CN101008000A - Rhodotorula mucilaginosa for producing beta-caroten, beta-caroten and its production method - Google Patents
Rhodotorula mucilaginosa for producing beta-caroten, beta-caroten and its production method Download PDFInfo
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Abstract
The invention relates to a viscous red torulin for beta- carotene production and the preparing method. The invention provides a viscous Rhodotorula mucilaginosa)CGMCC No.1536 for high- production of beta- carotene. The method comprises: activating viscous red torulin, fermentating for a certain of time, centrifuging and getting bacteria, disintegrating bacteria cell with thermal acid method, adding mixing liquid of petroleum ether and acetone with the proportion being 1: 1, oscillating at 28 Deg. C, extracting for one hour, and getting beta- carotene coarse extract. The invention is characterized by high productivity of beta- carotene, generally reaching 389 ug/ g, natural product of beta- carotene, no artificial colouring matter, and wide application of food and other industries.
Description
Invention field
The present invention relates to microorganism and microbial fermentation field.Specifically, the present invention relates to a kind of red torula bacterial strain, β-Hu Luobusu and production method thereof of producing β-Hu Luobusu.
Background technology
β-Hu Luobusu is a kind of important natural pigment that exists in the biologies such as plant, algae, fungi, but can not synthesize in human body and animal body, must take in from the external world.Think that at present β-Hu Luobusu has anti-oxidant, antitumor, the anti-ageing effect of waiting for a long time, as improving immunizing power to the AIDS patient.In addition, β-Hu Luobusu is the precursor of vitamin A, has the physiologically active of vitamin A, can treat the last somatocyte keratinization that causes owing to the A that is deficient in vitamin, xeropthalmus, nyctalopia etc.(Yang Ning, the progress of fermentative Production β-Hu Luobusu, food research and development, 2004,25 (3): 19-21.) β-Hu Luobusu is Food and Argriculture OrganizationFAO (Food and Agricultrure Organization ofthe United Nations), be called for short: FAO) and the World Health Organization (World HealthOrganization is called for short: WHO) the outstanding nourishing food additive of category-A (food additives) of foodstuff additive joint specialist council identification.Existing 52 countries in the whole world, area approval are used, and demand is more than 1000 tons in the world, and annual growth reaches 10～15%.Can be used as the reinforcer of oleomargarine, salad oil, sesame wet goods lipid food, to help the absorption of human body to β-Hu Luobusu.In addition; the extract of β-Hu Luobusu contains rich in amino acid, VITAMIN, natural moisturizing factor, trace element and other biologically active substance; add β-Hu Luobusu in daily cosmetics (lipstick, kermes etc.), the plentiful nature of color and luster can also play the effect of protection skin.It can also improve growth velocity and the meat quality of animal, improves the fecundity of ox, horse, pig, strengthens the color and luster of salmon, shrimp, deepens the color of birds, beasts and eggs.(Zhu Shiyong, the overview of β-Hu Luobusu and market outlook, grain and grease, 1998,1:20-24.)
In recent years, along with the needs and the progress of each side, the extraction of β-Hu Luobusu and preparation and the application in health care and medical field have obtained people's attention day by day.So research and development β-Hu Luobusu, particularly natural beta-carotin, health care has crucial meaning to human health to promote its application development.The demand of β-Hu Luobusu is anxious both at home and abroad increases, and locates the state that supply falls short of demand always.At present, the production of β-Hu Luobusu is based on synthesis method, and chemical synthesis is produced carotene technical sophistication, big, active low, the strong toxicity of difficulty, can not be absorbed by the body fully, and to human body generation toxic side effect to a certain degree, many countries have limited use.From natural material, extract β-Hu Luobusu, though have the report of lab scale aspect both at home and abroad, the present domestic exploitation commitment that still is in, there are shortcomings such as extraction output and purity are low in the suitability for industrialized production that always is unrealized.The year requirement of β-Hu Luobusu is 1200～1500t on the world market, and wherein 95% is synthetics, and the price of natural beta-carotin is the twice of synthetics, and domestic annual requirement is about 200t.Natural beta-carotin has enormous and latent market because of its Peak output and high profit.China's β-Hu Luobusu product is mainly from foreign procurement or buy raw material and carry out composite, need to strengthen the exploitation dynamics, make it to satisfy domestic growing natural pigment series products needs (Zhang Xiaobin Gao Yangzhe, the application of β-Hu Luobusu and progress, China's feed, 2003 (3): 22-24.).Can extract β-Hu Luobusu from plant, because of there being the cost height, price is expensive, and so weak points such as complex process and tinting strength difference are the Microbial resources that exploitation contains β-Hu Luobusu paid more and more attention.Microbe fermentation method utilizes microbial fermentation to produce β-Hu Luobusu, considers to be better than chemical synthesis and plant extract method from factors such as quality, technology, resource and costs, and will be the direction of Future Development.(virgin Hypon Cai Yang Xu is just big, the production method of β-Hu Luobusu, meticulous and specialty chemicals, 2005,15 (7): 1-6.)
