CN111995880A - Method for extracting biologically fermented carotenoid - Google Patents
Method for extracting biologically fermented carotenoid Download PDFInfo
- Publication number
- CN111995880A CN111995880A CN202010843264.9A CN202010843264A CN111995880A CN 111995880 A CN111995880 A CN 111995880A CN 202010843264 A CN202010843264 A CN 202010843264A CN 111995880 A CN111995880 A CN 111995880A
- Authority
- CN
- China
- Prior art keywords
- carotenoid
- powder
- wall breaking
- optionally
- wall
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C403/00—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
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/005—Processes comprising at least two steps in series
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/10—Purification; Separation; Use of additives by extraction, i.e. purification or separation of liquid hydrocarbons with the aid of liquids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/14—Purification; Separation; Use of additives by crystallisation; Purification or separation of the crystals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/148—Purification; Separation; Use of additives by treatment giving rise to a chemical modification of at least one compound
- C07C7/14833—Purification; Separation; Use of additives by treatment giving rise to a chemical modification of at least one compound with metals or their inorganic compounds
- C07C7/1485—Purification; Separation; Use of additives by treatment giving rise to a chemical modification of at least one compound with metals or their inorganic compounds oxides; hydroxides; salts
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Water Supply & Treatment (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for extracting carotenoid from microbial fermentation, which comprises the following steps: (1) carrying out spray drying on a microorganism fermentation liquor containing carotenoid to obtain first fungus powder; (2) breaking the wall of the dry fungus powder by using a wall breaking machine to obtain second fungus powder; (3) saponifying the wall-broken bacterium powder, and filtering to obtain third bacterium powder; (4) extracting the third bacterial powder, filtering and collecting filtrate; (5) concentrating the filtrate to obtain concentrated solution, stirring at low temperature for crystallization, adding a small amount of anhydrous ethanol for rinsing, filtering to obtain crystals, and vacuum drying the crystals to obtain pure carotenoid crystals. The method has the advantages of wall breaking rate of 99%, extraction rate of more than 98%, total yield of more than 90%, simple process operation, mild conditions, less carotenoid loss, stable product quality, high solvent recovery rate, effectively reduced cost, and suitability for industrial production.
Description
Technical Field
The invention relates to the technical field of extraction and separation of microbial active ingredients. More particularly, relates to a method for extracting carotenoid from microbial fermentation.
Background
Carotenoids (carotenoids) are a generic name for an important class of natural pigments, which are commonly found in yellow, orange-red or red pigments of animals, higher plants, fungi, algae. It is an isoprenoid polymer containing 40 carbons, i.e., a tetraterpene compound. Typical carotenoids are formed by 8 isoprene units joined end to end. The color of carotenoids varies depending on the number of conjugated double bonds. The greater the number of conjugated double bonds, the more red the color shifts. The natural carotenoids found so far are over 700 kinds, and can be divided into two groups according to the difference of chemical structures, one is carotene family (containing only two elements of carbon and hydrogen, and not containing oxygen element, such as beta-carotene and lycopene), and the other is xanthophyll family (containing oxygen-containing functional group of hydroxyl, keto, carboxyl, methoxyl, etc., such as xanthophyll and astaxanthin). Carotenoid is the main source of vitamin A in vivo, and also has effects of resisting oxidation, regulating immunity, resisting cancer, delaying aging, etc.
At present, the preparation method of carotenoid mainly comprises natural extraction method, chemical synthesis method and microbial fermentation method. The natural extraction method has low carotenoid yield and high cost, and is limited by the raw material source; the chemical synthesis method has potential safety hazard; the microbial fermentation method has the advantages of simple process, high production efficiency, consistent biological activity with natural plant extracts and the like, and is receiving more and more attention. With the development of modern molecular genetic technology, the separation of genes is promoted, the in-vivo function complementation, the characteristic optimization of recombinase and the creation of transgenic plants are realized, and the progress of microbial fermentation biosynthesis of carotenoid is promoted. Carotenoids produced by microbial fermentation have become a good alternative to industrially synthesized carotenoids. Among them, fungus microorganisms (such as Saccharomyces cerevisiae, Phaffia rhodozyma and Blakeslea trispora) are the best choice for producing carotenoids in microbial fermentation industrialization, as fermentation engineering bacteria widely applied in food and drug industries.
