CN103911297B - A kind of Rhodosporidium toruloides Y0 and application thereof - Google Patents
A kind of Rhodosporidium toruloides Y0 and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of Rhodosporidium toruloides Y0 and application thereof.Rhodosporidium toruloides (Rhodosporidium? toruloides) Y0 is preserved in China typical culture collection center (CCTCC) on December 29th, 2013, address: Wuhan University of Wuhan, China city, deposit number: CCTCC? No:M2013725.Rhodosporidium toruloides (Rhodosporidium of the present invention? toruloides) Y0 has abundant nutrition, can be applicable to produce the functional additive of bait of animal, can originate as main nutrient by marine culture wastewater simultaneously, when according to mass volume ratio add 10 ~ 14 ‰ sucrose as a supplement carbon source, not only grow in marine culture wastewater fast, thus reach and utilize marine culture wastewater, reduce the object that discharge of wastewater pollutes but also obtain high level aquatic animal additive of bait.
Description
Technical field:
The invention belongs to technical field of microbe application, be specifically related to a kind of Rhodosporidium toruloides Y0 and application thereof.
Background technology:
Yeast nutrition enriches, be of moderate size, a lot of biological excellent natural baits, yeast is also a class saprophytic microorganism simultaneously, many kinds of substance can be decomposed, utilize the livestock wastes such as the organism in aquaculture water, ammonia nitrogen, hydrogen sulfide, and form dominant population, effectively suppress other harmful microbe to grow, play the effect of biological control.In recent years along with the progress of aquaculture technology, the application of yeast in aquaculture is more and more extensive.
But the yeast can applying sea farming is in the market also considerably less, conventional yeast remains the bread yeast (Saccharomyces cerevisiae) coming from food brewing, this yeast coming from terrestial enviornment can not adapt to briny environment, therefore can not play the effect purified water, its impact for marine fishery growth of animal also lacks the report of science.Based on this kind of situation, be adapted to the unicellular yeast of ocean environment for marine aquaculture in the urgent need to separation.On the other hand, produce the foodstuffs material that the raw material of yeast mostly is the mankind or animal, cause production cost higher thus, the final yeast product price formed is also higher, limits its widespread use in aquaculture.Therefore the yeast production raw material exploring new cheapness is needed.
In addition, the strict restriction of breeding wastewater discharge is not synchronously carried out in the flourish and powerful expansion of China's sea farming, brings serious problem of environmental pollution thus.In today of social pay attention to day by day environment protection, development environment friendly aquaculture pattern, walking continuable aquaculture road for development becomes inexorable trend.At present, although livestock waste is separated by some cultivating wastewater purification methods from water body, effectively livestock waste can not be utilized.How to carry out breeding wastewater, the recycling of refuse is the problem that development environment friendly aquaculture pattern must solve.
Summary of the invention:
First object of the present invention is to provide a kind of ocean Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 providing a strain to have high nutritive value, efficiency utilization marine culture wastewater, this bacterium is preserved in China typical culture collection center (CCTCC) on December 29th, 2013, address: Wuhan University of Wuhan, China city, deposit number: CCTCCNo:M2013725.
Rhodosporidium toruloides of the present invention (Rhodosporidium toruloides) Y0 is located away from Zhanjiang prawn culturing field sewage draining exit.Yeast isolation substratum is YPD yeast special culture media.Rhodosporidium toruloides screening culture medium is prawn culturing waste media.The object of this screening obtains the yeast specie that can grow in prawn culturing waste media, and the yeast of separation will have the ability utilizing livestock waste under low sugar or sugar-free condition.
The colony morphology characteristic of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 and cell morphological characteristic: it 24 hours is white that bacterium colony is cultivated on YPD culture medium flat plate, it is light red after 72 hours, the circular projection of bacterium colony, smooth surface, quality thickness, butteriness, edge is comparatively neat, without wine flavour.Spherical or the elliposoidal of cellular form, single, see sprout (as shown in Figure 1).In the prawn culturing waste media of Rhodosporidium toruloides growth conditions and YPD substratum similar.
