CN103333746B - Method and bacteria for separating conjugated linoleic acid from oil tea meal - Google Patents
Method and bacteria for separating conjugated linoleic acid from oil tea meal Download PDFInfo
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- CN103333746B CN103333746B CN201310258358.XA CN201310258358A CN103333746B CN 103333746 B CN103333746 B CN 103333746B CN 201310258358 A CN201310258358 A CN 201310258358A CN 103333746 B CN103333746 B CN 103333746B
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Abstract
The invention discloses a method for separating conjugated linoleic acid from oil tea meal. The method comprises the following steps of: 1) fermenting the oil tea meal; 2) putting the fermented oil tea meal into diethyl ester for digestion; 3) evaporating the conjugated linoleic acid mixture extraction liquid to remove the diethyl ester to obtain a conjugated linoleic acid mixture; 4) mixing heptane, acetonitrile, acetic acid and methanol in a volume ratio of 4:4:1:1, and standing for layering to obtain the upper phase and lower phase of a solvent system respectively; 5) dissolving the conjugated linoleic acid mixture in the upper phase of the solvent system to obtain a sample solution; 6) injecting the lower phase of the solvent system into a countercurrent chromatograph, and pumping the sample solution into the countercurrent chromatograph, and performing elution; and 7) collecting the elution liquid, and respectively concentrating and freeze-drying the collected same elution liquid to obtain conjugated linoleic acid. The invention also discloses a bacterial strain lactobacillus sp. LL-ZSDS001 used in the fermentation process.
Description
Technical field
The invention belongs to functional foodstuff development technique field, relate to the extraction and separation method of functional component in food, particularly the method for the multiple conjugated linoleic acid isomers of separated preparation from conjugated linolic acid mixture.
Background technology
Conjugated linolic acid is flaxen oily matter, there is the functions such as the body fat of minimizing is piled up, enhancing body is immune and antitumor, can be widely used in medicine and food service industry, for the preparation for the treatment of obesity or reducing blood-fat or hypoglycemic medicine or food and feed additive, also can be used for preparing anti-oxidant and skin care external preparation whitening.Conjugated linolic acid can not synthesize people and most animal, must be taken in by the external world, therefore has important using value.
Current existing conjugated linolic acid, generally has following several:
。
Conjugated linolic acid in above formula is named as respectively: A:8 is anti-, 10 trans-conjugated linolic acids, and B:10 is anti-, 12 cis-conjugated linolic acids, C:9 is suitable, 11 trans-conjugated linolic acids.The existing separation purification method about conjugated linolic acid is mainly to adopt solvent crystallization, urea adduct method etc., and its limitation is that separation purity is low, complex operation, speed are slow.Secondly, sample recovery rate is low, recovery is difficult, is difficult to application in batches.High-speed countercurrent chromatography is a kind of continuous liquid luquid partition chromatography technology without solid carrier, its stationary phase is retained in separator column by gravity field and effect of centrifugal force, solid carrier and sample generation chemical reaction have been avoided and sex change and irreversible adsorption, sample recovery rate is high, can from rough sample, one step prepare high-purity monomer compound, both be applicable to trace analysis and also can be used for extensive preparation, be widely used in the fields such as food, medicine, natural product.Adopt the separation of high speed adverse current chromatogram method to prepare conjugated linoleic acid isomers and there is good application prospect.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for the conjugated linoleic acid isomers separation that a kind of cost is lower, efficiency is higher.
In order to solve the problems of the technologies described above, the invention provides a kind of from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, comprise the steps:
1), by Extracted From Oil-tea-cake fermentation, obtain the Extracted From Oil-tea-cake after fermentation;
2), lixiviate:
Extracted From Oil-tea-cake after fermentation is put into ether lixiviate 60 ~ 120min, obtain conjugated linolic acid mixture extracting solution;
3), by conjugated linolic acid mixture extracting solution (with Rotary Evaporators) evaporative removal ether, obtain conjugated linolic acid mixture;
4), heptane, acetonitrile, acetic acid, methyl alcohol are mixed according to the volume ratio of 4:4:1:1, standing rear layering, respectively phase mutually and under solvent systems on solvent systems;
5), conjugated linolic acid mixture is dissolved on solvent systems mutually according to the ratio of 0.8 ~ 1.2g/ (30 ~ 40) mL, obtain sample solution;
6), first in counter current chromatograph, inject phase under solvent systems (until chromatographic column is filled);
Then sample solution is pumped in counter current chromatograph, the flow velocity of sample size 30 ~ 41mL(sample introduction of sample solution is 0.8 ~ 1.0mL/min); Then with carrying out wash-out as moving phase under solvent systems, flow velocity is that 0.8 ~ 1.2mL/min(is preferably 1.0mL/min),
7), collect elutriant (elution fraction), the identical elutriant of collecting is concentrated respectively and lyophilize, obtain conjugated linolic acid.Remarks explanation: absorbancy (233nm).