Generally, the thickness of the fungal microbial cell wall is about 0.1 to 0.3 μm, and the thickness increases as the cell ages, the structure is tough and difficult to break, and the cell must be broken or decomposed by external force. The carotenoid belongs to an intracellular product, the structure of the cell wall of a fungus microorganism must be completely destroyed in the extraction process, so that the carotenoid can be fully released and effectively extracted, otherwise, the carotenoid is difficult to extract due to the obstruction of the cell wall. The unique structure of the cell wall of the fungal microorganism causes the cell wall to be difficult to break, the commonly used cell wall breaking methods such as a grinding method, a freeze-thaw method, an acid method, a high-pressure homogenization method, an ultrasonic method and the like have an unsatisfactory cell wall breaking effect on the cell wall of the fungal microorganism, and some methods can also destroy the activity of intracellular biomacromolecules.
In patent CN110407659A, a high-pressure homogenizer is adopted to carry out wall breaking on fungus microorganisms, the wall breaking pressure is 100MPa, the energy consumption is high, and the wall breaking rate is generally not more than 80%; in addition, the high-pressure homogenizer needs to break the wall in a liquid environment, after the wall is broken, a large amount of cell contents are gushed out, solid-liquid separation is troublesome, even if membrane concentration is adopted for solid-liquid separation, the water content is still as high as 40-50%, and the subsequent vacuum drying cost is greatly increased; patent CN201310116039.5 discloses a method for extracting astaxanthin, which adopts acidic aqueous solution to break the walls of algae, the wall-breaking step is simple, the wall-breaking rate is as high as more than 95%, but the wall-breaking temperature is high, which leads to unstable astaxanthin and loss of effective components (generally more than 25%); some patents adopt enzymatic wall breaking, but the time consumption of enzymatic treatment is long, generally for several hours, the carotenoid loss is about 20%, and the enzymatic wall breaking cost is high.
Therefore, the microbial wall breaking of the fungi is a difficult point for extracting the carotenoid through biological fermentation and is an important reason for restricting the extraction yield and the extraction cost. Incomplete wall breaking can increase the difficulty of the carotenoid extraction process and limit the extraction yield; in addition, since the carotenoid is a reductive substance, the carotenoid is very easy to oxidize, and the oxidation risk is increased and the purity is reduced when the carotenoid is exposed to an unstable environment for a long time in the wall breaking process.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art to some extent.
To this end, in one aspect of the invention, the invention proposes a process for extracting carotenoids from microbial fermentation sources. According to an embodiment of the invention, the method comprises: (1) spray drying the fermentation liquor containing carotenoid to obtain first bacterial powder; (2) performing wall breaking treatment on the first bacterium powder by using a low-temperature shearing type wall breaking machine to obtain second bacterium powder (3), adding an alkaline ethanol solution into the second bacterium powder for saponification, and filtering to obtain third bacterium powder; (4) adding an organic solvent into the third bacterial powder for extraction, filtering the bacterial powder and collecting filtrate; (5) and (3) carrying out vacuum reduced pressure concentration on the filtrate to obtain a concentrated solution, placing the concentrated solution in a low-temperature environment, stirring and crystallizing, adding a small amount of absolute ethyl alcohol for rinsing, filtering to obtain crystals, and placing the crystals in a vacuum drying oven for drying to obtain pure carotenoid crystals.
The inventor finds that by adopting the scheme of firstly spray drying and then wall breaking, the defect that the carotenoid is exposed in a high-temperature environment for a long time in the drying process after wall breaking is avoided, the oxidation and isomerization losses of the carotenoid are reduced, and the product yield is improved; meanwhile, a single solvent is adopted for extraction and crystallization, so that the problem that the solvent is difficult to recover due to extraction and crystallization of various mixed solvents is solved, the recovery rate of the solvent can reach over 90 percent, and the cost is reduced; more importantly, the low-temperature shearing type wall breaking machine is adopted to break the wall of the dry yeast cells, the wall breaking rate of the yeast cells is up to more than 99% by optimizing the wall breaking conditions, the wall breaking loss is small, the method is greatly beneficial to the subsequent extraction and purification of the carotenoid, the final extraction rate is up to more than 98%, the purity of the carotenoid crystals is up to 99.9%, the total yield is up to more than 90%, and the product quality is stable and suitable for industrial production.
According to an embodiment of the present invention, the extraction process of carotenoids may also have the following additional technical features:
according to the embodiment of the invention, the microorganism is a common eukaryotic microorganism engineering bacterium (including saccharomyces cerevisiae, phaffia rhodozyma and blakeslea trispora) for fermentation expression of carotenoid, and the fermentation solid content is between 5% and 20%.
According to an embodiment of the invention, the spray drying parameters are set as: the air inlet temperature is 150-200 ℃, and preferably 180-190 ℃; the air outlet temperature is 70-90 ℃, and preferably 80-90 ℃.
