CN103772255A - Method for extracting beta-carotene from algae by low-molecule organic matters/inorganic salt aqueous two-phase extraction - Google Patents
Method for extracting beta-carotene from algae by low-molecule organic matters/inorganic salt aqueous two-phase extraction Download PDFInfo
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Abstract
The invention discloses a method for extracting beta-carotene from algae by low-molecule organic matter/an inorganic salt aqueous two-phase extraction. The method comprises the following steps: (1), pretreating algae; (2), performing solid-liquid digestion; (3), performing aqueous two-phase extraction, extracting by using a low-molecule organic matter/inorganic salt aqueous two-phase system; (4), performing reduced pressure distillation and vacuum drying; and (5), recovering the inorganic salt. The method for extracting the beta-carotene from algae by the low-molecule organic matter/the inorganic salt aqueous two-phase extraction is simple and easy to operate, high in phase separation, high in extraction rate, small in organic solvent consumption, small in environment pollution, low in production cost, simple to operate, and capable of realizing large-scale industrialized production.
Description
Technical field
The invention belongs to the separation and purification field of active ingredient of natural product, be specifically related to the method for β-carotene in a kind of low molecule organic matter/inorganic salt binary aqueous two-phase extraction algae.
Technical background
Algae is a kind of emerging Dietotherapy medicament, in algae, contain a large amount of carotene, protein, vitamin B12 etc., can promote the compositions such as absorption of human body ferro element, have very high medicinal and nutritive value, united state grain and oil tissue is recommended as " classic food of 21st century ".Wherein the content of β-carotene is higher, and in the content beta-carotene in 6g algae and 28 bottles of cow's milk or 20 eggs, content beta-carotene is suitable.β-carotene can prevent the oxidative damage of DNA and lipoprotein, has anti-curing cancers, and the different physiological roles such as preventing cardiovascular disease can be applicable to function of blood sugar reduction food or nutritive health products, have good commercial value at health-product market.
In prior art, be mainly to adopt organic solvent extraction method, macroporous resin chromatography and SCF-CO 2 method to the extraction of β-carotene in algae.There is following technical problem in these extracting method: 1. extraction efficiency compared with low, solvent load is large, the production cycle is long, complex operation, unsuitable suitability for industrialized production; 2. equipment requirements is high, energy consumption is high, cost is high.Chinese patent CN102702059A discloses extract of a kind of spirulina β-carotene and preparation method thereof, adopts the mixture as solvent of ethanol-methyl alcohol to carry out supersound extraction to spirulina.There is above technical problem in this organic solvent extraction method.And double water-phase extractive technique is because mild condition, easy to operate, adjustable factor are many and can be by the successful experiences of conventional solvent extraction, and be considered to a kind of unit operation of biological downstream engineering initial gross separation.Separation and the purifying of many biological products have successfully been applied to take superpolymer/superpolymer or high polymer/inorganic salt as typical traditional double-aqueous phase system.But also there is following technical problem in traditional double-aqueous phase system: 1. energy consumption is high, production cost is high; 2. operating process complexity; 3. product yield is low, is unfavorable for the realization of heavy industrialization.
Summary of the invention
The object of this invention is to provide that a kind of method is simple, easy to operate, phase-splitting is fast, percentage extraction is high, consumption of organic solvent is little, environmental pollution is little, production cost is low, the method for β-carotene in simple to operate, the low molecule organic matter/inorganic salt binary aqueous two-phase extraction algae that can realize large-scale industrial production.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, carry out according to the following step:
(1) algae pre-treatment: algae is ground into powder, obtains algae powder;
(2) solid-liquid lixiviate: add low molecule organic matter to make solvent in described algae powder, carry out supersound process, and by centrifugal the mixed solution obtaining after supersound process removal of impurities, obtain β-carotene crude extract;
(3) aqueous two-phase extraction: get described β-carotene crude extract, add wherein low molecule organic matter and inorganic salt after concentrating, form low molecule organic matter/inorganic salt double-aqueous phase system, adjusting pH is 3-7, be mixed evenly, leave standstill phase-splitting, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure and vacuum-drying, obtain β-carotene in algae;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, then filter, obtain inorganic salt crystal.