Utilize microbial fermentation technology to produce natural beta-carotin, main good Blakeslea trispora and rhodotorula both at home and abroad.The U.S. adopts Blakeslea trispora both sexes strain fermentation to produce β-Hu Luobusu since nineteen sixty-five, and by the improvement to fermenting process, its fermentation production rate improves constantly, and has been in the industrialization development conceptual phase in recent years, and product is put on market.Domesticly utilize the research of trispore Bruce mould also by pilot scale.Chinese invention patent (number of patent application: CN98111199.8 as people such as Jiang Wen time application, publication number: CN1193048): a kind of method of producing beta-carotene by fermentation is the technology of cultivating filamentous fungus fermentative preparation β-Hu Luobusu with the trispore Bruce mould spore inoculating in airlift fermentor.Two Chinese invention patents of people such as virgin Hypon application: (number of patent application: 00116697.2, publication number: CN1331342A and number of patent application: CN00116698.0, publication number: CN1331343), two patents are that the positive and negative bacterial strain with trispore Bruce mould inserts and carries out in same the seeding tank that mixed seeds is cultivated and fermentation.Above technology is mainly concerned with the method for utilizing the trispore Bruce mould producing beta-carotene by fermentation and the product of acquisition thereof.But,, cause its proterties more easily to fail, and have characteristics such as fermentation technique complex process, fermentation period length, cost height because blakeslea trispora is the mode of reproduction of mould.Adopt the rhodotorula producing beta-carotene by fermentation, though the pigment fermentation level is not as the height of Blakeslea trispora at present, but advantage such as rhodotorula has that nutritional requirement is simple, growth cycle is short and thalline is nontoxic, nutritious has great practical value and DEVELOPMENT PROSPECT.The rhodotorula bacterial strain content beta-carotene of screening is all lower at present, and product is also promoted.
Summary of the invention
At not utilizing the red torula of viscosity (Rhodotorula mucilaginosa) to produce β-Hu Luobusu both at home and abroad, other rhodotorula of open report produces all lower present situation of productive rate of β-Hu Luobusu.The invention provides a kind of microbial strains, the β-Hu Luobusu of β-Hu Luobusu and method of producing this β-Hu Luobusu of producing.
The bacterial strain of β-Hu Luobusu is produced in a strain provided by the invention, separate, screen and cultivate by soil sample at gardens, Urumchi, Xinjiang black fallow, obtain a collection of microorganism strains, therefrom filter out a strain and be numbered the bacterial strain of XJU-1, thereby provide a kind of β-Hu Luobusu bacterial strain, it has the good characteristic of producing β-Hu Luobusu, identifies through microbiology, belongs to the red torula of viscosity (Rhodotorula mucilaginosa).
The present invention provides a kind of production method of β-Hu Luobusu on the basis of the red torula of viscosity (Rhodotorula mucilaginosa) that obtains.
Simultaneously, the present invention also provides a kind of β-Hu Luobusu, obtains through the concrete fermentation process that the present invention determines by utilizing bacterial strain of the present invention.