According to the embodiment of the invention, the wall breaking method is wall breaking by a wall breaking machine, and the wall breaking conditions are as follows: the temperature is 0-10 ℃, the water content of the first bacterial powder is 5-15%, the preferred water content is 10%, and the wall breaking rate is at least 98%.
According to an embodiment of the invention, the ethanolic solution of a base is an ethanolic solution of sodium hydroxide.
According to an embodiment of the invention, the sodium hydroxide ethanol solution has a concentration of 1% to 10%, preferably 2% to 5%.
According to the embodiment of the present invention, the sodium hydroxide ethanol solution is 2 to 10 times, preferably 5 to 10 times of the weight of the dry microbial cells.
According to an embodiment of the present invention, the second fungal powder is extracted using an organic solvent.
According to an embodiment of the present invention, the organic solvent is selected from the group consisting of ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate and isobutyl acetate, preferably ethyl acetate;
according to the embodiment of the invention, the total amount of the organic solvent added is 10-30 times of the weight of the first bacterial powder.
According to the embodiment of the invention, the extraction times are 2-3 times.
According to an embodiment of the present invention, in the step (5), the extract is concentrated under reduced pressure in vacuum.
According to the embodiment of the invention, the temperature of the vacuum reduced pressure concentration is set to be 40-50 ℃.
According to the embodiment of the invention, the concentration is carried out until the final concentration of the extract liquor is 10-50 g/L.
According to the embodiment of the invention, the concentrated solution is placed in a low-temperature environment and stirred for crystallization, and a small amount of absolute ethyl alcohol is added for rinsing and filtering.
According to an embodiment of the invention, the temperature of crystallization is between-10 ℃ and 10 ℃, preferably between-4 ℃ and 4 ℃.
According to the embodiment of the invention, the crystallization time is 2-24 h, preferably 4-10 h.
According to an embodiment of the present invention, the crystal is dried in a vacuum drying oven.
According to the embodiment of the invention, the drying temperature is 20-60 ℃, and preferably 40-50 ℃.
According to the embodiment of the invention, the drying time is 2-20 hours, preferably 10-15 hours.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows microscopic images of lycopene-expressing Saccharomyces cerevisiae cells before wall breaking;
FIG. 2 shows microscopic images of lycopene-expressing Saccharomyces cerevisiae after cell wall breaking.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The low temperature shear wall breaking machine used in the following examples, model JRG-3C, was a general purpose wall breaking machine with closed circulating water cooling.
The method for calculating the wall breaking rate used in the following examples was as follows:
(1) respectively putting A1 g of the bacterial powder after wall breaking and A2 g of the bacterial powder before wall breaking into a 15ml centrifuge tube;
(2) wherein A1 is directly added with a plurality of acetone for extraction, then transferred to a 20ml volumetric flask, added with acetone for extraction continuously until the acetone is colorless, and the volume is constant to 20ml, thus obtaining a solution A to be detected;
(3) a2 performing wall breaking extraction by extracting yeast carotenoid by hot acid method, adding acetone according to (2) and extracting until colorless, and metering to volume of 20ml to obtain solution B to be detected;
(4) diluting the solution A to be detected and the solution B to be detected by isopropanol for N times, and then measuring the absorbance value A by using an ultraviolet spectrophotometer with the isopropanol as a blank under the corresponding wavelength of each sample; the absorbance value is controlled between 0.2 and 0.8, otherwise, the dilution factor is adjusted.
(5) Calculation of the content of fungal powder
In the formula:
a is the absorbance of the diluent at the corresponding wavelength, wherein the wavelength of lycopene is 470nm, the wavelength of beta-carotene is 450nm, and the wavelength of astaxanthin is 480 nm; n is the dilution multiple of the solution when the absorbance is measured;
v is the volume (ml) of acetone extract, here the number is 20;
A1％is extinction coefficient at 1% concentration, wherein lycopene is 3450AU, beta-carotene is 2500AU, astaxanthin is 2100 AU;
m is the weighed mass (mg) of the fungus powder;
10 is A1％The concentration of the solution (mg/ml).
