CN107021894A - Arctic Sea fuchsin coccus B7740 produces the isolation and purification method of isoprenoid - Google Patents
Arctic Sea fuchsin coccus B7740 produces the isolation and purification method of isoprenoid Download PDFInfo
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- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
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Abstract
The invention discloses the isolation and purification method that Arctic Sea fuchsin coccus B7740 produces isoprenoid, this method isolates and purifies Arctic Sea fuchsin coccus B7740 production isoprenoids using high speed adverse current chromatogram.This method produces isoprenoid material to Rhodococcus sp B7740 using high-speed counter-current chromatograph and carries out efficiently separating purifying, obtains three kinds of rare marine source carotenoid, and three kinds of rare marine source carotenoid are further identified with high-precision mass spectrum.
Description
Technical field
The invention belongs to the technical field that Rhodococcus sp B7740 production isoprenoids are isolated and purified, and in particular to arctic ocean
Rhodococcus sp B7740 produces the isolation and purification method of isoprenoid.
Background technology
Rhod is the gram-positive bacteria that a class is distributed widely in nature, in soil, bottom sediment and food grass
Rich content in animal wastes, and it is most for battalion's saprogenesis.Rhod (Rhodococcus sp.) be 1891 by
What Zoof was set up, from setting up so far, classification position is not known always.At present, Rhodococcus sp belongs to actinomyces door
(Actinobacteria), Actinomycetes (Actinobacteria), actinomyces subclass (Actinobacteridae), actinomyces
Mesh (Actinomycetales), bar bacterium suborder (Croynebacterineae), Nocardiaceae (Nocardiaceae) is red
Coccus (Rhodococcus).
At present, the research both at home and abroad to rhodococcus erythropolis is more, is concentrated mainly on the research of its biodegradability and molecule
Research is learned, and isoprenoid material is produced to Rhodococcus sp and studies less, class especially is produced to the Rhodococcus sp B7740 that polar ocean is originated
The identification of isoprene material is still very deficient with studying, and Rhodococcus sp B7740 is arctic scientific expedition of China in third time arctic section
Found when examining from the surface seawater of 20 meters of B77 websites, not yet have any document report for the purifying of the bacterium institute product matter.It is red
The unique metabolic pathway of the coccus B7740 production carotenoids procatarxis microorganism, compared with common higher plant carrys out source carotenoid,
Structure difference is larger, and structure species are not single, and its unique structure not give only its unique activity, also increases its purifying
Difficulty.
The content of the invention
To solve the problem of above-mentioned prior art is present, class isoamyl is produced the invention provides Arctic Sea fuchsin coccus B7740
The isolation and purification method of diene, the separation method is easy to operate, and isoprenoid material can be produced to Rhodococcus sp B7740 and is entered
Row efficiently separates purifying.
Realize technical scheme that above-mentioned purpose of the present invention used for：
Arctic Sea fuchsin coccus B7740 produces the isolation and purification method of isoprenoid, is separated using high speed adverse current chromatogram pure
Change Arctic Sea fuchsin coccus B7740 production isoprenoids.
Arctic Sea fuchsin coccus B7740 produces the isolation and purification method of isoprenoid, comprises the following steps：
1st, it is 10 by the volume ratio of n-hexane, acetonitrile and dichloromethane:6-8:2-4 prepares dicyandiamide solution, fully shakes up, treats
Dicyandiamide solution starts layering, and stratification is simultaneously separated, and obtains upper liquid and subnatant, upper liquid is used as stationary phase, subnatant conduct
Mobile phase；
2nd, isoprenoid extract solution section bottom liquid is dissolved, is configured to 100-1000ug/ml sample solutions；
3rd, water bath with thermostatic control is opened, is 18-22 DEG C by temperature setting；
4th, with the spiral column jecket in washes of absolute alcohol pump；
5th, it is pumped into stationary phase；
6th, two phase solvent system is balanced：
UV-detector is opened, waits after the completion of preheating, wavelength is set to 450nm；Rotate forward and rotate main frame, while with
10ml/min flow pumps enter mobile phase, device port of export outflow mobile phase to be detected and when UV signal stablizes constant, then solvent body
System is in a basic balance；
7th, sample introduction；
8th, flow point is received.