The DNA that test kit (purchased from Takara, China) extracts Y0 bacterial strain is extracted with Yeast genome.Pcr amplification the 18S rDNA that checks order, 26S rDNA, ITS rDNA fragment, its 18S rDNA, 26S rDNA, ITS rDNA sequence are respectively as shown in SEQ ID NO.1 ~ 3, show through blast comparison and Phylogenetic Analysis: recently, it is one that the supporting rate with 100% is gathered for the 18S rDNA sequence of Y0 bacterial strain and Rhodosporidium toruloides sequence relation.The yeast reaching 99% with the 26S rDNA of Y0 bacterial strain, ITS rDNA highest similarity only has Rhodosporidium toruloides, all with 100% supporting rate to gather be one.Draw according to above polygene homologous sequence result and Phylogenetic Analysis result, Y0 bacterial strain belongs to the new strains of Rhodosporidium toruloides (Rhodosporidium toruloides), called after Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, this bacterial classification is preserved in China typical culture collection center (CCTCC) on December 29th, 2013, address: Wuhan University of Wuhan, China city, deposit number: CCTCC No:M2013725.
Through measuring Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, there is abundant nutrition, therefore, second object of the present invention is to provide the application of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in the functional additive of bait producing aquatic animal.
Through sucrose gradient test, determine that in breeding wastewater, cultivate Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 reaches stationary phase in 48h.Add suitable amount of sucrose and significantly can increase cell concn, the optimum addition of sucrose is 14gL
-1, cell concn 6.1 × 10
8individual ml
-1.
3rd object of the present invention is to provide the application of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in marine culture wastewater disposal of resources.
First Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 is incubated in YPD substratum, forms seed liquor.Again seed liquor is added in concentrated and sterile-processed marine culture wastewater with the ratio of 2 ~ 5% (V/V), for the feature that carbon source in marine culture wastewater lacks relatively, add the sucrose of 10 ~ 14 ‰ as carbon source according to mass volume ratio.Room temperature was cultivated through 48 ~ 72 hours, more after filtration, substep sedimentation, the red winter spore yeast-raised products of circle can be obtained, thus reach breeding wastewater higher value application object.
Rhodosporidium toruloides of the present invention (Rhodosporidium toruloides) Y0 is preserved in China typical culture collection center (CCTCC) on December 29th, 2013, address: Wuhan University of Wuhan, China city, deposit number: CCTCC No:M2013725.
accompanying drawing illustrates:
Fig. 1 is that Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 cultivates and cellular form figure.Wherein, a: Rhodosporidium toruloides (Rhodosporidium toruloides) the Y0 colonial morphology on YPD flat board; B: Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 cellular form, the spherical or elliposoidal of cell, gemmation, scale=10 μm; C: Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 bacterium liquid is orange red; D: the colonial morphology of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 bacterial strain on prawn culturing refuse flat board;
Fig. 2 is the growth curve of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in YPD liquid nutrient medium;
Fig. 3 is that the growth of Rhodosporidium toruloides under different sucrose (Rhodosporidium toruloides) Y0 in wastewater medium is bent.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.Method used and technology are conventional method and technology if no special instructions.
Embodiment 1:
One, the separation of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, screening and qualification.
(1), the separation of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, screening
Get Zhanjiang prawn culturing field water port mud 1g, add sterilizing seawater 10ml, get 200 μ L after mixing and be coated with yeast isolation substratum (YPD) flat board, YPD is yeast special culture media, preparation method is: glucose (independent sterilizing) 20g, peptone 20g, yeast extract 10g, seawater 1L, after sterilizing, cooling adds the paraxin of 1ml50mg/ml.Cultivate after 48 hours for 30 DEG C, picking growth bacterium colony, purifying is also preserved, and obtains isolate.
Be incubated at by above-mentioned isolate in liquid YPD, get 1 μ L dibbling respectively in yeast screening culture medium, this screening culture medium is that prawn culturing waste media is dull and stereotyped, preparation method is: shrimp argol powder 20g, agar powder 20g, seawater 1L, slightly cold after sterilizing, add the paraxin of 1ml50mg/ml.The object of this screening obtains the yeast specie that can grow in prawn culturing waste media, and the yeast of separation will have the ability utilizing livestock waste under low sugar or sugar-free condition.Cultivate after 72 hours for 30 DEG C, record growth and bacterium colony is ruddy bacterial strain, choose wherein that a strain growth is fast, bacterium colony is large, color is that ruddy bacterial strain is recorded as Y0.