(6) Calculation of wall breaking Rate
After the wall of the yeast cell is broken, acetone can be used for directly extracting and measuring the content of the carotenoid; the cells which are not wall-broken can not be directly extracted, and the content needs to be determined by wall breaking and then acetone extraction, so the wall-breaking rate is defined by the ratio of the carotenoid content of the cells after wall breaking to the carotenoid content of the cells before wall breaking, and the calculation formula of the wall-breaking rate is as follows:
the wall breaking rate is X1/X2 × 100%, X1 is the carotenoid content of the cell after wall breaking, and X2 is the carotenoid content of the cell before wall breaking. Method for extracting carotenoid from microbial fermentation
In one aspect of the invention, the invention provides a method for extracting carotenoids from microbial fermentation. According to an embodiment of the invention, the method comprises: s1 drying treatment; s2 wall breaking treatment; s3 saponification and filtration treatment; s4 extraction and filtration treatment; s5 concentration, crystallization, rinsing and drying. The extraction method adopts the method of drying firstly and breaking the wall later, reduces the risk of exposing the carotenoid to a high-temperature environment, reduces the loss of oxidation and isomerization, and greatly improves the purity of subsequent finished products. Adopt low temperature shear type broken wall machine to carry out the broken wall, when not causing the degradation loss, the broken wall rate also reaches more than 98%, has reduced subsequent extraction purification's the degree of difficulty. The single solvent is adopted for extraction and crystallization, the problem that solvent recovery is difficult due to the fact that various mixed solvents are adopted for extraction and crystallization is avoided, the solvent recovery rate reaches over 90%, and cost is reduced. The method has the advantages of simple process, mild conditions, high product purity, no heavy metal pollution, total yield of over 90 percent, stable product quality and suitability for industrial production.
The specific technical scheme is as follows:
a method for extracting biologically fermented carotenoid comprises the following steps:
s1 drying treatment
In this step, a carotenoid-containing microbial fermentation broth is spray-dried to obtain a first bacterial powder. Therefore, most of water in the fermentation liquor is removed, and the subsequent wall breaking treatment is facilitated.
According to the embodiment of the invention, the spray drying parameters are set to be that the inlet air temperature is 150-200 ℃, preferably 180-190 ℃, and the outlet air temperature is 70-90 ℃, preferably 80-90 ℃.
S2 breaking cell wall
In the step, the first bacterial powder is adjusted to a water content of about 10%, and a low-temperature shear type wall breaking machine is used for wall breaking. Thereby, the intracellular pigment is exposed to facilitate the subsequent extraction.
The low temperature shear type broken wall machine that this scheme used utilizes the compound mechanical force field that produces when two roller relative motion, makes the dry fungus powder of adding sheared broken wall at low temperature, and broken wall rate is 98% at least.
According to the embodiment of the invention, in the step S2, the wall breaking temperature is 0-10 ℃, so that the carotenoid is prevented from degradation loss in the wall breaking process.
According to the embodiment of the invention, in the step S2, the wall breaking time is 30-60 min. The invention does not strictly limit the wall breaking time, only needs the wall breaking rate to reach more than 98 percent, and can be flexibly adjusted according to the actual situation.
S3 saponification and filtration treatment
In the step, the second bacterial powder is added with an ethanol solution of alkali for stirring and saponification, and then the third bacterial powder is obtained after filtration. Therefore, fat-soluble impurities and other miscellaneous pigments in the dry bacterial powder can be removed conveniently.
According to the embodiment of the present invention, the concentration of the ethanol solution of sodium hydroxide used for saponification is 1% to 10%, preferably 2% to 5%, and the amount added is 2 to 10 times, preferably 5 to 10 times the weight of the dry microbial cells. Saponifying by adding sodium hydroxide ethanol solution to remove fat-soluble impurities and other heterochromyl, and filtering to retain the carotenoid-containing bacterial powder.
According to the embodiment of the invention, the saponification treatment is carried out at 40-50 ℃ for 30-60 min, the specific saponification time and temperature are determined according to the content of fat-soluble impurities and heteropigments, and the destruction and degradation of the carotenoid are reduced as much as possible.
S4 extraction and filtration treatment
In this step, the third bacterial powder is subjected to repeated extraction and filtration treatment. Therefore, the organic solvent can fully extract the carotenoid in the bacterial powder, so that the impurities such as inorganic salt and the like can be removed by filtering along with the bacterial powder.
According to the embodiment of the invention, the carotenoid is extracted from the crushed fungus powder by utilizing the characteristic that the carotenoid is easily dissolved in an organic solvent. Filtering to separate bacteria powder and extractive solution, repeatedly extracting the separated bacteria powder, and mixing the extractive solutions.
According to the embodiment of the invention, the organic solvent used for extraction is selected from ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate and isobutyl acetate, preferably ethyl acetate, and the total amount of the organic solvent added is 10-30 times of the weight of the first bacterial powder.