Further, the volume ratio of n-hexane, acetonitrile and dichloromethane is 10:8:2、10:6.5:3.5、10:7:3 or 10:
6.75:3.25。
Compared with prior art, the advantages of the present invention are：
The present invention produces isoprenoid material using TBE-300C high-speed counter-current chromatographs to Rhodococcus sp B7740 to be had
Isolating and purifying for effect, obtains three kinds of rare marine source carotenoid.The rare carotenoid of this three kinds of arctic marine sources,
With the entirely different end group of source carotenoid is carried out with common higher plant, three Carotenoids are identified as aromatics recklessly
Radish element, unique structure assigns its unique activity, with applied to food, medicine, cosmetics value, and because its is only
Special natural origin, market favor is more susceptible to than artificial synthesized carotenoid.
Figure of description
Fig. 1 is (n-hexane, acetonitrile and the dichloro in dicyandiamide solution of Rhodococcus sp B7740 productions isoprenoid material in embodiment 1
Methane volumetric ratios are 10:8:When 2) HSCCC chromatograms.
Fig. 2 is (n-hexane, acetonitrile and the dichloro in dicyandiamide solution of Rhodococcus sp B7740 productions isoprenoid material in embodiment 1
Methane volumetric ratios are 10:6.5:When 3.5) HSCCC chromatograms.
Fig. 3 is (n-hexane, acetonitrile and the dichloro in dicyandiamide solution of Rhodococcus sp B7740 productions isoprenoid material in embodiment 1
Methane volumetric ratios are 10:7:When 3) HSCCC chromatograms.
Fig. 4 is (n-hexane, acetonitrile and the dichloro in dicyandiamide solution of Rhodococcus sp B7740 productions isoprenoid material in embodiment 1
Methane volumetric ratios are 10:6.75:When 3.25) HSCCC chromatograms.
Fig. 5 is the high-precision mass spectrogram of separation product one in embodiment 1.
Fig. 6 is the high-precision mass spectrogram of separation product two in embodiment 1.
Fig. 7 is the high-precision mass spectrogram of separation product three in embodiment 1.
Embodiment
With reference to embodiment, the present invention is described in detail.
Arctic Sea fuchsin coccus B7740 is inventor during the 7-9 months in 2008 Chinese third time Chinese Arctic Research Expeditions,
With Seabird911Plus CTD systems from Sea Surface deep Arctic Ocean B77 websites (146 ° of 49.28 ' W, 76 ° of 58.08 ' N) 25m
Isolated in water sample, Ningbo City microorganism is compared online with environmental project key lab by 16S rDNA sequence Bs last
It is right, judge the bacterium as Rhod.
Embodiment 1
Arctic Sea fuchsin coccus B7740 production isoprenoid isolates and purifies experiment
1st, experiment material
1.1st, experiment reagent
Lysozyme (20000U/mg) is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, zinc acetate, sodium chloride, methanol and two
Chloromethanes is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and n-hexane, dichloromethane are purchased from the limited public affairs of Chinese medicines group chemical reagent
Department；Hplc grade methanol, acetonitrile are purchased from silent winged scientific and technological (China) Co., Ltd of generation that of match；Chromatographic grade methyl tertiary butyl ether(MTBE) is purchased from Shanghai
Aladdin biochemical technology Ji Gu Co., Ltds.
1.2nd, laboratory apparatus
TBE-300C high-speed counter-current chromatographs are purchased from Shanghai with field bio tech ltd；2695 high performance liquid chromatographs
Purchased from Waters, US；LTQ-Orbitrap Elite mass spectrographs are purchased from silent winged scientific and technological (U.S.) Co., Ltd of generation that of match.
1.3rd, experimental raw
According to Chinese patent (a kind of microcapsule preparation method of Rhodococcus sp B7740 carotenoid, 201510016005.8)
Disclosed method prepares isoprenoid extract solution：
The lyophilized bacterium powders (Wanli College, Zhejiang's offer) of 0.8g Arctic Sea fuchsin coccuses B7740 are weighed to be placed in centrifuge tube, plus
Enter 16ml lysozyme solns (1mg/ml), be placed in 37 DEG C of water-baths, avoid light place 1h, then add 24ml saturated acetic acid zinc solutions
The Rhodococcus sp of broken wall is settled, by the centrifuge tube in centrifuging 10min at 7000rpm, 4 DEG C, supernatant is abandoned, then 64ml is mixed
Close organic solvent (methanol：Dichloromethane=4:1, volume ratio) it is added in Rhodococcus sp precipitation, stirred with glass bar, from
The heart, isolates supernatant, continues to add mixed organic solvents (methanol into centrifuge tube：Dichloromethane=4:1, volume ratio), such as
This is extracted 2-3 times repeatedly, is merged supernatant, is obtained isoprenoid extract solution.