The invention provides a kind of red torula bacterial strain, called after XJU-1, it can produce β-Hu Luobusu with high yield.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Microbial Culture Preservation Commission common micro-organisms center (CGMCC).Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100080.Preservation date is on November 23rd, 2005, and preserving number is CGMCC.No1536.Be accredited as the red torula of viscosity (Rhodotorula mucilaginosa) through microbiology.This bacterial strain preferred growth is in the YPD media surface, under 4～45 ℃ of culture temperature and pH4.0～11.0 scopes, grow, alkaline-resisting heat resistance obviously is better than the rhodotorula bacterial strain reported, 100 power microscopes are observed down, the thalline ellipse, cell size (5.3～6.5) * (3.7～4.5) μ m budding, monolateral sprouting, on YPD substratum plate, bacterium colony is rounded, smooth surface, quality evenly, neat in edge, easily provoke.Assimilation glucose, maltose, sucrose, semi-lactosi, cellobiose, wood sugar do not assimilate lactose, melibiose, inositol.For further accurately identifying this bacterial strain, having carried out with the base sequence in D1/D2 district among the big subunit 26SrDNA and small subunit 5.8SrDNA and two internal transcribed spacer districts (ITS1 and ITS2) base sequence analysis to it is the chemotaxonomy research of foundation, according to the similarity of sibling species type strain D1/D2 regional sequence and 5.8S-ITS regional sequence relatively, the result shows with the red torula of viscosity (Rhodotorulamucilaginosa) CBS17 and reaches 100% similarity that we are with the red torula of its called after viscosity (Rhodotorula mucilaginosa) XJU-1.The GenBank number of registration of the D1/D2 zone base sequence of this bacterial strain and 5.8S-ITS zone base sequence is respectively DQ132885 and DQ132886.
The β-Hu Luobusu that this bacterial classification produces, optimal culture condition is pH8.0,28 ℃ of temperature.The present invention further sets up the system process technology of culture presevation, rejuvenation and seed selection.
The red torula bacterial classification of viscosity of the present invention, its bacteria characteristic is as shown in the table.Its 26SrDNAD1/D2 district and 5.8S-ITS regional sequence are entrusted Shanghai to give birth to worker's order-checking portion and are measured, and the red torula CBS17 of its result and viscosity (Rhodotorula mucilaginosa) homology reaches 100%.
The red torula XJU-1 of viscosity bacterial strain physio-biochemical characteristics
Generation bacterial classification as β-Hu Luobusu of the present invention; the bacterial strain that both can be the present invention and protected also can be the original strain of nature screening, or the dissociant that makes a variation by natural variation or artificial induction; by method of the present invention, all can realize technique effect set forth in the present invention.
Method of production as above-mentioned dissociant comprises physical mutagenesis, handles as various rays such as treatment with uv radiation, cobalt 60 radiation treatment, ion implantation processing, laser irradiation; Chemomorphosis is handled, and optimizes the bacterial strain of production performance excellence with conventional β-Hu Luobusu generation bacterium separation screening substratum and method.
In addition, also can pass through Protocols in Molecular Biology, from original strain or induce variation bacterial classification, obtain the β-Hu Luobusu gene, with former sclerotium microorganism, as intestinal bacteria, subtilis etc., eukaryotic microorganisms, as yeast etc., as the genetic recipient bacterium, make up genetically engineered and produce bacterial strain, also can be used as β-Hu Luobusu of the present invention and produce bacterial classification.
Produce bacterial classification XJU-1 as β-Hu Luobusu of the present invention, can adopt following method that it is preserved, activates and screens, and shake flask fermentation obtains β-Hu Luobusu of the present invention.
Preservation method goes down to posterity on β-Hu Luobusu generation bacterium XJU-1 employing of the present invention conventional inclined-plane, this method is in any slant culture primary surface that is suitable for the yeast growth with bacterial classification inoculation of the present invention, the present invention preferentially uses YPD substratum (glucose 20%, peptone 20%, yeast extract paste 10%), its culture condition is pH7.0-9.0, temperature 25-30 ℃.
The bacterial classification of long-term preservation activates as follows in use, screens.The bacterial classification inoculation of the present invention of long-term preservation is suitable for the substratum of yeast growth in YPD substratum or other, as wort solid medium and potato agar substratum etc., at pH7.0-9.0, under the temperature 25-30 ℃ of condition, carry out fermentation culture behind the solid slant culture 48h, obtain β-Hu Luobusu of the present invention.In the fermentation culture process, also the slant strains direct inoculation can be carried out fermentation culture in fermention medium.Definite through testing, glucose helps the generation of β-Hu Luobusu most in the carbon source commonly used, and its most suitable concentration is 2%; Peptone is an optimum nitrogen source of producing β-Hu Luobusu, and its optimal concentration is 2%; Na
+, Mn
2+, Zn
2+And Mg
2+Promote the generation of β-Hu Luobusu; Ca
2+The red torula of strongly inhibited viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 generates β-Hu Luobusu, but does not influence the growth of yeast cell.
