following step: described Rhodotorula mucilaginose Rhodotorulamucilaginosa is carried out slant culture and seed culture, obtains activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in product fermentative carotenoid substratum, in 27-30 DEG C, pH6.5-7.5, carbon-nitrogen ratio 25-35:1,190rpm shaking culture 96h, namely obtaining the fermented liquid containing carotenoid, wherein carbon source is glucose, 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. application in fermentative production carotenoid of Rhodotorula mucilaginose Rhodotorulamucilaginosa according to claim 3 or 4, it is characterized in that: described slant culture temperature 28 DEG C, 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. application in fermentative production carotenoid of Rhodotorula mucilaginose Rhodotorulamucilaginosa according to claim 3 or 4, it is characterized in that: described seed culture temperature 28 DEG C, 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 mucilaginose Rhodotorulamucilaginosa according to claim 1 in fermentative production grease.
8. the application of Rhodotorula mucilaginose Rhodotorulamucilaginosa according to claim 7 in fermentative production grease, it is characterized in that being realized by following step: described Rhodotorula mucilaginose Rhodotorulamucilaginosa is carried out slant culture and seed culture, obtains activated seed liquid; According to the inoculum size of volume percent 10%, seed liquor is inoculated in product oil fermentation substratum, in 26-30 DEG C, pH4.5-8.0, carbon-nitrogen ratio 30-130:1,190rpm shaking culture 96h, namely obtains the fermented liquid containing grease, wherein carbon source is glucose, described product oil fermentation medium component is counted with g/L: glucose 85.6, yeast leaching 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 mucilaginose Rhodotorulamucilaginosa according to claim 8 in fermentative production grease, it is characterized in that: described slant culture temperature 28 DEG C, 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 mucilaginose Rhodotorulamucilaginosa according to claim 8 in fermentative production grease, it is characterized in that: described seed culture temperature 28 DEG C, 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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| 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 |
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| 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 |
| CN111500475B (en) * | 2019-12-13 | 2022-06-03 | 燕山大学 | Rhodotorula mucilaginosa ZOR1 and method for producing mannan and carotenoid |
| CN114874940B (en) * | 2022-05-10 | 2023-07-14 | 吉林省农业科学院 | Composite microbial agent, fermented product thereof and application thereof |
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