Described low molecule organic matter includes but not limited to one or more in ethanol, methyl alcohol, acetone;
Described inorganic salt include but not limited to one or more in phosphoric acid salt, vitriol, carbonate, nitrate.
Come separation and Extraction biological substance or active ingredient of natural product with the double-aqueous phase system that low molecule organic matter and inorganic salt form, energy efficient greatly, reduces costs, and flow process simplifies the operation, improve product yield, for the realization of heavy industrialization provides possibility.
In low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, the applicable algae of method of β-carotene comprises spirulina, salt algae, chlorella.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (2), (3), described low molecule organic matter is acetone, the massfraction of described acetone is 18%-26%.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (2), (3), described low molecule organic matter is the mixed solution of ethanol and acetone, the massfraction of described mixed solution is 18%-26%, and in described mixed solution, the volume ratio of ethanol and acetone is 1:1-1:2.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (3), described inorganic salt are ammonium sulfate, the massfraction of described ammonium sulfate is 18%-26%.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (3), in described step (3), the massfraction after described β-carotene crude extract is concentrated is 3%-11%.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (3), phase separation temperature is room temperature, the phase-splitting time is 21-30 minute.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (2), the volume of described low molecule organic matter is 8 times of algae powder volume.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (2), when described ultrasonication, ultrasonic power is 400W, ultrasonic time is 10-15 minute.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (2), when described centrifugal removal of impurities is processed, centrifugal rotational speed is 3000r/min, centrifugation time is 10 minutes.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, in described step (5), when ice bath cooling, reduces to 4 ℃ by temperature.
The temperature of underpressure distillation is 70-80 ℃, and pressure is 0.1MPa.
In algae, the yield of β-carotene is calculated as follows:
Wherein, system is compared R=V
on/ V
under, V
on, V
underit is respectively the volume (mL) of upper and lower phase;
The double water-phase partition ratio K=C of target substance
on/ C
under, C
on, C
underit is respectively the mass concentration (ug/mL) of target substance in upper and lower phase.
Beneficial effect of the present invention is:
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, method is simple, easy to operate, phase-splitting is fast, percentage extraction is high, consumption of organic solvent is little, environmental pollution is little, production cost is low, simple to operate, can realize large-scale industrial production.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
In low molecule organic matter/inorganic salt aqueous two-phase extraction spirulina of the present embodiment, the method for β-carotene, comprises the steps:
(1) spirulina pre-treatment: spirulina powder is broken into powder, obtains spirulina powder;
(2) solid-liquid lixiviate: get spirulina powder described in 5g, add the ethanol-acetone mixed solution of 40ml, supersound process 15 minutes under the power of 400W, then centrifugal removal of impurities in the situation that rotating speed is 3000r/min, processes 10 minutes, obtains β-carotene crude extract; The massfraction of described ethanol-acetone mixed solution is 22%, and in described ethanol-acetone mixed solution, the volume ratio of ethanol and acetone is 1:1.5.
(3) aqueous two-phase extraction: get described β-carotene crude extract, being concentrated into massfraction is 6%, adding wherein massfraction is that 22% ethanol-acetone mixed solution and massfraction are 22% ammonium sulfate, form ethanol-acetone/ammonium sulfate double-aqueous phase system, regulating pH is 5, be mixed evenly, under room temperature, leave standstill phase-splitting 25 minutes, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure at 75 ℃, then vacuum-drying, obtains β-carotene in spirulina, and yield is 98.54%;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, be down to 4 ℃, then filter, obtain ammonia sulfate crystal.
Embodiment 2
In low molecule organic matter/inorganic salt aqueous two-phase extraction salt algae of the present embodiment, the method for β-carotene, comprises the steps:
(1) salt algae pre-treatment: salt algae powder is broken into powder, obtains salt algae powder end;
(2) solid-liquid lixiviate: get salt algae powder end described in 5g, add the ethanol-acetone mixed solution of 40ml, supersound process 10 minutes under the power of 400W, then centrifugal removal of impurities in the situation that rotating speed is 3000r/min, processes 10 minutes, obtains β-carotene crude extract; The massfraction of described ethanol-acetone mixed solution is 18%, and in described ethanol-acetone mixed solution, the volume ratio of ethanol and acetone is 1:1.