Usually, can select following condition to cultivate.That is, culture temperature is scope 25-30 ℃, is preferably 28 ℃ scope; Incubation time 48h is as long as finish fermentation culture when the turnout of β-Hu Luobusu of the present invention reaches the highest; The pH of substratum can particularly be more suitable for the production of β-Hu Luobusu of the present invention in the production range of pH7.0-9.0 in the pH8.0 scope.Through aforesaid fermentation culture method, produce β-Hu Luobusu of the present invention.
The present invention provides a kind of method of producing β-Hu Luobusu simultaneously, comprises the steps: 1) strain culturing: with the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 the activation, the centrifugal thalline that obtains of fermentation culture certain hour; 2) adopt the broken somatic cells of acid heat method, add sherwood oil then: the mixed solution of acetone (1: 1), 28 ℃ of vibration lixiviate 1h obtain the β-Hu Luobusu crude extract.
From the nutrient solution of as above gained, extract β-Hu Luobusu, can be according to the method for routine, as organic solvent extraction etc., by microorganism collection, bacterial cell disruption, thalline drying, β-Hu Luobusu extraction, concentrate, crystallization and carrying out.
By the present invention's elaboration as above, obtain back embodiment and further verify, learn microbiological property of the present invention.
By implementing the concrete technical indicator of the present invention, realize content of the present invention, can reach following beneficial effect.
The productive rate of the red torula of viscosity of the present invention (Rhodotorula mucilaginosa) bacterial strain production β-Hu Luobusu is higher, generally can reach 389 μ g/g, the β-Hu Luobusu that obtains belongs to all-natural product, do not have artificial color potential harm, can be widely used in each field such as foodstuffs industry.Bacterial strain of the present invention has the growth cycle weak point, and product β-Hu Luobusu temperature and pH scope are wide, and the productive rate advantages of higher is convenient to suitability for industrialized production.
Description of drawings
Fig. 1: the red torula CGMCC of viscosity No.1536 produces the technical process of β-Hu Luobusu
Fig. 2: the red torula sample of β-Hu Luobusu standard substance (a) and viscosity (b) HPLC color atlas
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, then % refers to that all weight writes.
Embodiment
Embodiment 1: the isolation identification of producing the red torula of viscosity (Rhodotorulamucilaginosa) the CGMCC No.1536 of β-Hu Luobusu
1, sample collecting
Gather soil sample from gardens, Urumchi, Xinjiang black fallow, through technique means such as screening, optimizations, at suitable yeast culture primary surface, as select YPD substratum (glucose 20% for use, peptone 20%, yeast extract paste 10%) on the plate, 28 ℃ of separation obtain the red torula of viscosity (Rhodotorulamucilaginosa) XJU-1 bacterial strain.
2, strain identification
It has been carried out with the base sequence in D1/D2 district among the big subunit 26S rDNA and small subunit 5.8SrDNA and two internal transcribed spacer districts (ITS1 and ITS2) base sequence analysis is that the chemotaxonomy of foundation is identified, combining form is learned and is observed and physio-biochemical characteristics mensuration again, and it accurately is accredited as the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536.
Embodiment 2: the cultivation of the red torula of viscosity (Rhodotorulamucilaginosa) CGMCC No.1536 bacterial strain and the determining of zymotechnique of producing β-Hu Luobusu.
Select for use substratum as follows:
1. slant activation substratum: glucose 2%; Peptone 2%; Yeast powder 1%; PH9.0.
2. liquid seed culture medium: glucose 2%; Peptone 2%; Yeast powder 1%; PH8.5.
3. liquid fermentation medium: glucose 4%; Peptone 2%; Yeast powder 1%; PH8.0.
The present invention adopts conventional substratum, just pH is wanted particular requirement, and conventional fermentation is to carry out under the slant acidity environment, and bacterial strain of the present invention is a high yield β-Hu Luobusu under the meta-alkalescence condition.
Fermentation culture method (referring to accompanying drawing 1):
1. slant activation is cultivated: bacterial strain is received on the activated inclined plane substratum from the preservation inclined-plane, cultivated 48h for 28 ℃.
2. liquid seeds is cultivated: receive in the first order seed substratum, 24h is cultivated in 28 ℃ of vibrations (150r/min, down together).