As the improvement of the method for separated conjugated linolic acid from Extracted From Oil-tea-cake of the present invention:
The conjugated linolic acid of step 7) gained is divided into following 3 kinds: conjugated linoleic acid isomers I; Conjugated linoleic acid isomers II, conjugated linoleic acid isomers III;
Conjugated linoleic acid isomers I is
,
Conjugated linoleic acid isomers II is
,
Conjugated linoleic acid isomers III is
.
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
The method of Extracted From Oil-tea-cake fermentation is for comprising the following steps:
1., bacterial strain activation:
The bacterial strain Lactobacillus sp. LL-ZSDS001 that is CCTCC No:M2013148 by preserving number activates; Must activate rear bacterium liquid;
2., Extracted From Oil-tea-cake is mixed according to the mass ratio of 15:2 with water;
3., the inoculum size that after activation, bacterium liquid is 5% according to volume ratio is inoculated in step gains 2., after regulating pH to be 6.0 ~ 6.5, in 36.8 ~ 37.2 ℃ of cultivation 68 ~ 76h; Extracted From Oil-tea-cake after must fermenting.
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
The activation 1. of described step is: in the inoculum size access MRS liquid nutrient medium that the bacterium liquid that is the bacterial strain Lactobacillus sp. LL-ZSDS001 of CCTCC No:M2013148 by preserving number is 5% according to volume ratio, in 36.8 ~ 37.2 ℃, cultivate 23 ~ 25h; In the inoculum size access MRS liquid nutrient medium that gains are 2% according to volume ratio again, in 36.8 ~ 37.2 ℃, cultivate 23 ~ 25h; Must activate rear bacterium liquid.
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
Described step 2) in: the Extracted From Oil-tea-cake after fermentation and the solid-liquid ratio of ether are: 1g/ (1 ~ 5) mL, is preferably 1g/2mL.
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
Thereby described step 3) adopts Rotary Evaporators evaporation to realize and removes ether, and evaporation technology parameter is: temperature is 30 ~ 35 ℃, vacuum tightness≤100Pa(80-100Pa).
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
The simmer down to of described step 7): adopt Rotary Evaporators evaporation to realize concentrated, evaporation technology parameter is: temperature is 30 ~ 35 ℃, vacuum tightness≤100Pa(80-100Pa).
As of the present invention from Extracted From Oil-tea-cake the further improvements in methods of separated conjugated linolic acid:
Described step 1) is: the Extracted From Oil-tea-cake after fermentation is put into ether lixiviate 60 ~ 120min, after filtration with centrifugal, obtain conjugated linolic acid mixture extracting solution;
Described 200 mesh sieves (object is in order to remove insolubles) that were filtered into; Describedly centrifugally be: by the filtrate of filtering gained, by the centrifugal 15min(object of 3000rpm, be in order to remove impurity).
The bacterial strain that the present invention is used, preservation name is called: Lactobacillus sp. LL-ZSDS001, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University; Preservation date: on April 17th, 2013, preserving number: CCTCC No:M2013148.
This bacterial strain belongs to the Lactobacillus sp. LL-ZSDS001 milk-acid bacteria of Single ion microbeam seed selection.
In the present invention, with volume pump, by filling with mutually under solvent systems after chromatographic column, then sample solution is pumped in counter current chromatograph, then with certain flow, from chromatographic instrument head end, pump into moving phase, with graduated cylinder, at chromatographic tail end, receive the stationary phase flowing out; When chromatographic instrument tail end no longer includes stationary phase outflow, show that system is stable, now enter sample liquid 30 ~ 41mL, with every 3 minutes, collect one and manage.