2nd, experimental method：
2.1st, it is 10 by the volume ratio of n-hexane, acetonitrile and dichloromethane with 1000ml separatory funnels:8:2 prepare 2L solvents
System, fully vibration are shaken up, and treat that dicyandiamide solution starts layering, and 15min is stood under room temperature (25 DEG C) and is layered, separates, obtains
Upper liquid (stationary phase) and subnatant (mobile phase), upper liquid and subnatant are respectively put into blue lid bottle, ultrasound exhaust
20min, it is standby；
2.2nd, isoprenoid extract solution 20ml subnatants are dissolved, is configured to 300ug/ml sample solutions；
2.3rd, water bath with thermostatic control is opened, is 20 DEG C by temperature setting；
2.4th, with the spiral column jecket in washes of absolute alcohol pump：
Absolute ethyl alcohol is pumped into 50ml/min flow velocitys, is pumped into after 1min and stops, then opens air pump, 5min is vented, by spiral
Residual liquid drying in column jecket；Repeat this process 3 times (last time exhaust 1h)；
2.5th, it is pumped into stationary phase：
Stationary phase is pumped into 50ml/min flow velocitys, during device port of export outflow about 30-50ml stationary phases to be detected, termination of pumping；
2.6th, two phase solvent system is balanced：
UV-detector is opened, waits after the completion of preheating, wavelength is set to 450nm；Rotating forward rotation main frame (REV is reversion,
FWD is rotating forward), rotating speed is 800r/min, while entering mobile phase, device port of export outflow flowing to be detected with 10ml/min flow pumps
Mutually and UV signal is when stablizing constant, then dicyandiamide solution is in a basic balance；
2.7th, sample introduction；
Sample introduction six-way valve is switched to load, sample solution 20ml is poured into the syringe of loading sample column, is pushed away after exhaust bubble,
Sample liquid is drawn into sample introduction circle, inject is switched to untill sample liquid residue 1ml, then by load, and by detector, work
Stand zeroing, record again；
2.8th, flow point is received：
Work station start recording is treated, flow point is collected by the order of the signal of chromatogram appearance；
2.9th, HSCCC instruments are cleaned：
The connection of main frame and pump is disconnected, main frame is closed, is pumped into absolute ethyl alcohol with 50ml/min flow velocitys, borded pile is rinsed
Pipe, is pumped into after 1min and stops, then the tracheae of the import of main frame and air pump is connected, and is opened air pump, 5min is vented, by spiral column jecket
In residual liquid drying；Repeat this process 3 times (last time exhaust 30min)；
2.10th, each shunting of collection is spin-dried for Rotary Evaporators, then redissolved with methyl tertiary butyl ether(MTBE), dried up through nitrogen
Afterwards, each flow point sample to be measured is obtained, refrigerator negative one layer is positioned over and carries out low temperature, lucifuge storage；
2.11st, repeat step 2.1-2.10 tri- times, repeat only to change the volume of n-hexane, acetonitrile and dichloromethane every time
Than the volume ratio of n-hexane, acetonitrile and dichloromethane is changed into 10 respectively:6.5:3.5、10:7:3 or 10:6.75:3.25, other
Operation is constant.
3rd, one-level is detected：
Each testing sample obtained by each operation is detected with high performance liquid chromatography and UV absorption：
High performance liquid chromatography detection condition is as follows：
Chromatographic column YMCC30 (5um × 4.6mm × 150mm), 25 DEG C of column temperature, ultraviolet absorption detector, Detection wavelength
450nm, sample size 10uL；
Mobile phase：Mobile phase A is methanol, and B is methyl tertiary butyl ether(MTBE), flow velocity：1ml/min.