3. liquid fermentation and culture: inoculum size 10%, 500mL triangular flask, liquid amount 100mL, 28 ℃ of oscillation and fermentation cultivation 36h.
The mensuration of cellular biomass: with fermented liquid centrifugal (3000rPmin) 10min, abandon supernatant liquor, precipitation washing back recentrifuge, the gained yeast slurry is weighed in 55 ℃ of oven dry, concentrates, aftertreatment such as crystallization obtains β-Hu Luobusu, and its productive rate reaches 389 μ g/g.
Embodiment 3: the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 strain fermentation is produced the technology of β-Hu Luobusu.
Repeat zymotechnique step among the embodiment 2, verify the concrete parameter in the zymotechnique repeatedly.With the bacterial classification inoculation of the present invention of long-term preservation in the potato agar substratum, at pH7.0,25 ℃ of temperature, hunting speed are under the 150r/min condition, carry out fermentation culture behind the solid slant culture 48.2h, with fermented liquid centrifugal (3000rPmin) 10min, abandon supernatant liquor, precipitation washing back recentrifuge, the oven dry of gained yeast slurry is weighed, concentrate, aftertreatment such as crystallization obtains β-Hu Luobusu, its productive rate reaches 368 μ g/g.
Embodiment 4: the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 strain fermentation is produced the technology of β-Hu Luobusu.
Repeat zymotechnique step among the embodiment 2, verify the concrete parameter in the zymotechnique repeatedly.With the bacterial classification inoculation of the present invention of long-term preservation in the wort solid medium, at pH7.0,25 ℃ of temperature, hunting speed are under the 150r/min condition, carry out fermentation culture behind the solid slant culture 52h, with fermented liquid centrifugal (3000rPmin) 10min, abandon supernatant liquor, precipitation washing back recentrifuge, the oven dry of gained yeast slurry is weighed, concentrate, aftertreatment such as crystallization obtains β-Hu Luobusu, its productive rate reaches 352 μ g/g.
Embodiment 5: the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 strain fermentation is produced the technology of β-Hu Luobusu.
Repeat zymotechnique step among the embodiment 2, verify the concrete parameter in the zymotechnique repeatedly.With the bacterial classification inoculation of the present invention of long-term preservation in the substratum that is suitable for the yeast growth, at pH8.5,27 ℃ of temperature, hunting speed are under the 150r/min condition, carry out fermentation culture behind the solid slant culture 52h, with fermented liquid centrifugal (3000rPmin) 10min, abandon supernatant liquor, precipitation washing back recentrifuge, the oven dry of gained yeast slurry is weighed, concentrate, aftertreatment such as crystallization obtains β-Hu Luobusu, its productive rate reaches 388 μ g/g.
Embodiment 6: the red torula of viscosity (Rhodotorula mucilaginosa) CGMCC No.1536 strain fermentation is produced the technology of β-Hu Luobusu.
Repeat zymotechnique step among the embodiment 2, verify the concrete parameter in the zymotechnique repeatedly.With the bacterial classification inoculation of the present invention of long-term preservation in the substratum that is suitable for the yeast growth, at pH9.0,30 ℃ of temperature, hunting speed are under the 150r/min condition, carry out fermentation culture behind the solid slant culture 42.1h, with fermented liquid centrifugal (3000rPmin) 10min, abandon supernatant liquor, precipitation washing back recentrifuge, the oven dry of gained yeast slurry is weighed, concentrate, aftertreatment such as crystallization obtains β-Hu Luobusu, its productive rate reaches 376 μ g/g.
Embodiment 7: the extraction of β-Hu Luobusu
The centrifugal somatic cells that obtains of nutrient solution 4000r/min that will contain the red torula of toughness (Rhodotorula mucilaginosa) CGMCC No.1536 bacterial strain, deionized water wash once adds 5mol/L hydrochloric acid behind the centrifugal removal moisture content, vibration 1h, boiling water bath 4min, chilling.Centrifugal removal hydrochloric acid, behind the deionized water wash cell, centrifugal again removal moisture content.Add sherwood oil: acetone (1: 1), vibration, centrifugal (10000r/min) gets supernatant liquor.40 ℃ of temperature, concentrate under the condition of vacuum tightness 0.1Mpa, till having the part crystal to separate out.Use the dehydrated alcohol crystallization of 10 times of volumes at last, temperature is controlled at 0-5 ℃, and crystallization time obtains the higher β-Hu Luobusu of purity about 12h.