According to the embodiment of the invention, in the step S4, the number of times of extraction is 2-3. The extraction times are not strictly limited, the carotenoid in the fungus powder is completely extracted, and the extraction method can be flexibly adjusted according to actual conditions.
According to the embodiment of the invention, the filtering mode is not strictly limited, as long as the fungus powder and the extract liquid are separated under the condition of not causing the damage and the degradation of the carotenoid, and the fungus powder and the extract liquid can be flexibly selected according to the actual situation.
S5 concentration, crystallization, rinsing and drying treatment
In this step, the extract obtained in step S4 is concentrated under reduced pressure in vacuo, and the extract is concentrated to an appropriate concentration. Therefore, most of the organic solvent can be removed, and the organic solvent can be recycled, so that the process cost is reduced.
According to the embodiment of the invention, the temperature of vacuum reduced pressure concentration is 40-50 ℃, and the extract liquid is concentrated to 10-50 g/L.
In this step, the concentrated solution is placed in a low-temperature environment and crystallized by stirring. Thereby, the carotenoid is crystallized and precipitated, and the purpose of separating and removing impurities is achieved.
According to the examples of the present invention, after completion of the crystallization treatment, a small amount of anhydrous ethanol was added for rinsing, and filtration was carried out to collect carotenoid crystals. So as to further remove residual fat-soluble impurities and further improve the purity.
According to the embodiment of the invention, the crystallization is carried out in a low-temperature environment of 0-10 ℃, and the crystallization time is 2-24 h. Thereby, the carotenoid crystals are fully precipitated, and the yield and the purity are further improved.
According to an embodiment of the present invention, the crystallization is completed and then a filtration treatment is performed. Thereby facilitating separation and removal of the organic solvent to obtain carotenoid crystals for subsequent drying.
According to the embodiment of the invention, the amount of the absolute ethyl alcohol added is not strictly limited, so long as the residual fat-soluble impurities in the crystals are effectively removed, and the method can be flexibly adjusted according to actual conditions.
According to an embodiment of the invention, the invention uses a filter membrane to filter the crystals. The filtering mode is not strictly limited, so long as the carotenoid crystals and the organic solvent are separated under the condition of not causing the destruction and degradation of the carotenoid, and the filtering mode can be flexibly selected according to the actual situation.
According to an embodiment of the present invention, in this step, the carotenoid crystals are dried in a vacuum drying oven. Thereby, the residual organic solvent and moisture are removed.
According to the embodiment of the invention, the drying temperature is 20-60 ℃, and preferably 40-50 ℃; the drying time is 2-20h, preferably 10-15 h. Thereby, the residual organic solvent and water can be removed sufficiently, and the high-purity carotenoid finished product can be obtained.
Example 1
A method for extracting biologically fermented carotenoid, which takes saccharomyces cerevisiae fermentation liquor expressing lycopene as a raw material for extraction, specifically comprises the following steps:
(1) spray drying 20L lycopene fermentation broth after fermentation and canning, setting air inlet temperature of 190 ℃ and air outlet temperature of 90 ℃, and spray drying to obtain 1kg of dry yeast powder;
(2) humidifying the dry yeast powder by purified water to adjust the water content to 10%, then performing wall breaking treatment by a low-temperature shearing type wall breaking machine, breaking the wall for 30min, wherein the cooling mode is closed circulating water cooling, the temperature is 8 ℃, collecting the wall-broken yeast powder, and detecting that the wall breaking rate is more than 98% and the lycopene content loss is less than 2%;
(3) adding 10L ethanol solution with sodium hydroxide content of 1% into the dried cell powder after cell wall breaking for saponification, stirring at 40 deg.C for 60min, filtering, collecting cell powder, and draining ethanol to the greatest extent;
(4) adding 10L ethyl acetate into the bacterial powder obtained in the step (3) for extraction, stirring and extracting for 30min at the temperature of 50 ℃, repeating for 3 times, combining the extract liquor, filtering the thalli and collecting the extract liquor;
(5) vacuum concentrating the extractive solution at 45 deg.C until final lycopene concentration is 50g/L, and recovering ethyl acetate for recycling;
(6) freezing and crystallizing the concentrated solution, slowly stirring and crystallizing for 8 hours at the low temperature of 0 ℃, filtering crystallization mother liquor by using a filter membrane to recover a solvent after crystallization is finished, adding a small amount of absolute ethyl alcohol into the crystals to rinse and filter;
(7) and drying the filtered crystal in a vacuum drying oven at 50 ℃ for 10 h. 90g of pure lycopene crystal with the content of 98.86 percent, the extraction rate of 98 percent, the total yield of the product of 90 percent and the total recovery rate of the ethyl acetate solvent of 92 percent is obtained.