Adopt the separated conjugated linolic acid of method of the present invention, tool has the following advantages:
1), owing to not needing solid support, the separation of material realizes according to the difference of its partition ratio in two-phase, thereby avoided, because of sample loss that irreversible adsorption causes, inactivation, sex change etc., not only making sample all reclaim, the sample of recovery more can reflect the characteristic that it is original.
2), present method with respect to traditional solid-liquid isolation technique have applied widely, flexible operation, efficient, fast, the advantage such as preparation amount is large, expense is low.
3), present method compares simple to operately with existing solvent crystallization, urea adduct method etc., preparation efficiency is high, the advantage that output is large, and can linear amplification.
4), present method employs new technology, novel method, is a kind of new technology of separated conjugated linolic acid, level of automation is high, has a extensive future;
5), resulting conjugated linolic acid purity is high.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the HSCCC separating spectrum of embodiment 1.
Fig. 2 is the HSCCC separating spectrum of embodiment 2.
Fig. 3 is the HSCCC separating spectrum of embodiment 3.
Fig. 4 is the HPLC collection of illustrative plates of separating obtained conjugated linoleic acid isomers.
Embodiment
Embodiment 1, a kind of from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, carry out successively following steps:
1), Extracted From Oil-tea-cake fermentation, carry out successively following steps:
1., bacterial strain activation:
The bacterial strain Lactobacillus sp. LL-ZSDS001 that is CCTCC No:M2013148 by preserving number activates; Must activate rear bacterium liquid; Be specially:
The bacterial strain Lactobacillus sp. LL-ZSDS001 that the preserving number of take is CCTCC No:M2013148 is starting strain, and the bacterium liquid of drawing lmL accesses in the 50mL triangular flask of the MRS liquid nutrient medium that 20mL is housed, 37 ℃ of standing cultivation 24h; Then by gains, be in 2% inoculum size access MRS liquid nutrient medium by volume, 37 ℃ of standing cultivation 24h(, reactivate 24h); Must activate rear bacterium liquid.
Remarks explanation: MRS liquid nutrient medium is conventional medium, for: peptone 10.0g, extractum carnis 10.0g, yeast extract paste 5.0g, glucose 10.0g, distilled water are settled to 1000ml, and pH 6.5.
2., Extracted From Oil-tea-cake is mixed according to the mass ratio of 15:2 with water;
3., the step inoculum size that 1. bacterium liquid is 5% according to volume ratio after the activation of gained is inoculated in step gains 2., utilize NaH
2pO
4after regulating pH to be 6.4 left and right, in 37 ℃, cultivate 72h; Extracted From Oil-tea-cake after must fermenting.
2), lixiviate:
First the Extracted From Oil-tea-cake 500g after fermentation is put into 1000ml ether lixiviate 80min, then cross 200 mesh sieves to remove insolubles, gained filtrate is in order to remove precipitated impurities by the centrifugal 15min(object of 3000rpm), the supernatant liquor of gained is conjugated linolic acid mixture extracting solution.
3), by conjugated linolic acid mixture extracting solution with Rotary Evaporators in the vacuum tightness of 80 ~ 90Pa, evaporate to dryness, to constant weight (removal ether), obtains conjugated linolic acid mixture 11.27g at 35 ℃.
4), measure heptane 400mL, acetonitrile 400mL, acetic acid 100mL, methyl alcohol 100mL shake and mix in separating funnel, and standingly make it layering, respectively phase mutually and under solvent systems on solvent systems; By on solvent systems mutually with under solvent systems, be respectively charged into mutually in reagent bottle standby.That is, solvent systems adopts heptane/acetonitrile/acetic acid/methyl alcohol (4:4:1:1, the v/v) system under at room temperature (25 ~ 35 ℃).
5), take during 0.8 gram of conjugated linolic acid mixture is dissolved on 30mL solvent systems mutually, ultrasonic oscillation 10min, thus conjugated linolic acid mixture is fully dissolved, obtain sample solution.
6), counter current chromatograph is selected TBE-1000A type high-speed counter-current chromatograph (Shanghai Tongtian Biotechnology Co., Ltd.)
By inject the chromatographic column of high speed adverse current chromatogram under solvent systems with the flow velocity of 2.0mL/min, until chromatographic column is filled.
Open counter current chromatograph to 900rpm, then with the flow velocity of 0.8mL/min, input above-mentioned sample solution, after sample introduction finishes, (flow pump with 1.0mL/min enters moving phase again, phase under solvent systems) carry out wash-out, with collector, collect elution fraction (elutriant), application Ag
+-HPLC chromatography (4.6mm i.d. * 250mm Stainless steel; 5 μ m particle size) detect the separation case of conjugated linoleic acid isomers.