Embodiment 8: the mensuration of β-Hu Luobusu
The measuring method of β-Hu Luobusu is with reference to the high phase liquid phase chromatography of GB/T 5009.83-2003 standard determination method.The mensuration of β-Hu Luobusu of the present invention is entrusted the test analysis center check of Xinjiang Academy of Agricultural Sciences, adopt 5g rhodotorula smudge cells extracting solution 50mL, detected result represents that with absolute magnitude β-Hu Luobusu is 1946 μ g, and the content that last calculation result gets β-Hu Luobusu is 389.2 μ g/g.Concrete experimental procedure is as follows:
Extract β-Hu Luobusu with sherwood oil 80ml and acetone 20ml mixed solution, through three oxidations and the aluminium column purification is qualitative with retention time then with high phase liquid chromatography for measuring, peak area quantification.
Reagent: sherwood oil (boiling range 30-60 ℃); Aluminium sesquioxide (it is standby that moisture eliminator is put in taking-up for 100-200 order, 140 ℃ of activation 2h); Methyl alcohol (chromatographically pure); Second eyeball (chromatographically pure); The β-Hu Luobusu standard substance adopt Sigma company to produce, and content is greater than 99%; All the other agents useful for same are analytical pure or the above rank of analytical pure.
The β-Hu Luobusu extracting solution is evaporated to dried (40 ℃ of bath temperatures) on rotatory evaporator.Use a small amount of petroleum ether dissolution, carry out alumina column chromatography then.Alumina column is 1.5cm (internal diameter) * 1cm (height), the first alumina column of washing with elutriant acetone 5ml and sherwood oil 95ml, and then the solution of adding dissolved samples extracting solution, with acetone 5ml and sherwood oil 95ml wash-out β-Hu Luobusu, the control flow velocity is 20/min, be collected in the 10ml volumetric flask, be settled to scale with elutriant.With 0.45 μ m filtering with microporous membrane, filtrate is made HPLC and is analyzed usefulness.
HPLC reference conditions: chromatographic column: Spherisord C18 post 4.6mm * 150mm; Moving phase: methyl alcohol 90ml and second eyeball 10ml; Flow velocity: 1.2ml/min; Wavelength; 448nm.
Typical curve: advance the β-Hu Luobusu standard and use liquid 20 μ l, carry out HPLC and analyze, β-Hu Luobusu concentration is made typical curve with peak area.
β-Hu Luobusu is measured: draw the β-Hu Luobusu solution 20 μ l of purifying, carry out HPLC and analyze, check in the amount of contained β-Hu Luobusu from typical curve.
The content that last calculation result gets β-Hu Luobusu is 389.2 μ g/g.The red torula sample of β-Hu Luobusu standard substance (a) and viscosity (b) HPLC color atlas is referring to accompanying drawing 2.
Make the mensuration of parallel sample simultaneously.
Embodiment 9: the thermal stability property of the β-Hu Luobusu that the present invention extracts detects
Get an amount of Beta Carotene Powder, be dissolved in sherwood oil (90～120 ℃ of boiling ranges), respectively get 15mL and place the 25mL colorimetric cylinder, in the water bath with thermostatic control of differing temps, be incubated then, respectively do three repetitions, timing sampling is cooled to room temperature with tap water, supply the absorbance of measuring 448nm behind the solvent, calculate the pigment survival rate.The result is as shown in the table.As can be seen from the table, β-Hu Luobusu is strong to thermostability, 90 ℃ of water-bath 3h rate of loss only about 19%.