(3) aqueous two-phase extraction: get described β-carotene crude extract, being concentrated into massfraction is 3%, adding wherein massfraction is that 18% ethanol-acetone mixed solution and massfraction are 18% ammonium sulfate, form ethanol-acetone/ammonium sulfate double-aqueous phase system, regulating pH is 3, be mixed evenly, under room temperature, leave standstill phase-splitting 21 minutes, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure at 70 ℃, then vacuum-drying, obtains β-carotene in salt algae, and yield is 97.55%;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, be down to 4 ℃, then filter, obtain ammonia sulfate crystal.
Embodiment 3
In low molecule organic matter/inorganic salt aqueous two-phase extraction chlorella of the present embodiment, the method for β-carotene, comprises the steps:
(1) chlorella pre-treatment: chlorella powder is broken into powder, obtains chlorella powder;
(2) solid-liquid lixiviate: get chlorella powder described in 5g, add the acetone of 40ml, supersound process 10 minutes under the power of 400W, then centrifugal removal of impurities in the situation that rotating speed is 3000r/min, processes 10 minutes, obtains β-carotene crude extract; The massfraction of described acetone is 26%.
(3) aqueous two-phase extraction: get described β-carotene crude extract, being concentrated into massfraction is 11%, adding wherein massfraction is that 26% acetone and massfraction are 26% saltpetre, form acetone/saltpetre double-aqueous phase system, regulating pH is 7, be mixed evenly, under room temperature, leave standstill phase-splitting 30 minutes, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure at 80 ℃, then vacuum-drying, obtains β-carotene in chlorella, and yield is 95.28%;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, be down to 4 ℃, then filter, obtain potassium nitrate crystals.
Embodiment 4
In low molecule organic matter/inorganic salt aqueous two-phase extraction salt algae of the present embodiment, the method for β-carotene, comprises the steps:
(1) salt algae pre-treatment: salt algae powder is broken into powder, obtains salt algae powder end;
(2) solid-liquid lixiviate: get salt algae powder end described in 5g, add the methanol-acetone mixed solution of 40ml, supersound process 10 minutes under the power of 400W, then centrifugal removal of impurities in the situation that rotating speed is 3000r/min, processes 10 minutes, obtains β-carotene crude extract; The massfraction of described methanol-acetone mixed solution is 24%, and in described ethanol-acetone mixed solution, the volume ratio of ethanol and acetone is 1:2.
(3) aqueous two-phase extraction: get described β-carotene crude extract, being concentrated into massfraction is 9%, adding wherein massfraction is the ammonium sulfate that 24% methanol-acetone mixed solution and massfraction are 24%, form methanol-acetone/ammonium sulfate double-aqueous phase system, regulating pH is 4, be mixed evenly, under room temperature, leave standstill phase-splitting 28 minutes, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure at 78 ℃, then vacuum-drying, obtains β-carotene in salt algae, and yield is 96.28%;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, be down to 4 ℃, then filter, obtain ammonia sulfate crystal.
Embodiment 5 amplifies the amount in embodiment 1.
In low molecule organic matter/inorganic salt aqueous two-phase extraction spirulina of the present embodiment, the method for β-carotene, comprises the steps:
(1) spirulina pre-treatment: spirulina powder is broken into powder, obtains spirulina powder;
(2) solid-liquid lixiviate: get spirulina powder described in 5kg, add the ethanol-acetone mixed solution of 40L, supersound process 15 minutes under the power of 400W, then centrifugal removal of impurities in the situation that rotating speed is 3000r/min, processes 10 minutes, obtains β-carotene crude extract; The massfraction of described ethanol-acetone mixed solution is 22%, and in described ethanol-acetone mixed solution, the volume ratio of ethanol and acetone is 1:1.5.