When without elution peak, stop wash-out.
Reality is: elution time is 240 min; Within every 3 minutes, collect 1 pipe.The elutriant of 12nd ~ 19 pipes is merged, obtain elutriant I; The elutriant of 31st ~ 37 pipes is merged, obtain elutriant II; The elutriant of 47th ~ 54 pipes is merged, obtain elutriant III.
Elutriant I, elutriant II, elutriant III are proceeded as follows respectively:
First, 30 ~ 35 ℃ of temperature, on the Rotary Evaporators of 80 ~ 90Pa vacuum tightness, concentrate, until absence of liquid distills out, then in-40 ℃ of lyophilizes to constant weight.
Obtain conjugated linoleic acid isomers I (shown in formula I) 0.2462g.
Obtain conjugated linoleic acid isomers II (shown in formula II) 0.1921g.
Obtain conjugated linoleic acid isomers III (shown in formula III) 0.1548g.
Through Ag
+-HPLC method detects, purity>=98% of conjugated linoleic acid isomers I; Purity>=96% of conjugated linoleic acid isomers II; Purity>=97% of conjugated linoleic acid isomers III.
Conjugated linoleic acid isomers I is
(formula I),
Conjugated linoleic acid isomers II is
(formula II),
Conjugated linoleic acid isomers III is
(formula III).
Embodiment 2, a kind of from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, carry out successively following steps:
Step 1) ~ step 3) is with embodiment 1.
4), measure heptane 800mL, acetonitrile 800mL, acetic acid 200mL, methyl alcohol 200mL shake and mix in separating funnel, and standingly make it layering, respectively phase mutually and under solvent systems on solvent systems; By on solvent systems mutually with under solvent systems, be respectively charged into mutually in reagent bottle standby.That is, solvent systems adopts heptane/acetonitrile/acetic acid/methyl alcohol (4:4:1:1, the v/v) system under at room temperature (25 ~ 35 ℃).
5), take during 1.2 grams of conjugated linolic acid mixtures are dissolved on 40mL solvent systems mutually, ultrasonic oscillation 10min, thus conjugated linolic acid mixture is fully dissolved, obtain sample solution.
6), counter current chromatograph is selected TBE-1000A type high-speed counter-current chromatograph (Shanghai Tongtian Biotechnology Co., Ltd.)
By inject the chromatographic column of high speed adverse current chromatogram under solvent systems with the flow velocity of 2.0mL/min, until chromatographic column is filled.
Open counter current chromatograph to 1000rpm, then with the flow velocity of 1.0mL/min, input above-mentioned sample solution, after sample introduction finishes, then with the flow pump of 1.0mL/min, enter moving phase (phase under solvent systems) and carry out wash-out, with collector, collect elution fraction (elutriant), application Ag
+-HPLC chromatographic column (4.6 mm i.d. * 250 mm Stainless steel; 5 μ m particle size) detect the separation case of conjugated linoleic acid isomers.
When without elution peak, stop wash-out.
Reality is: elution time is 330min; Within every 3 minutes, collect 1 pipe.The elutriant of 22nd ~ 34 pipes is merged, obtain elutriant I; The elutriant of 48th ~ 57 pipes is merged, obtain elutriant II; The elutriant of 72nd ~ 85 pipes is merged, obtain elutriant III.
Elutriant I, elutriant II, elutriant III are proceeded as follows respectively:
In temperature, be first 30 ~ 35 ℃, on the Rotary Evaporators of 80 ~ 90Pa vacuum tightness, concentrate, until absence of liquid distills out; Then in-40 ℃ of lyophilizes to constant weight.
Obtain conjugated linoleic acid isomers I (shown in formula I) 0.3261g.
Obtain conjugated linoleic acid isomers II (shown in formula II) 0.2733g.
Obtain conjugated linoleic acid isomers III (shown in formula III) 0.2116g.
Through Ag
+-HPLC method detects, purity>=98% of conjugated linoleic acid isomers I; Purity>=95% of conjugated linoleic acid isomers II; Purity>=96% of conjugated linoleic acid isomers III.
Embodiment 3, a kind of from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, carry out successively following steps:
Step 1) ~ step 3) is with embodiment 1.