Differing temps is to the influence of β-Hu Luobusu
| Temperature (℃) | Test event | t/h | |||
| 0 | 1 | 2 | 3 | ||
| 30 | Absorbancy | 0.674 | 0.674 | 0.674 | 0.673 |
| Survival rate (%) | 100 | 100 | 100 | 99.9 | |
| 50 | Absorbancy | 0.674 | 0.648 | 0.645 | 0.640 |
| Survival rate (%) | 100 | 96.1 | 95.7 | 95.1 | |
| 70 | Absorbancy | 0.674 | 0.598 | 0.598 | 0.597 |
| Survival rate (%) | 100 | 88.7 | 88.7 | 88.6 | |
| 90 | Absorbancy | 0.674 | 0.551 | 0.549 | 0.548 |
| Survival rate (%) | 100 | 81.8 | 81.5 | 81.3 | |
Embodiment 10: the ph stability Characteristics Detection of the β-Hu Luobusu that the present invention extracts
Get an amount of β-Hu Luobusu powder and be dissolved in the methyl alcohol, respectively get 10mL, go into 0.1%, 0.5%, 1%, 2% in respectively, 4% hydrochloric acid and KOH solution 10mL place in indoor dark place, and timing sampling repeats sample three times, measure absorbancy and change.The result is as shown in the table.As can be seen from the table, acid has strong destruction to β-Hu Luobusu, and concentration is high more, and the stability of class Radix Dauci Sativae is poor more.Alkali is little to the influence of β-Hu Luobusu, and, time lengthening, absorbancy changes also little.
The influence of acid base pair β-Hu Luobusu stability
| Reagent | Time t/h | Massfraction (%) | |||||
| 0.1 | 0.5 | 1 | 2 | 3 | 4 | ||
| HCl | 0 | 0.655 | 0.655 | 0.655 | 0.655 | 0.655 | 0.655 |
| 2 | 0.532 | 0.475 | 0.436 | 0.379 | 0.201 | 0.174 | |
| 24 | 0.158 | 0.129 | 0.124 | 0.096 | 0.040 | 0.032 | |
| NaOH | 0 | 0.655 | 0.655 | 0.655 | 0.655 | 0.655 | 0.655 |
| 2 | 0.653 | 0.639 | 0.628 | 0.592 | 0.565 | 0.478 | |
| 24 | 0.653 | 0.638 | 0.626 | 0.588 | 0.559 | 0.452 |
Embodiment 11: the light stabilising characteristic of the β-Hu Luobusu that the present invention extracts detects
The extracting solution of β-Hu Luobusu is dissolved in the sherwood oil, respectively gets the 25mL triplicate, place indoor dark place respectively, under room scattering light and the illumination of 40W fluorescent lamp, regularly measure absorbancy.The result is as shown in table 3: the red torula of viscosity (Rhodotorula mucilaginosa) XJU-1 β is relatively poor to the anti-high light of carotene as can be seen from following table, one week of 40W fluorescent lamp illumination, with a toll of about 40%, but if can be kept at the dark place, then comparatively desirable.
Light is to the influence of β-Hu Luobusu stability
| Illuminate condition and test event | Time t/d | |||
| 0 | 1 | 3 | 7 | |
| Indoor dark place | 0.674 | 0.674 | 0.669 | 0.664 |
| Survival rate (%) | 100 | 100 | 99.3 | 98.5 |
| Room scattering light | 0.674 | 0.671 | 0.658 | 0.603 |
| Survival rate (%) | 100 | 99.6 | 97.6 | 89.5 |
| Under the 40W fluorescent lamp | 0.674 | 0.633 | 0.567 | 0.422 |
| Survival rate (%) | 100 | 93.9 | 84.1 | 62.6 |
Embodiment 12: the selection of the nutritional factor of the β-Hu Luobusu that the present invention extracts
Through test of many times, glucose helps the generation of β-Hu Luobusu most in the carbon source commonly used, and its most suitable concentration is 2%; Peptone is an optimum nitrogen source of producing β-Hu Luobusu, and its optimal concentration is 2%; Na
+, Mn
2+, Zn
2+And Mg
2+Promote the generation of β-Hu Luobusu; Ca
2+The red torula of strongly inhibited viscosity (Rhodotorula mucilaginosa) XJU-1 generates β-Hu Luobusu, but does not influence the growth of yeast cell.
Embodiment 13: the determining of the optimum solvent of the β-Hu Luobusu that the present invention extracts
It is centrifugal to get fermented liquid 40mL, wash to fermented liquid with physiological saline colourless, with the acetone of equal volume, methyl alcohol, sherwood oil, ethyl acetate, acetone: sherwood oil (1: 1) soaks 1h respectively, measures OD 448nm, the result shows when adopting single solvent, acetone is best to the extraction effect of β-Hu Luobusu, and methyl alcohol and sherwood oil take second place, and ethyl acetate works hardly to extracting.(acetone: in the time of sherwood oil), extraction effect is apparently higher than single solvent, and with acetone: sherwood oil (1: 1) is as the optimum extraction solvent when adopting mixed solvent.