Elution program：
First stage：30 minutes, the wherein volume basis of mobile phase A are eluted using the mixed liquor of mobile phase A and Mobile phase B
Content is changed into 70% from 95, and the volumn concentration of Mobile phase B is changed into 30% from 5；
Second stage：20 minutes, the wherein volume basis of mobile phase A are eluted using the mixed liquor of mobile phase A and Mobile phase B
Content is changed into 50% from 70, and the volumn concentration of Mobile phase B is changed into 50% from 30；
Phase III：10 minutes, the wherein volume basis of mobile phase A are eluted using the mixed liquor of mobile phase A and Mobile phase B
Content is changed into 95% from 50, and the volumn concentration of Mobile phase B is changed into 5% from 50.
4th, one-level experimental result：
4.1st, n-hexane, acetonitrile and methylene chloride volume ratio are 10 in dicyandiamide solution:8:Separating effect when 2
Dicyandiamide solution is in n-hexane：Acetonitrile：The volume ratio of dichloromethane is 10:8:Under conditions of 2, isoprenoid is extracted
The HSCCC chromatograms of liquid are as shown in figure 1, as shown in Figure 1, occur in that three peaks, three peaks are respectively peak one, peak by peak sequence
Two and peak three.
The HPLC and UV absorbance detection result of flow point sample to be measured corresponding to peak one, peak two and peak three are as shown in table 1：
The isoprenoid extract solution of table 1 n-hexane, acetonitrile and methylene chloride volume ratio in dicyandiamide solution are 10:8:When 2
The HPLC and UV absorbance detection result of separation product
As it can be seen from table 1 the separating effect at peak one is bad, containing three kinds of main components, although the separating effect at peak two
Better, purity has reached 80.33%, but miscellaneous peak is too many, and the separating effect at peak three is best, high purity 95.21%, and it is maximum
UV absorption wavelength is 452nm.
4.2nd, n-hexane, acetonitrile and methylene chloride volume ratio are 10 in dicyandiamide solution:6.5:Separating effect when 3.5
Dicyandiamide solution is in n-hexane：Acetonitrile：The volume ratio of dichloromethane is 10:6.5:Under conditions of 3.5, isoprenoid
The HSCCC chromatograms of extract solution are as shown in Fig. 2 as shown in Figure 2, occur in that eight peaks, eight peaks are respectively peak by peak sequence
First, peak two, peak three, peak four, peak five, peak six, peak seven and peak eight.
The HPLC and UV absorbance detection result of flow point sample to be measured corresponding to one-peak of peak eight are as shown in table 2：
The isoprenoid extract solution of table 2 n-hexane, acetonitrile and methylene chloride volume ratio in dicyandiamide solution are 10:6.5:
The HPLC of separation product and UV absorbance detection result when 3.5
| HSCCC separation products | HPLC appearance times | HPLC goes out peak area | HPLC goes out peak area % | UV absorption λ max |
| Peak one | Disorderly, peak is miscellaneous | _ | _ | |
| Peak two | Disorderly, peak is miscellaneous | _ | _ | |
| Peak three | 22.0 | 361307 | 81.62 | 274,452,468 |
| Peak four | 26.8 | 1791658 | 56.79 | 434,458,484 |
| Peak five | 32.2 | 4562003 | 93.41 | 282,452,476 |
| Peak six | 29.5 | 831778 | 93.92 | 284,434,460,486 |
| Peak seven | Disorderly, peak is miscellaneous | _ | _ | |
| Peak eight | 24.1 | 2537273 | 80.36 | 250,274 |
As shown in Table 2, the separating effect at peak one, peak two and peak seven is very poor, is miscellaneous peak entirely, and the purity at peak three reaches
81.62%, but also have some miscellaneous peaks, as separating effect is general, the separating effect at peak four is also bad, and purity only has 56.79%, miscellaneous
Peak is too many, and the separating effect at peak five is very good, and purity has reached 93.41%, and its uv-absorption maximum wavelength is 452nm, peak six
Purity although reached 93.41%, there is conditions of streaking, it is but single without occurring although the purity of peak eight has reached 80.36%
Peak, two peaks have been connected in together, and separating effect is also general.
4.3rd, n-hexane, acetonitrile and methylene chloride volume ratio are 10 in dicyandiamide solution:7:Separating effect when 3
Dicyandiamide solution is in n-hexane：Acetonitrile：The volume ratio of dichloromethane is 10:7:Under conditions of 3, isoprenoid is extracted
The HSCCC chromatograms of liquid are as shown in figure 3, from the figure 3, it may be seen that occurring in that four peaks, four peaks are respectively peak one, peak by peak sequence
2nd, peak three and peak four, while (curved portion before peak one), two (curved portion between peak three and peak four) one between marking
With three (curved portion after peak four).