4), measure heptane 800mL, acetonitrile 800mL, acetic acid 200mL, methyl alcohol 200mL shake and mix in separating funnel, and standingly make it layering, respectively phase mutually and under solvent systems on solvent systems; By on solvent systems mutually with under solvent systems, be respectively charged into mutually in reagent bottle standby.That is, solvent systems adopts heptane/acetonitrile/acetic acid/methyl alcohol (4:4:1:1, the v/v) system under at room temperature (25 ~ 35 ℃).
5), take during 1.0 grams of conjugated linolic acid mixtures are dissolved on 35mL spectrum solvent systems mutually, ultrasonic oscillation 10min, thus conjugated linolic acid mixture is fully dissolved, obtain sample solution.
6), counter current chromatograph is selected TBE-1000A type high-speed counter-current chromatograph (Shanghai Tongtian Biotechnology Co., Ltd.)
By inject the chromatographic column of high speed adverse current chromatogram under solvent systems with the flow velocity of 6.0mL/min, until chromatographic column is filled.
Open counter current chromatograph to 600rpm, then with the flow velocity of 0.8 L/min, input above-mentioned sample solution, after sample introduction finishes, then with the flow pump of 1.0mL/min, enter moving phase (phase under solvent systems) and carry out wash-out, with collector, collect elution fraction (elutriant), application Ag
+-HPLC chromatographic column (4.6 mm i.d. * 250 mm Stainless steel; 5 μ m particle size) detect the separation case of conjugated linoleic acid isomers.
When without elution peak, stop wash-out.
Reality is: elution time is 300 min; Within every 3 minutes, collect 1 pipe.The elutriant of 23rd ~ 31 pipes is merged, obtain elutriant I; The elutriant of 48th ~ 57 pipes is merged, obtain elutriant II; The elutriant of 70th ~ 76 pipes is merged, obtain elutriant III.
Elutriant I, elutriant II, elutriant III are proceeded as follows respectively:
In temperature, be first 30 ~ 35 ℃, on the Rotary Evaporators of (80 ~ 90Pa) vacuum tightness, concentrate, until absence of liquid distills out; ; Then in-40 ℃ of lyophilizes to constant weight.
Obtain conjugated linoleic acid isomers I (shown in formula I) 0.2835g.
Obtain conjugated linoleic acid isomers II (shown in formula II) 0.2630g.
Obtain conjugated linoleic acid isomers III (shown in formula III) 0.2153g.
Through Ag
+-HPLC method detects, purity>=98% of conjugated linoleic acid isomers I; Purity>=97% of conjugated linoleic acid isomers II; Purity>=96% of conjugated linoleic acid isomers III.
In invention process, contriver has also carried out following comparative example:
Comparative example 1, by the methyl alcohol in heptane/acetonitrile/acetic acid/methyl alcohol (4:4:1:1, v/v), change solvent for use system into ethanol, all the other contents are equal to embodiment 1.
Through Ag
+-HPLC method detects, the purity < 50% of conjugated linoleic acid isomers I; The purity < 50% of conjugated linoleic acid isomers II; The purity < 50% of conjugated linoleic acid isomers III.
Comparative example 2, make heptane/acetonitrile/acetic acid/methyl alcohol (4:4:1:1, v/v) system into heptane/acetonitrile/acetic acid/methyl alcohol (3:3:2:2, v/v) system; All the other contents are equal to embodiment 1.
Through Ag
+-HPLC method detects, the purity < 30% of conjugated linoleic acid isomers I; The purity < 30% of conjugated linoleic acid isomers II; The purity < 30% of conjugated linoleic acid isomers III.
Comparative example 3, for embodiment 1, do to change as follows:
Step 5): during 0.8 gram of conjugated linolic acid mixture is dissolved under 30mL solvent systems mutually, obtain sample solution.
6), by inject the chromatographic column of high speed adverse current chromatogram on solvent systems with the flow velocity of 2.0mL/min, until chromatographic column is filled; Select on solvent systems as moving phase simultaneously;
All the other contents are equal to embodiment 1.