Claims (8)
1, a kind of red torula of viscosity (Rhodotorulamucilaginosa) CGMCC.No.1536 with generation β-Hu Luobusu ability.
2, a kind of production method of β-Hu Luobusu, it comprises,
A: CGMCC.No.1536 carries out the strain activation and culture step to the red torula of viscosity (Rhodotorula mucilaginosa);
B: the activated spawn of utilizing steps A to obtain is carried out fermentation step;
C: the bacterium liquid that utilizes steps A and B to obtain carries out the back extraction step.
3, production method as claimed in claim 2 is characterized in that the red torula of viscosity is carried out in the strain activation and culture step, and glucose is optimum carbon source, and its most suitable concentration is 2%, and peptone is an optimum nitrogen source, and its optimal concentration is 2%, Na
+, Mn
2+, Zn
2+And Mg
2+Promote the generation of β-Hu Luobusu; Ca
2+Suppress the red torula of viscosity and generate β-Hu Luobusu.
4, production method as claimed in claim 2 is characterized in that, in fermentation culture method, bacterial strain is received on the activated inclined plane substratum from the preservation inclined-plane, and at pH7.0-9.0, temperature 25-30 ℃, under the hunting speed 150r/min culture condition, fermentation culture 48h.
5, production method as claimed in claim 2 is characterized in that, the bacterium liquid that utilizes steps A and B to obtain carries out in the extraction step of back, adopt the broken somatic cells of acid heat method, add sherwood oil: the mixed solution of acetone (1: 1), 28 ℃ of vibration lixiviate 1h obtain the β-Hu Luobusu crude extract.
6, production method as claimed in claim 4 is characterized in that, in fermentation culture method, optimal culture condition is pH8.0,28 ℃ of temperature, hunting speed 150r/min.
7, a kind of β-Hu Luobusu, it utilizes the red torula of the described viscosity of claim 1 (Rhodotorula mucilaginosa) CGMCC.No.1536, is prepared from by the described production method of claim 2.
8, β-Hu Luobusu as claimed in claim 7 is characterized in that, β-Hu Luobusu is strong to thermostability, 90 ℃ of water-bath 3h rate of loss only 19%, and acid has strong destruction to it, and concentration is high more, and its stability is poor more, and alkali is to its not influence.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115716A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Sporidiobolus pararoseus bacterial strain and application thereof |
| KR20160133593A (en) * | 2015-05-12 | 2016-11-23 | 가천대학교 산학협력단 | Novel microorganism producing carotinoid |
| CN109868227A (en) * | 2019-03-22 | 2019-06-11 | 吉林农业大学 | A kind of method of producing beta-carotene by fermentation |
| CN113845456A (en) * | 2021-08-23 | 2021-12-28 | 山东微研生物科技有限公司 | Process and equipment for filtering and extracting beta-carotene fermentation liquor |
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2006
- 2006-01-25 CN CNB2006100084854A patent/CN100543128C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115716A (en) * | 2010-12-07 | 2011-07-06 | 江南大学 | Sporidiobolus pararoseus bacterial strain and application thereof |
| CN102115716B (en) * | 2010-12-07 | 2013-03-13 | 江南大学 | Sporidiobolus pararoseus bacterial strain and application thereof |
| KR20160133593A (en) * | 2015-05-12 | 2016-11-23 | 가천대학교 산학협력단 | Novel microorganism producing carotinoid |
| KR101718611B1 (en) * | 2015-05-12 | 2017-03-22 | 가천대학교 산학협력단 | Novel microorganism producing carotinoid |
| CN109868227A (en) * | 2019-03-22 | 2019-06-11 | 吉林农业大学 | A kind of method of producing beta-carotene by fermentation |
| CN109868227B (en) * | 2019-03-22 | 2021-09-24 | 吉林农业大学 | Method for producing beta-carotene by fermentation |
| CN113845456A (en) * | 2021-08-23 | 2021-12-28 | 山东微研生物科技有限公司 | Process and equipment for filtering and extracting beta-carotene fermentation liquor |
| CN113845456B (en) * | 2021-08-23 | 2023-05-09 | 山东微研生物科技有限公司 | Filtering extraction process and equipment for beta-carotene fermentation liquor |
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| CN100543128C (en) | 2009-09-23 |
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