The HPLC and UV absorbance detection result such as table 3 of flow point sample to be measured corresponding to one-peak of peak four and one-three
It is shown：
The isoprenoid extract solution of table 3 n-hexane, acetonitrile and methylene chloride volume ratio in dicyandiamide solution are 10:7:When 3
The HPLC and UV absorbance detection result of separation product
| HSCCC separation products | HPLC appearance times | HPLC goes out peak area | HPLC goes out peak area % | UV absorption λ max |
| Indirect liquid one | _ | _ | _ | |
| Peak one | 22.0 | 18926349 | 92.82 | 274,452,468 |
| Peak two | _ | _ | _ | |
| Peak three | 26.8 | 2834863 | 85.1 | 434,458,484 |
| Indirect liquid two | 32.2 | 1235196 | 83.79 | 282,452,476 |
| Peak four | 32.2 | 3291241 | 78.96 | 282,452,476 |
| Indirect liquid three | 29.5 | 1996169 | 91.79 | 284,434,460,486 |
As known from Table 3, indirect liquid one, peak two separating effect it is very poor, the separating effect of peak one and indirect liquid three is relatively good,
Purity has respectively reached 92.82%, 91.79%, and its uv-absorption maximum wavelength is respectively 452nm, 460nm, the purity at peak four
Although purity has reached 78.96%, occur without unimodal, two peaks have been connected in together, separating effect is general, though the purity at peak three
85.1% so has been reached, has been occurred without unimodal, two peaks have been connected in together, and separating effect is bad, although indirect liquid two reaches
83.79%, but there is conditions of streaking, and have more small miscellaneous peak.
4.4th, n-hexane, acetonitrile and methylene chloride volume ratio are 10 in dicyandiamide solution:6.75:Separating effect when 3.25
Dicyandiamide solution is in n-hexane：Acetonitrile：The volume ratio of dichloromethane is 10:6.75:Under conditions of 3.25, class isoamyl two
The HSCCC chromatograms of alkene extract solution are as shown in figure 4, as shown in Figure 4, occur in that six peaks, six peaks are respectively by peak sequence
Peak one, peak two, peak three, peak four, peak five, peak six.
The HPLC and UV absorbance detection result of flow point sample to be measured corresponding to one-peak of peak six are as shown in table 4：
The isoprenoid extract solution of table 4 n-hexane, acetonitrile and methylene chloride volume ratio in dicyandiamide solution are 10:6.75:
The HPLC of separation product and UV absorbance detection result when 3.25
| HSCCC separation products | HPLC appearance times | HPLC goes out peak area | HPLC goes out peak area % | UV absorption λ max |
| Peak one | 22.0 | 3262502 | 72.56 | 274,452,468 |
| Peak two | 24.1 | 1441997 | 62.6 | 250,274 |
| Peak three | 32.3 | 9490917 | 92.87 | 282,452,476 |
| Peak four | 29.5 | 4359151 | 96.39 | 284,434,460,486 |
| Peak five | Disorderly, peak is miscellaneous | _ | _ | |
| Peak six | 26.8 | 7564664 | 89.7 | 434,458,484 |
As known from Table 4, peak one, peak two separating effect it is general, purity only has 72.56% and 62.6%, the He of peak three respectively
Preferably, purity has respectively reached 92.87% and 96.39% to the separating effect at peak four, and its uv-absorption maximum wavelength is respectively
452nm, 460nm, the separating effect at peak five are very poor, and preferably, purity is 89.7% to the separating effect at peak six, its maximal ultraviolet absorption
Wavelength is 458nm, through analysis, and peak six is beta carotene.