Through Ag
+-HPLC method detects, the purity < 20% of conjugated linoleic acid isomers I; The purity < 20% of conjugated linoleic acid isomers II; The purity < 20% of conjugated linoleic acid isomers III.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (7)
1. the method for separated conjugated linolic acid from Extracted From Oil-tea-cake, is characterized in that comprising the steps:
1), by Extracted From Oil-tea-cake fermentation, obtain the Extracted From Oil-tea-cake after fermentation;
The method of Extracted From Oil-tea-cake fermentation is for comprising the following steps:
1., bacterial strain activation:
It by preserving number, is the bacterial strain of CCTCC No:M2013148
lactobacillus sp.LL-ZSDS001 activate bacterium liquid after must activating;
2., Extracted From Oil-tea-cake is mixed according to the mass ratio of 15:2 with water;
3., by bacterium liquid after activation, being 5% according to volume ratio, inoculum size is inoculated in step gains 2., after regulating pH to be 6.0～6.5, cultivates 68～76h in 36.8～37.2 ℃, obtains the Extracted From Oil-tea-cake after fermentation;
2), lixiviate:
Extracted From Oil-tea-cake after fermentation is put into ether lixiviate 60～120min, obtain conjugated linolic acid mixture extracting solution;
3), by conjugated linolic acid mixture extracting solution evaporative removal ether, obtain conjugated linolic acid mixture;
4), heptane, acetonitrile, acetic acid, methyl alcohol are mixed according to the volume ratio of 4:4:1:1, standing rear layering, respectively phase mutually and under solvent systems on solvent systems;
5), on the solvent systems of every 30～40mL, dissolve the conjugated linolic acid mixture of 0.8～1.2g in mutually, obtain sample solution;
6), first in counter current chromatograph, inject phase under solvent systems,
Then sample solution is pumped in counter current chromatograph, the sample size of sample solution is 30～41mL; Then with carrying out wash-out as moving phase under solvent systems, flow velocity is 0.8～1.2mL/min;
7), collect elutriant, the elutriant of collection is concentrated and lyophilize, obtain conjugated linolic acid.
According to claim 1 from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, it is characterized in that:
The bacterial strain activation 1. of described step is: by preserving number, be the bacterial strain of CCTCC No:M2013148
lactobacillus sp.LL-ZSDS001, in the inoculum size access MRS liquid nutrient medium that bacterium liquid is 5% according to volume ratio, in 36.8～37.2 ℃, cultivate 23～25h; In the inoculum size access MRS liquid nutrient medium that gains are 2% according to volume ratio again, in 36.8～37.2 ℃, cultivate 23～25h, bacterium liquid after must activating.
According to claim 2 from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, it is characterized in that:
Described step 2) in: the Extracted From Oil-tea-cake after every 1g fermentation adopts the ether lixiviate of 1～5mL.
According to claim 3 from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, it is characterized in that:
Described step 3) thus adopting Rotary Evaporators evaporation to realize removes ether, evaporation technology parameter is: 30～35 ℃ of temperature, vacuum tightness 80～100Pa.
According to claim 4 from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, it is characterized in that:
Described step 7) simmer down to: adopt Rotary Evaporators evaporation to realize concentrated, evaporation technology parameter is: 30～35 ℃ of temperature, vacuum tightness 80～100Pa.
According to claim 5 from Extracted From Oil-tea-cake the method for separated conjugated linolic acid, it is characterized in that:
Described step 2) be: the Extracted From Oil-tea-cake after fermentation is put into ether lixiviate 60～120min, after filtration with centrifugal, obtain conjugated linolic acid mixture extracting solution;
Described 200 mesh sieves that were filtered into; Describedly centrifugally be: the filtrate that will filter gained is with the centrifugal 15min of 3000rpm.
7. a milk-acid bacteria, is characterized in that: preservation name is called:
lactobacillus sp.LL-ZSDS001, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan University, preservation date: on April 17th, 2013, preserving number: CCTCC No:M2013148.
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| CN101092603A (en) * | 2007-05-25 | 2007-12-26 | 中国农业科学院农产品加工研究所 | Method for producing conjugated linoleic acid, and dedicated bacterial strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101092603A (en) * | 2007-05-25 | 2007-12-26 | 中国农业科学院农产品加工研究所 | Method for producing conjugated linoleic acid, and dedicated bacterial strain |
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| Title |
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| Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography;Xueli Cao等;《Journal of Chromatography A》;20031231;第1021卷;第117-124页 * |
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| 孙磊等.应用QuikPREP HSCCC系统实现共轭亚油酸及其异构体的分离.《分析试验室》.2008,第27卷 * |
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