Example 2
A method for extracting biologically fermented carotenoid, which takes saccharomyces cerevisiae fermentation liquor expressing beta-carotene as a raw material for extraction, and specifically comprises the following steps:
(1) spray drying 22L of beta-carotene fermentation liquor after fermentation and canning, setting the air inlet temperature at 180 ℃ and the air outlet temperature at 80 ℃, and spray drying to obtain 1.32kg of dry yeast powder;
(2) humidifying the dry yeast powder by purified water to adjust the water content to 10%, then performing wall breaking treatment by a low-temperature shearing type wall breaking machine, breaking the wall for 45min, wherein the cooling mode is closed circulating water cooling, the temperature is 4 ℃, collecting the wall-broken yeast powder, and detecting that the wall breaking rate is more than 99% and the loss of the beta-carotene content is less than 1.5%;
(3) adding 6.6L ethanol solution with sodium hydroxide content of 2% into the dried cell powder after cell wall breaking for saponification, stirring at 40 deg.C for 60min, filtering, collecting cell powder, and draining ethanol to the greatest extent;
(4) adding 16.5L ethyl acetate into the bacterial powder obtained in the step (3) for extraction, stirring and extracting for 60min at the temperature of 40 ℃, repeating for 2 times, combining the extract liquor, and filtering the thallus collecting liquid;
(5) vacuum concentrating the extractive solution at 40 deg.C under reduced pressure until the final concentration of beta-carotene is 40g/L, and recovering ethyl acetate for recycling;
(6) and (3) performing freeze crystallization on the concentrated solution, and slowly stirring and crystallizing for 4 hours at the low temperature of-4 ℃. After crystallization is finished, filtering crystallization mother liquor by using a filter membrane to recover the solvent, adding a small amount of absolute ethyl alcohol into the crystals to rinse and filter;
(7) and (3) drying the filtered crystal in a vacuum drying oven at the drying temperature of 50 ℃ for 12 h. 120g of pure beta-carotene crystal with the content of 99.90 percent, the extraction rate of 98.5 percent, the total yield of the product of 91 percent and the total recovery rate of the ethyl acetate solvent of 91 percent is obtained.
Example 3
A method for extracting biologically fermented carotenoid, which takes saccharomyces cerevisiae fermentation liquor expressing astaxanthin as a raw material for extraction, specifically comprises the following steps:
(1) spray drying 25L astaxanthin fermentation liquid after fermentation and tank placing, setting air inlet temperature of 190 ℃ and air outlet temperature of 90 ℃, and spray drying to obtain 1.4kg of dry yeast powder;
(2) humidifying the dry yeast powder by purified water to adjust the water content to 9%, then performing wall breaking treatment by a low-temperature shearing type wall breaking machine for wall breaking for 60min, wherein the cooling mode is closed circulating water cooling, the temperature is 0 ℃, collecting the wall-broken yeast powder, and detecting that the wall breaking rate is more than 99% and the astaxanthin content loss is less than 2.5%;
(3) adding 8.4L ethanol solution with sodium hydroxide content of 3% into the dried cell powder after cell wall breaking for saponification, stirring at 40 deg.C for 30min, filtering, collecting cell powder, and draining ethanol to the greatest extent;
(4) adding 7L ethyl acetate into the bacterial powder obtained in the step (3) for extraction, stirring and extracting for 30min at the temperature of 50 ℃, repeating for 3 times, combining the extract liquor, filtering the thalli and collecting the extract liquor;
(5) vacuum concentrating the extractive solution at 40 deg.C until final lycopene concentration is 45g/L, and recovering ethyl acetate for recycling;
(6) and (3) performing freeze crystallization on the concentrated solution, and slowly stirring and crystallizing for 10 hours at a low temperature of 4 ℃. After crystallization is finished, filtering crystallization mother liquor by using a filter membrane to recover the solvent, adding a small amount of absolute ethyl alcohol into the crystals to rinse and filter;
(7) and drying the filtered crystals in vacuum, wherein the drying temperature is set to be 40 ℃, and the drying time is 15 h. 26.94g of pure astaxanthin crystal is obtained, the content is 97.89%, the extraction rate is 96.7%, the total yield of the product is 89.8%, and the total recovery rate of ethyl acetate solvent is 95%.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A method for extracting carotenoid from microbial fermentation is characterized by comprising the following steps:
(1) drying a microbial fermentation broth containing carotenoid to obtain first bacterial powder;
(2) performing wall breaking treatment on the first bacterium powder to obtain second bacterium powder;
(3) saponifying the second bacterial powder to obtain third bacterial powder;
(4) extracting the third bacterial powder by using a single organic solvent to obtain an extract liquid;
(5) concentrating, crystallizing, rinsing and drying the extract liquor to obtain pure carotenoid crystals;
the method is characterized in that: the wall breaking treatment in the step (2) needs to ensure that the wall breaking rate of microbial cells reaches at least 98%; water is not introduced in the steps (3), (4) and (5); the purity of the obtained carotenoid crystals is at least 97%, and the total yield is at least 89%.