5th, secondary detection
Take separation product one (the corresponding flow point in peak one in 4.3), separation product two (the corresponding flow point in peak four in 4.4), divide
High-precision mass spectral analysis is carried out from product three (the corresponding flow point in peak three in 4.1)：
Mass Spectrometry Conditions：
ESI ion guns, scanning range m/z400-700, resolution ratio 60000, ion trap DDA patterns, with collision-induced solution
3 signals most strong ion is chosen from (CID) and 35% collision energy, during scanning and carries out Tandem Mass Spectrometry Analysis, heating-up temperature
250 DEG C, 350 DEG C of capillary temperature, sheath gas：35, auxiliary gas flow speed：15, spray voltage：3.5kV, S-lens radio frequency water
Flat 60%, sample is dissolved in chromatogram methanol, sample size 2ul, flow velocity 0.2ml/min.
6th, two grades of experimental results：
As shown in figure 5, its molecular ion peak in high-precision mass spectrum of separation product one is m/z=589.3283, it is it
The actual high-precision hydrogenated molecule value of measurement, the deviation of itself and theoretical value is shown in Table 5, and its error is identification separation within 4.9ppm
Product one is synechoxanthin.
As shown in fig. 6, its molecular ion peak in high-precision mass spectrum of separation product two is m/z=533.4095, it is it
The actual high-precision hydrogenated molecule value of measurement, the deviation of itself and theoretical value is shown in Table 5, and its error is 8.8ppm, identifies separation product
Two be chlorobactene.
As shown in fig. 7, its molecular ion peak in high-precision mass spectrum of separation product three is m/z=529.3835, it is it
The actual high-precision hydrogenated molecule value of measurement, the deviation of itself and theoretical value is shown in Table 5, and its error is 1.1ppm, identifies that No. 21 peaks are
isorenieratene。
The high-precision mass spectrometric data of separation product one-three is as shown in Figure 5：
| product | Formula | Calculated mass | Error(ppm) | Experimental mass |
| Separation product one | C40H4404+H+ | 589.3312 | 4.9 | 589.3283 |
| Separation product two | C40H52+H+ | 533.4142 | 8.8 | 533.4095 |
| Separation product three | C40H48+H+ | 529.3829 | 1.1 | 529.3835 |
Claims (3)
1. Arctic Sea fuchsin coccus B7740 produces the isolation and purification method of isoprenoid, it is characterised in that：Utilize high-speed counter-current color
Spectrum isolates and purifies Arctic Sea fuchsin coccus B7740 production isoprenoids.
2. Arctic Sea fuchsin coccus B7740 according to claim 1 produces the isolation and purification method of isoprenoid, its feature
It is to comprise the following steps：
2.1st, it is 10 by the volume ratio of n-hexane, acetonitrile and dichloromethane:6-8:2-4 prepares dicyandiamide solution, fully shakes up, treats molten
Agent system starts layering, and stratification is simultaneously separated, and obtains upper liquid and subnatant, upper liquid is used as stream as stationary phase, subnatant
Dynamic phase；
2.2nd, isoprenoid extract solution section bottom liquid is dissolved, is configured to 100-1000ug/ml sample solutions；
2.3rd, water bath with thermostatic control is opened, is 18-22 DEG C by temperature setting；
2.4th, with the spiral column jecket in washes of absolute alcohol pump；
2.5th, it is pumped into stationary phase；
2.6th, two phase solvent system is balanced：
UV-detector is opened, waits after the completion of preheating, wavelength is set to 450nm；Rotate forward and rotate main frame, while with 10ml/min
Flow pump enters mobile phase, device port of export outflow mobile phase to be detected and when UV signal stablizes constant, then dicyandiamide solution has been balanced；
2.7th, sample introduction；
2.8th, flow point is received.
3. Arctic Sea fuchsin coccus B7740 according to claim 2 produces the isolation and purification method of isoprenoid, its feature
It is：The volume ratio of n-hexane, acetonitrile and dichloromethane is 10:8:2、10:6.5:3.5、10:7:3 or 10:6.75:3.25.
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| Title |
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| 陈亚淑等: "北极海洋红球菌（Rhodococcus sp.）B7740产类胡萝卜素的提取条件优化及甲基萘醌类类胡萝卜素鉴定", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593021A (en) * | 2018-12-27 | 2019-04-09 | 湖北省农业科学院农产品加工与核农技术研究所 | The extraction separation method of colorless carotenoid in a kind of citrus pomace |
| CN109593021B (en) * | 2018-12-27 | 2021-04-30 | 湖北省农业科学院农产品加工与核农技术研究所 | Method for extracting and separating colorless carotenoid from citrus pomace |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107021894B (en) | 2019-04-26 |
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