(3) aqueous two-phase extraction: get described β-carotene crude extract, being concentrated into massfraction is 6%, adding wherein massfraction is that 22% ethanol-acetone mixed solution and massfraction are 22% ammonium sulfate, form ethanol-acetone/ammonium sulfate double-aqueous phase system, regulating pH is 5, be mixed evenly, under room temperature, leave standstill phase-splitting 25 minutes, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure at 75 ℃, then vacuum-drying, obtains β-carotene in spirulina, and yield is 98.24%;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, be down to 4 ℃, then filter, obtain ammonia sulfate crystal.
Scale-up shows, the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, and in the situation that increasing charging capacity, the yield of β-carotene does not almost decline, and this shows that the method is suitable for large-scale industrial production.
The method of β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae of the present invention, method is simple, easy to operate, phase-splitting is fast, percentage extraction is high, consumption of organic solvent is little, environmental pollution is little, production cost is low, simple to operate, can realize large-scale industrial production.
The present invention includes but be not limited to above-mentioned embodiment, any product, method that meets this patent description, within all falling into protection scope of the present invention.
Claims (10)
1. a method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae, is characterized in that, comprises the following steps:
(1) algae pre-treatment: algae is ground into powder, obtains algae powder;
(2) solid-liquid lixiviate: add low molecule organic matter to make solvent in described algae powder, carry out supersound process, and by centrifugal the mixed solution obtaining after supersound process removal of impurities, obtain β-carotene crude extract;
(3) aqueous two-phase extraction: get described β-carotene crude extract, add wherein low molecule organic matter and inorganic salt after concentrating, form low molecule organic matter/inorganic salt double-aqueous phase system, adjusting pH is 3-7, be mixed evenly, leave standstill phase-splitting, above contain mutually a large amount of β-carotenes;
(4) underpressure distillation, vacuum-drying: get the upper phase that contains a large amount of β-carotenes that step (3) obtains, it is carried out to concentrating under reduced pressure and vacuum-drying, obtain β-carotene in algae;
(5) reclaim inorganic salt: get the lower phase that step (3) obtains, first it is carried out to ice bath cooling, then filter, obtain inorganic salt crystal.
2. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, it is characterized in that, in described step (2), (3), described low molecule organic matter is acetone, and the massfraction of described acetone is 18%-26%.
3. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, it is characterized in that, in described step (2), (3), described low molecule organic matter is the mixed solution of ethanol and acetone, the massfraction of described mixed solution is 18%-26%, and in described mixed solution, the volume ratio of ethanol and acetone is 1:1-1:2.
4. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (3), described inorganic salt are ammonium sulfate, and the massfraction of described ammonium sulfate is 18%-26%.
5. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (3), the massfraction after described β-carotene crude extract is concentrated is 3%-11%.
6. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (3), phase separation temperature is room temperature, and the phase-splitting time is 21-30 minute.
7. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (2), the volume of described low molecule organic matter is 8 times of algae powder volume.
8. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (2), when described ultrasonication, ultrasonic power is 400W, and ultrasonic time is 10-15 minute.
9. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (2), when described centrifugal removal of impurities is processed, centrifugal rotational speed is 3000r/min, and centrifugation time is 10 minutes.
10. the method for β-carotene in low molecule organic matter/inorganic salt aqueous two-phase extraction algae according to claim 1, is characterized in that, in described step (5), when ice bath cooling, temperature is reduced to 4 ℃.
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| CN110156655A (en) * | 2019-06-04 | 2019-08-23 | 河西学院 | The method of ultrasonic wave added hydrophilic-hydrophobic two-phase system preparation microalgae beta carotene |
| CN110627698A (en) * | 2019-10-12 | 2019-12-31 | 河北农业大学 | Method for preparing astaxanthin by applying aqueous two-phase system separation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110156655A (en) * | 2019-06-04 | 2019-08-23 | 河西学院 | The method of ultrasonic wave added hydrophilic-hydrophobic two-phase system preparation microalgae beta carotene |
| CN110627698A (en) * | 2019-10-12 | 2019-12-31 | 河北农业大学 | Method for preparing astaxanthin by applying aqueous two-phase system separation |
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