2. The method of claim 1, wherein the microorganism is a eukaryotic microorganism-engineered bacterium;
optionally, the eukaryotic microorganism engineering bacteria are selected from saccharomyces cerevisiae, phaffia rhodozyma and blakeslea trispora.
3. The method according to claim 1, wherein the carotenoid is a carotenoid representative of the types commonly found in food and feed additives;
optionally, the carotenoid is selected from lycopene, beta-carotene and astaxanthin.
4. The method of claim 1, wherein the drying is spray drying;
optionally, the temperature of the spray drying inlet air is set to be 150-200 ℃, and preferably 180-190 ℃;
optionally, the temperature of the spray drying air outlet is set to be 70-90 ℃, and preferably 80-90 ℃.
5. The method according to claim 1, wherein the wall breaking treatment is to break the wall of the microorganism by using a shear wall breaking machine;
optionally, the water content of the first bacterial powder before the wall breaking treatment is adjusted to 5-15%, preferably 10%.
Optionally, the temperature of the wall breaking treatment is 0-10 ℃.
6. The method according to claim 1, wherein the saponification treatment is saponification using an ethanol solution of sodium hydroxide;
optionally, the concentration of the sodium hydroxide ethanol solution is 1-10%, preferably 2-5%.
Optionally, the addition amount of the sodium hydroxide ethanol solution is 2-10 times, preferably 5-10 times of the weight of the first bacterial powder.
7. The method of claim 1, wherein the third fungal powder is subjected to an extraction treatment using a single organic solvent;
optionally, said organic solvent is selected from the group consisting of ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate and isobutyl acetate, preferably ethyl acetate;
optionally, the total amount of the organic solvent added is 10-30 times of the weight of the first bacterial powder.
8. The method according to claim 1, wherein the crystals are rinsed in step (5) with anhydrous ethanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010843264.9A CN111995880B (en) | 2020-08-20 | 2020-08-20 | Method for extracting biologically fermented carotenoid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010843264.9A CN111995880B (en) | 2020-08-20 | 2020-08-20 | Method for extracting biologically fermented carotenoid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111995880A true CN111995880A (en) | 2020-11-27 |
| CN111995880B CN111995880B (en) | 2022-04-29 |
Family
ID=73472261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010843264.9A Active CN111995880B (en) | 2020-08-20 | 2020-08-20 | Method for extracting biologically fermented carotenoid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111995880B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114133348A (en) * | 2021-11-09 | 2022-03-04 | 湖北广济药业股份有限公司 | Method for extracting high-purity all-trans beta-carotene |
| CN115010636A (en) * | 2022-06-30 | 2022-09-06 | 山东微研生物科技有限公司 | Extraction method of cantharis yellow fermentation liquid |
| CN115160201A (en) * | 2022-07-29 | 2022-10-11 | 浙江可明生物医药有限公司 | Method for purifying beta-carotene in fermentation liquor |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732049A (en) * | 2012-06-04 | 2012-10-17 | 华中科技大学 | Method for preparing and extracting carotenoid from microbial thalli |
| CN102787158A (en) * | 2011-05-20 | 2012-11-21 | 浙江医药股份有限公司新昌制药厂 | Method for producing natural beta-carotene by fermentation and application |
| CN103012230A (en) * | 2013-01-06 | 2013-04-03 | 大连医诺生物有限公司 | Novel process for high-effective extraction of carotenoid in Blakeslea trispora |
| CN106220541A (en) * | 2016-08-10 | 2016-12-14 | 大连医诺生物有限公司 | The method preparing bata-carotene for raw material with trispore Bruce mould mycelium |
| CN106554984A (en) * | 2016-10-13 | 2017-04-05 | 嘉必优生物技术（武汉）股份有限公司 | The method for extracting bata-carotene |
| CN106565399A (en) * | 2016-10-13 | 2017-04-19 | 嘉必优生物技术（武汉）股份有限公司 | A lycopene extracting method |