(2), the heterogeneous Molecular Identification of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0
Adopt the primer being anchored to fungi 18S rDNA, 26S rDNA, ITS rDNA gene fragment, carry out pcr amplification, and PCR primer is checked order.Primer sequence is as shown in table 1.
The title of table 1. primer and sequence
Bacterial strain Y018S rDNA, 26S rDNA, ITS rDNA sequence are respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.Show through blast comparison and Phylogenetic Analysis: recently, it is one that the supporting rate with 100% is gathered for the 18S rDNA sequence of Y0 bacterial strain and Rhodosporidium toruloides sequence relation.The yeast reaching 99% with the 26S rDNA of Y0 bacterial strain, ITS rDNA highest similarity only has Rhodosporidium toruloides, all with 100% supporting rate to gather be one.Draw according to above polygene homologous sequence result and Phylogenetic Analysis result, Y0 bacterial strain belongs to the new strains of Rhodosporidium toruloides (Rhodosporidium toruloides), called after Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, this bacterial classification is preserved in China typical culture collection center (CCTCC) on December 29th, 2013, address: Wuhan University of Wuhan, China city, deposit number: CCTCC No:M2013725.
Two, the Analysis of Nutritional of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0
Kjeldahl determination (GB/T6432-94) protein content to Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 bacterial strain is adopted to measure, be crude protein content be 32.55% when cultivating 2d, when cultivating 5d, crude protein content reaches 44.10%, to illustrate after stationary phase in Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 cell still at cumulative protein.Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 protein content is higher than the content (29%) being generally used for the middle protein of bread yeast (Saccharomyces cerevisiae) that single cell protein is produced.
Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 bacterial strain amino acid composition is measured with liquid phase chromatography (GB/T18246-2000).Its amino acid composition is in table 2, and result shows: total hydrolysis amino acid accounts for 27.3% of dry cell weight, wherein must account for 50.8% of total amino acid by amino acid.Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 is similar to the amino acid compositional model of Stichopus variegatus (Sempen) (Stichopus herrmanni), Environment of Litopenaeus vannamei Low (Litopenaeus vannamei), amino acid composition calculating biological value (BV) of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 to Stichopus variegatus (Sempen) and Environment of Litopenaeus vannamei Low is respectively 83.6 and 83.75, and this result shows that Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 has good application potential as aquatic feed.
The red winter spore yeast Y0 bacterial strain amino acid of table 2. circle forms and compares with aquatic living things
aten kinds in aquatic animal necessary amino acid;
bnecessary amino acid whose summation;
cthe aminoacids content reference (Wen etc., 2010) of Stichopus variegatus (Sempen);
dthe aminoacids content reference (Tacon etc., 2002) of Environment of Litopenaeus vannamei Low.
Measure Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 lipid acid composition with vapor-phase chromatography (GB/T17377-2008), the results are shown in Table 3.From table, Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 oleic acid (C18:1) content is the highest, accounts for 54.55% of total fatty acids; Next is palmitinic acid (C16:0), accounts for 18.90% of total fatty acids.Total unsaturated fatty acid content is respectively up to 61.63%, and the lipid acid proportion of composing of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 is suitable as can be seen here, has higher nutritive value.
Lipid acid composition (accounting for the per-cent of total fatty acids) of the red winter spore yeast Y0 of table 3. circle
athe summation of saturated fatty acid;
bthe summation of monounsaturated fatty acids;
cthe summation of polyunsaturated fatty acid;
dthe summation of unsaturated fatty acids; N.D. do not detect.
Three, the growth curve of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in breeding wastewater substratum and the suitableeest sugared addition are determined
Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 YPD substratum growth curve as shown in Figure 2.The growth curve of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in the wastewater medium containing different sucrose as shown in Figure 3.As seen from the figure: the growth curve of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in wastewater medium is identical in YPD substratum general trend with it, and lag phase is not observed, and reaches stationary phase during 48h.Statistical study shows: when utilizing wastewater medium to cultivate Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, the optimum addition of sucrose is 14gL
-1, under this sucrose concentration, after cultivating 48h, the cell concn of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 reaches 6.06 × 10
8individual ml
-1, higher than inoculum density 53.6 times.Record, when sucrose concentration is 14gL after experiment terminates
-1time, yeast raw product 16.04gL can be obtained in wastewater medium
-1.During 48h, the cell concn of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in YPD substratum is 2.33 × 10
8cfuml
-1, higher than inoculum density 25.2 times.Cultivate the cell concn of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in wastewater medium after 48h and can reach its 2.6 times of cell concn in YPD substratum.Therefore, compared with YPD commercially available culture medium, the breeding wastewater substratum added after sucrose demonstrates more excellent culture effect.
Four, the application of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 in marine culture wastewater disposal of resources
Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 is inoculated in YPD substratum, and 30 DEG C of incubated overnight, as seed culture fluid.From the concentrated breeding wastewater that industrial circulating water marine fish culture systematic position is collected, focus on recycling treatment pond, pass into ozone disinfection, after detoxicating process, place 24 hours, period stirs Chi Shui repeatedly, to help into water body aeration.With 2%(V/V) ratio add seed culture fluid, then add the sucrose of 10 ‰ according to mass volume ratio, fully stir mixing, room temperature is fermented 72 hours, and period repeatedly stirs as far as possible.
After fermentation ends, stirring Chi Shui, extracting Chi Shui out respectively by binding round the cylinder mould having 10 orders, 20 orders, 40 mesh sieve thin,tough silk with water pump.Individual relative bacterium due to Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 wants large, water body after filtration was through natural subsidence 8 hours, Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 natural subsidence is to container bottom, absorb upper ocean water, the light red lotion of lower floor's thickness is the tunning of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0.
The tunning of this Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 can be directly used in aquatic animal of throwing something and feeding, and also can be packaged into merchandise sales after drying further.
Embodiment 2:
Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 is inoculated in YPD substratum, and 30 DEG C of incubated overnight, as seed culture fluid.The waste water that prawn intensive culture CS central drainage is discharged, focuses on recycling treatment pond, until natural subsidence after 24 hours, absorbs the unnecessary clear water in upper strata, only leave a small amount of sewage of bottom and residual bait ight soil with water pump.Pass into ozone disinfection, after detoxicating process, place 24 hours, period stirs Chi Shui repeatedly, to help into water body aeration.With 5%(V/V) ratio add seed culture fluid, then add the sucrose of 14 ‰ according to mass volume ratio, fully stir mixing, room temperature is fermented 48 hours, and period repeatedly stirs as far as possible.
After fermentation ends, stirring Chi Shui, extracting Chi Shui respectively out respectively by binding round the cylinder mould having 10 orders, 20 orders, 40 mesh sieve thin,tough silk with water pump.Individual relative bacterium due to Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 wants large, water body after filtration was through natural subsidence 8 hours, Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 natural subsidence is to container bottom, absorb upper ocean water, the white lotion of lower floor's thickness is the tunning of Rhodosporidium toruloides (Rhodosporidium toruloides) Y0.
The tunning of this Rhodosporidium toruloides (Rhodosporidium toruloides) Y0 can be directly used in aquatic animal of throwing something and feeding, and also can be packaged into merchandise sales after drying further.
Claims (4)
1. Rhodosporidium toruloides (Rhodosporidium toruloides) Y0, its deposit number: CCTCC No:M2013725.
2. the application of Rhodosporidium toruloides according to claim 1 (Rhodosporidium toruloides) Y0 in the functional additive of bait producing aquatic animal.
3. the application of Rhodosporidium toruloides according to claim 1 (Rhodosporidium toruloides) Y0 in marine culture wastewater disposal of resources.
4. the application of Rhodosporidium toruloides according to claim 3 (Rhodosporidium toruloides) Y0 in marine culture wastewater disposal of resources, and be characterised in that, concrete grammar is: be first incubated in YPD substratum by Rhodosporidium toruloides (Rhodosporidiumtoruloides) Y0, form seed liquor, again by seed liquor with 2 ~ 5%(V/V) ratio add in concentrated and sterile-processed marine culture wastewater, the sucrose of 10 ~ 14 ‰ is added as carbon source according to mass volume ratio, room temperature was cultivated through 48 ~ 72 hours, again after filtration, substep sedimentation, the red winter spore yeast-raised products of circle can be obtained.
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| CN111662833B (en) * | 2020-06-12 | 2021-10-26 | 中国科学院南海海洋研究所 | Bottom-settling rhodosporidium toruloides microbial preparation, preparation method and application thereof |
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