| CN108017568A (en) * | 2016-11-04 | 2018-05-11 | 嘉必优生物技术（武汉）股份有限公司 | The method for extracting beta carotene |
-
2020
- 2020-08-20 CN CN202010843264.9A patent/CN111995880B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787158A (en) * | 2011-05-20 | 2012-11-21 | 浙江医药股份有限公司新昌制药厂 | Method for producing natural beta-carotene by fermentation and application |
| CN102732049A (en) * | 2012-06-04 | 2012-10-17 | 华中科技大学 | Method for preparing and extracting carotenoid from microbial thalli |
| CN103012230A (en) * | 2013-01-06 | 2013-04-03 | 大连医诺生物有限公司 | Novel process for high-effective extraction of carotenoid in Blakeslea trispora |
| CN106220541A (en) * | 2016-08-10 | 2016-12-14 | 大连医诺生物有限公司 | The method preparing bata-carotene for raw material with trispore Bruce mould mycelium |
| CN106554984A (en) * | 2016-10-13 | 2017-04-05 | 嘉必优生物技术（武汉）股份有限公司 | The method for extracting bata-carotene |
| CN106565399A (en) * | 2016-10-13 | 2017-04-19 | 嘉必优生物技术（武汉）股份有限公司 | A lycopene extracting method |
| CN108017568A (en) * | 2016-11-04 | 2018-05-11 | 嘉必优生物技术（武汉）股份有限公司 | The method for extracting beta carotene |
Non-Patent Citations (3)
| Title |
|---|
| 庞昆云: "基于多参数相关分析的番茄红素发酵过程优化及提取工艺研究", 《华东理工大学硕士学位论文》 * |
| 庞昆云等: "三孢布拉霉菌发酵番茄红素提取工艺的优化", 《华东理工大学学报(自然科学版)》 * |
| 谭新国等: "三孢布拉霉发酵生产β-胡萝卜素 ", 《中南民族学院学报(自然科学版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114133348A (en) * | 2021-11-09 | 2022-03-04 | 湖北广济药业股份有限公司 | Method for extracting high-purity all-trans beta-carotene |
| CN115010636A (en) * | 2022-06-30 | 2022-09-06 | 山东微研生物科技有限公司 | Extraction method of cantharis yellow fermentation liquid |
| CN115160201A (en) * | 2022-07-29 | 2022-10-11 | 浙江可明生物医药有限公司 | Method for purifying beta-carotene in fermentation liquor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111995880B (en) | 2022-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111995880B (en) | Method for extracting biologically fermented carotenoid | |
| CN107473999B (en) | Microbial wall breaking method and carotenoid product | |
| WO2012159446A1 (en) | METHOD FOR PRODUCING NATURAL β-CAROTENE BY FERMENTATION AND USE THEREOF | |
| CN101381337A (en) | Astaxanthin extraction method | |
| CN102796610A (en) | Production technology for extracting eucommia oil and eucommia protein from eucommia kernels | |
| CN103012230A (en) | Novel process for high-effective extraction of carotenoid in Blakeslea trispora | |
| CN110407659B (en) | Carotenoid extraction method | |
| US8921615B2 (en) | Process for preparing xanthophyll crystal | |
| EP3143155A1 (en) | Process for preparing carotenoids by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus blakeslea trispora | |
| CN1830959A (en) | Method for preparing astraxantbin using phytoxanthin extraction | |
| CN102676605A (en) | Method for producing xylose and L-arabinose using xylose mother liquor | |
| JPH04197192A (en) | Production of xylose and reduced xylose | |
| CN116970018B (en) | Ergosterol preparation and extraction method | |
| CN101643758B (en) | Method for extracting monensin sodium by methanol extraction method | |
| CN101891774B (en) | Production process of rhamnose | |
| CN108017505B (en) | Method for extracting lycopene | |
| CN107602434B (en) | Astaxanthin crystal derived from haematococcus pluvialis and preparation process thereof | |
| CN109485559A (en) | A method of extracting shikimic acid from illiciumverum | |
| CN108440359A (en) | One kind extracting astaxanthin from haematococcus pluvialis（Oil）Method | |
| CN108017568B (en) | Method for extracting β -carotene | |
| CN106554273B (en) | Method for purifying long-chain dicarboxylic acid in fermentation liquor | |
| CN107266512A (en) | A kind of purification process of spiramvcin | |
| CN113912480A (en) | Extraction method of coenzyme Q10 | |
| CN109280685B (en) | Method for producing natural carotenoid by increasing dissolved oxygen in fermentation process of natural carotenoid | |
| CN104327082B (en) | A kind of method that high-efficiency environment friendly reclaims riboflavin from crystalline mother solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |