CN113862210A - Method for promoting high yield of astaxanthin by phaffia rhodozyma - Google Patents

Method for promoting high yield of astaxanthin by phaffia rhodozyma Download PDF

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CN113862210A
CN113862210A CN202110510672.7A CN202110510672A CN113862210A CN 113862210 A CN113862210 A CN 113862210A CN 202110510672 A CN202110510672 A CN 202110510672A CN 113862210 A CN113862210 A CN 113862210A
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astaxanthin
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phaffia rhodozyma
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胡鹏
张小芳
李勇
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Suzhou Jishilai Amine Biotechnology Co ltd
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Abstract

The invention discloses a method for promoting high yield of astaxanthin by phaffia rhodozyma by TiO2/Al2O3, which is characterized by comprising the following steps: the method comprises the following steps: inoculating Phaffia yeast seed liquid with nano TiO2/Al2O3Fermenting and culturing in sterile fermentation medium of mother liquor to obtain nanometer TiO2/Al2O3The mother liquor is a nano titanium dioxide mixed liquor taking a nano alumina water dispersion liquid as a carrier, and the pH value inside and outside the phaffia rhodozyma cells is kept unbalanced during fermentation, so that the stress reaction of the phaffia rhodozyma is excited, and the metabolic pathway in the phaffia rhodozyma is oriented to the direction of producing astaxanthin. The invention has the advantages that: by using nano alumina water dispersion as carrier, treating and dissolvingThe nanometer titanium dioxide is added into a fermentation medium, the pH value imbalance inside and outside the phaffia rhodozyma cell is kept during fermentation, the phaffia rhodozyma stress reaction is excited, the metabolic pathway in the phaffia rhodozyma body is enabled to be carried out towards the direction of producing the astaxanthin, and the content of the astaxanthin is increased.

Description

Method for promoting high yield of astaxanthin by phaffia rhodozyma
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for promoting high yield of astaxanthin by phaffia rhodozyma by TiO2/Al2O 3.
Background
Astaxanthin (Astaxanthin) has a chemical name of 3,3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene and a molecular formula of C40H52O4The compound has a relative molecular mass of 596.86, belongs to ketocarotenoid, and is a terpene unsaturated compound. The crystal astaxanthin is pink, has a melting point of 215/216 ℃, is insoluble in water, has fat solubility, and is easily soluble in most organic solvents such as chloroform, acetone, benzene and the like. The conjugated double bond chain in the astaxanthin molecular structure and the unsaturated ketone group and the hydroxyl group at the tail end of the conjugated double bond chain can attract unpaired electrons of free radicals or provide electrons for the free radicals, so that the free radicals are removed, the antioxidation effect is realized, the structure also enables the astaxanthin molecular structure to easily react with light, heat and oxides, and the astaxanthin molecular structure is degraded into the astaxanthin after the structure is changed. The chemical structural formula of astaxanthin is shown in figure 1.
Astaxanthin exists mainly in both free and esterified forms. Astaxanthin in the free state is extremely unstable and easily oxidized, and astaxanthin is generally chemically synthesized in the free form. The esterified astaxanthin is stable because each hydroxyl group in the terminal ring structure of astaxanthin is easy to form ester with fatty acid, astaxanthin on skin and outer shell of aquatic animal is mainly in lipidated form, astaxanthin on meat and viscera is mainly in free form, and astaxanthin in rhodotorula rubra and haematococcus pluvialis is mainly in esterified form.
Astaxanthin has a strong antioxidant activity. Animal experiments show that astaxanthin can remove NO2Sulfides and disulfides, and also can reduce lipid peroxidation and effectively inhibit lipid peroxidation initiated by free radicals. Simultaneously has anticancer activity, can significantly influence animal immunity, and enhance aerobic metabolismIt can obviously enhance human muscle strength and tolerance, and has anti-infectious activity. In the feed industry, astaxanthin is mainly used as a feed additive for crustaceans and poultry such as fish (salmon, sturgeon, rainbow trout, red sea bream, etc.) and shrimp and crab. The astaxanthin can effectively protect the hepatopancreas of aquatic animals and the like, so that the aquatic animals have healthy and bright colors. Astaxanthin has strong functions in preventing and treating diseases of fishes, shrimps, crabs and poultry, can improve immunity and survival rate, and has important effects on normal growth and healthy culture, and survival rate and reproduction rate improvement. Astaxanthin also can increase flavor of fish, and can be directly used as precursor compound for forming food flavor of salmon, reeves shad, etc., and can also promote fatty acid or other lipid precursor to be converted into flavor compound of salmon. The astaxanthin is added into the poultry feed to increase the yolk pigment content of eggs, and the egg laying rate of hens can be improved, and the health of the laying hens is promoted.
At present, two modes of chemical synthesis and microbial synthesis extraction are mainly used for producing astaxanthin, and chemically synthesized astaxanthin is expensive, and has obvious differences from natural astaxanthin in the aspects of molecular structure biological function, application effect and biological safety performance. At present, methods for the fermentative synthetic extraction of astaxanthin using microorganisms are increasingly dominating. Astaxanthin is derived from algae, bacteria, and phytoplankton. Crustaceans of some aquatic species, including shrimps and crabs, appear red due to long-term consumption of these algae, bacteria and phytoplankton, which are also preyed by fishes such as salmon, afterburns, and the like, birds such as flamingo, chickens, ducks, and the like, and poultry, and pigments are stored in skin and adipose tissues to make their skin and feathers also appear red. Research shows that a variety of algae such as snow algae, chlamydomonas, euglena, and umbrella algae contain astaxanthin, wherein the accumulation amount of astaxanthin by haematococcus pluvialis is higher than that of other green algae, and the haematococcus pluvialis is a well-accepted biological source for producing natural astaxanthin. The bacteria are influenced by the factors of the bacteria, so the utilization value is low. Phaffia is considered to be the most suitable source of astaxanthin currently produced by fermentation of fungi. Extraction of astaxanthin from Phaffia rhodozyma is one of the major routes for the production of astaxanthin.
Phaffia can be astaxanthin-producing yeast in nature, and trans-astaxanthin has been approved by the FDA in 2000 for use as a food additive. Phaffia yeast is mainly unicellular and sometimes forms pseudo hypha, the propagation mode is vegetative propagation bud propagation and has no sexual propagation, the cells not only contain rich protein, lipid and vitamin, but also contain a large amount of unsaturated fatty acid and a plurality of astaxanthin precursors, and the growth temperature range of the Phaffia yeast is 0-27 ℃. The method for producing astaxanthin by phaffia rhodozyma has the characteristics that: the Phaffia rhodozyma does not need illumination, can utilize various sugars as carbon sources to carry out rapid heterotrophic metabolism, has short fermentation period and high production speed, can realize high-density culture in a fermentation tank, and can be used as bait, feed additives and the like after pigment extraction.
Microorganisms such as yeast or Escherichia coli can synthesize astaxanthin in vivo by using energy substances such as glucose or oil and fat. A schematic diagram of the synthetic pathway of astaxanthin in Phaffia rhodozyma is shown in FIG. 2. After the microorganisms take Glucose (Glucose) or other energy substances, the microorganisms are converted into a key metabolic intermediate Acetyl coenzyme A (Acetyl/CoA), isoprene pyrophosphoric acid (IPP) is synthesized through a mevalonate pathway (MVA), IPP generates farnesyl pyrophosphoric acid (FPP) through condensation reaction under the action of related enzymes, two molecules of FPP can be further condensed to generate geranium peronopyrum pyrophosphoric acid (GGPP), two molecules of GGPP further condenses to generate Phytoene (Phytoene), Phytoene is dehydrogenated and cyclized to generate beta/carotene, and the final product Astaxanthin (Astaxanthin) is generated through oxidation, ketonization and hydroxylation.
It is noted that the phaffia strain astaxanthin cultured under natural conditions has low astaxanthin production and is easy to degrade, and industrial production cannot be realized. Therefore, increasing astaxanthin production using various mutagenesis methods has been the first goal of research. The currently adopted methods include an ultraviolet ray mutagenesis method, a high-energy ray mutagenesis method, a chemical reagent mutagenesis method, a genetic engineering modification method and the like. For example, Gong 2012 (Gong, J., Duan, N., Zhao, X., 2012. Evaporation engineering of Phaffia rhodozyma for astaxanthin/overproducing strain. Front. chem. Sci. Eng. 6 (2), 174. one. 178.) the addition of micron-sized titanium dioxide (TiO 2) to the culture medium of Phaffia yeast to induce a stress reaction in the yeast, thereby overexpressing certain specific genes to increase astaxanthin production, is mentioned. Although the literature reports that the addition of titanium dioxide can promote the research of producing astaxanthin by phaffia rhodozyma, the solid particle size of the titanium dioxide mentioned in the literature is only micron grade, and the content of the astaxanthin in the phaffia rhodozyma cultured by the method reported in the literature is low because yeast fermentation is usually in aqueous solution, and the micron grade titanium dioxide is extremely low in solubility and hardly soluble in water, so that the micron grade titanium dioxide cannot be efficiently delivered to yeast cells during the fermentation.
Disclosure of Invention
The invention aims to provide a method for effectively promoting high yield of astaxanthin by phaffia rhodozyma.
In order to achieve the purpose, the invention adopts the following technical scheme: the method for promoting high yield of astaxanthin by phaffia rhodozyma by TiO2/Al2O3 comprises the following steps: inoculating Phaffia yeast seed liquid with nano TiO2/Al2O3Carrying out fermentation culture in a sterile fermentation culture medium of mother liquor, wherein the nano TiO is2/Al2O3The mother liquor is a nano titanium dioxide mixed liquor taking a nano alumina water dispersion liquid as a carrier, and the pH value inside and outside the phaffia rhodozyma cells is kept unbalanced during fermentation, so that the stress reaction of the phaffia rhodozyma is excited, and the metabolic pathway in the phaffia rhodozyma is oriented to the direction of producing astaxanthin.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: adding 100-200 mg of nano TiO into each liter of sterile fermentation medium2/Al2O3The pH value of the sterile fermentation medium of the mother liquor is kept between 5.2 and 7.8, the pH value in the Phaffia rhodozyma cells during fermentation is lower than the pH value outside the cells, and the pH difference between the pH value in the Phaffia rhodozyma cells and the pH value outside the cells during fermentation is kept between-2.0 and-1.4.
Further, the aforementioned TiO2/Al2O3 promoting methodA method for high yield of astaxanthin by phaffia rhodozyma comprises the following steps: nano TiO22/Al2O3The preparation method of the mother liquor comprises the following steps: mixing nano titanium dioxide into nano alumina water dispersion liquid, and uniformly stirring to obtain nano TiO2/Al2O3The mother liquor of the nano TiO2/Al2O3 contains 18-22 wt% of nano alumina particles, 1-2 wt% of nano titanium dioxide and 76-81 wt% of water, wherein the size of the solid nano titanium dioxide particles is 40-80 nanometers, and the particle size of the nano alumina particles is 5-10 nanometers.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: the pH value of the sterile fermentation medium is controlled and adjusted by using 2-5 mol/L NaOH aqueous solution and 1-2 mol/L hydrochloric acid aqueous solution.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: the preparation method of the Phaffia yeast seed liquid comprises the following steps: the method comprises the following steps of inoculating Phaffia rhodozyma into a seed culture medium for fermentation to prepare a Phaffia rhodozyma seed solution, wherein the seed culture medium usually adopts a yeast extract peptone glucose culture medium, and the seed culture medium comprises the following components: 1-3% of glucose, 1-3% of yeast extract and 1-2% of peptone.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: the seed fermentation process is carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-25 ℃.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: the sterile fermentation medium comprises the following components: 10-100 g/L of glucose, 0.5-1 g/L of yeast extract, 0.5-1 g/L of magnesium sulfate, 2-6 g/L of ammonium sulfate, 1-3 g/L of monopotassium phosphate, 1-3 g/L of dipotassium phosphate and 0.1-0.2 g/L of calcium chloride.
Further, the TiO2/Al2O3 promotes the high yield of astaxanthin by phaffia rhodozyma, wherein: inoculating the phaffia yeast seed liquid into an aseptic fermentation culture medium, and then performing fermentation culture in a fermentation tank, wherein the fermentation culture period is 48-72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled at 20 ℃, the initial stirring speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial aeration in the fermentation tank is 0.5-2 vvm, and the stirring speed and the aeration are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process.
Through the implementation of the technical scheme, the invention has the beneficial effects that: the invention uses nano-alumina water dispersion as a carrier to process and dissolve nano-titanium dioxide into a fermentation culture medium, so that the nano-titanium dioxide can be efficiently delivered into yeast cells during fermentation, the pH value in the cells can be rapidly regulated, the pH value imbalance between the inside and the outside of the phaffia yeast cells is kept during fermentation, namely the pH environment inside and outside the phaffia yeast cell membranes is changed, the pH value gradient inside and outside the phaffia yeast cell membranes is caused, the stress reaction of the phaffia yeast is further excited, the metabolic pathway inside the phaffia yeast is carried out towards the direction of producing astaxanthin, and the content of the astaxanthin in the phaffia yeast is effectively increased.
Drawings
FIG. 1 shows the chemical structural formula of astaxanthin.
FIG. 2 is a schematic diagram showing the synthetic pathway of astaxanthin in Phaffia rhodozyma.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the preferred embodiments.
Comparative example 1
The fermentation method can be applied to batch fermentation occasions and also can be applied to continuous fermentation occasions. This example illustrates the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermentor.
Firstly, Phaffia rhodozyma (ATCC 24202) is inoculated into 100ml of seed culture medium for fermentation to prepare a Phaffia rhodozyma seed solution; the seed culture medium is usually a yeast extract peptone glucose medium, which generally comprises the following components: 2% of glucose, 1% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-20 ℃;
2L of carbon-source-free sterile fermentation medium autoclaved at 121 ℃ is put into a 5L fermentation tank, the autoclaving time is 30 minutes, and the pH value of the fermentation medium is controlled and adjusted to 7.2 by using 5mol/L NaOH aqueous solution and 2mol/L hydrochloric acid aqueous solution; the composition of the sterile fermentation medium described in this example includes: 80g/L glucose, 1g/L yeast extract, 1g/L magnesium sulfate, 6g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L dipotassium phosphate and 0.2g/L calcium chloride; in particular, 500mg/L of titanium dioxide (Aladdin, CAS 13463/67/7, SKU # T164497) is additionally added to the culture medium, the solid particle size of the titanium dioxide is micron grade;
inoculating the obtained Phaffia rhodozyma seed liquid into a sterile fermentation culture medium with the pH value constant at 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled to be 15-25 ℃, the initial stirring rotating speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation volume in the fermentation tank is 0.5-2 vvm, and the stirring rotating speed and the ventilation volume are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process;
when 72 hours of fermentation was completed and samples were taken, Intracellular pH at that time point was measured using an Intracellular pH test kit (purchased from abcam, Intracellular pH Assay kit ab 228552), and the pH at the cell membrane inner wall at that time point was 5.8; because the pH of the fermentor was kept constant at 7.2 at all times, a pH gradient Δ pH (intracellular pH/extracellular pH) of about-1.4 was calculated inside and outside the cell membrane, i.e., the pH difference between the pH inside the Phaffia yeast cells and the pH outside the cells during fermentation was kept at-1.4;
cell dry weight determination was performed after the entire 72 hour fermentation was completed: weighing the fermentation liquor, centrifuging at the rotating speed of 4000rpm for 5 minutes, washing the obtained precipitate with water once, centrifuging at the rotating speed of 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃, weighing, and measuring the dry weight concentration of cells in the fermentation liquor to be 39 g/L.
1g of strain sample after 72 hours of fermentation is taken, after centrifugal washing, 3mol/L hydrochloric acid aqueous solution with the same volume is added into the strain sample, and the strain sample is heated in boiling water bath for 5 minutes to break cell walls. And extracting the cell after wall breaking by using acetone to obtain an acetone solution of astaxanthin. All acetone residues were removed by distillation at 45 ℃ in the absence of light. Then measuring the astaxanthin content by High Performance Liquid Chromatography (HPLC) to be about 3.2mg/g cell dry weight; the astaxanthin content 3.2mg/g obtained by the measurement is multiplied by the cell dry weight concentration 39g/L obtained by the fermentation liquid, and the concentration of the astaxanthin obtained by the fermentation of the batch is 124.8 mg/L.
Example two
The fermentation method can be applied to batch fermentation occasions and also can be applied to continuous fermentation occasions. This example illustrates the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermentor.
Firstly, 200mg of nano titanium dioxide (Allatin, CAS 13463/67/7, SKU # T104936) is weighed, and the solid particle size of the titanium dioxide is 60 nanometers; in addition, 10mL of nano alumina water dispersion (Aladdin, CAS 1344/28/1, SKU # A119404) is prepared, wherein the nano alumina water dispersion contains 20wt% of nano alumina particles, and the particle size of the nano alumina particles is 5-10 nanometers; mixing the 200mg nano titanium dioxide into 10mL nano alumina water dispersion, and stirring uniformly to obtain 10mL nano TiO containing 200mg nano titanium dioxide2/Al2O3Mother liquor; (since the amount of 200mg is small, the volume is hardly increased by adding 10ml of nano-alumina aqueous dispersion, so that nano-TiO is obtained2/Al2O3Mother liquor was also 10 mL);
phaffia rhodozyma (ATCC 24202) is inoculated into 100ml of seed culture medium for fermentation to prepare a Phaffia seed solution; the seed culture medium is usually a yeast extract peptone glucose medium, which generally comprises the following components: 1% of glucose, 1% of yeast extract, 1% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of carbon-source-free sterile fermentation medium autoclaved at 121 ℃ is put into a 5L fermentation tank, the autoclaving time is 30 minutes, and the pH value of the fermentation medium is controlled and adjusted to 7.2 by using 2mol/L NaOH aqueous solution and 1mol/L hydrochloric acid aqueous solution; the composition of the sterile fermentation medium described in this example includes: 60g/L glucose, 0.5g/L yeast extract, 0.5g/L magnesium sulfate, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate and 0.1g/L calcium chloride; in particular, 10mL of prepared nano TiO containing 200mg of nano titanium dioxide is added into the culture medium2/Al2O3Mother liquor, so that 100mg/L of nano TiO is added into the fermentation liquor2；
Inoculating the obtained Phaffia rhodozyma seed liquid into a sterile fermentation culture medium with the pH value constant at 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled to be 15-25 ℃, the initial stirring rotating speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation volume in the fermentation tank is 0.5-2 vvm, and the stirring rotating speed and the ventilation volume are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process;
when the 72 hour fermentation was completed and a sample was taken, the Intracellular pH at this time point was measured using an Intracellular pH test kit (purchased from abcam, Intracellular pH Assay kit ab 228552), and the pH at the inner wall of the cell membrane at this time point was 5.3. Since the pH of the fermenter was kept constant at 7.2 at all times, it was calculated that the pH gradient Δ pH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-1.9, i.e., the pH difference between the pH inside the Phaffia yeast cells and the pH outside the cells during fermentation was kept at-1.9;
cell dry weight determination was performed after the entire 72 hour fermentation was completed: weighing the fermentation liquor, centrifuging at the rotating speed of 4000rpm for 5 minutes, washing the obtained precipitate with water once, centrifuging at the rotating speed of 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃, weighing, and measuring the dry weight concentration of cells in the fermentation liquor to be 36 g/L.
1g of strain sample after 72 hours of fermentation is taken, after centrifugal washing, 3mol/L hydrochloric acid aqueous solution with the same volume is added into the strain sample, and the strain sample is heated in boiling water bath for 5 minutes to break cell walls. And extracting the cell after wall breaking by using acetone to obtain an acetone solution of astaxanthin. All acetone residues were removed by distillation at 45 ℃ in the absence of light. Then measuring the astaxanthin content by High Performance Liquid Chromatography (HPLC) to be about 12.3mg/g cell dry weight; the astaxanthin content 12.3mg/g obtained by the measurement is multiplied by the cell dry weight concentration 36g/L obtained by the fermentation liquid, and the concentration of the astaxanthin in the fermentation batch is 442.8 mg/L.
EXAMPLE III
The fermentation method can be applied to batch fermentation occasions and also can be applied to continuous fermentation occasions. This example illustrates the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermentor.
Firstly, 400mg of nano titanium dioxide (Allatin, CAS 13463/67/7, SKU # T104936) is weighed, and the solid particle size of the titanium dioxide is 60 nanometers; in addition, 10mL of nano alumina water dispersion (Aladdin, CAS 1344/28/1, SKU # A119404) is prepared, wherein the nano alumina water dispersion contains 20wt% of nano alumina particles, and the particle size of the nano alumina particles is 5-10 nanometers; mixing 300mg of the nano titanium dioxide into 10mL of nano alumina water dispersion, and uniformly stirring to obtain 10mL of nano TiO containing 300mg2Of nano TiO2/Al2O3Mother liquor; (since the amount of 300mg is small, the volume is hardly increased by adding 10ml of nano-alumina aqueous dispersion, so that nano-TiO is obtained2/Al2O3Mother liquor was also 10 mL);
phaffia rhodozyma (ATCC 24202) is inoculated into 100ml of seed culture medium for fermentation to prepare a Phaffia seed solution; the seed medium is usually YPD medium (yeast extract peptone glucose medium) and its composition usually comprises: 3% of glucose, 3% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of the fermented liquid is put into a 5L fermentation tank and is heated to 121 DEG CAutoclaving a carbon-source-free sterile fermentation medium for 30 minutes, and controlling and adjusting the pH value of the fermentation medium to 7.2 by using a 5mol/L NaOH aqueous solution and a 2mol/L hydrochloric acid aqueous solution; the composition of the sterile fermentation medium described in this example includes: 100g/L glucose, 1g/L yeast extract, 1g/L magnesium sulfate, 6g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L dipotassium phosphate and 0.2g/L calcium chloride; in particular, 10mL of prepared medium containing 300mg of nano TiO was added2Of nano TiO2/Al2O3Mother liquor, so that 150 mg/L of nano TiO is added into the fermentation liquor2；
Inoculating the obtained Phaffia rhodozyma seed liquid into a sterile fermentation culture medium with the pH value constant at 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled to be 15-25 ℃, the initial stirring rotating speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation volume in the fermentation tank is 0.5-2 vvm, and the stirring rotating speed and the ventilation volume are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process;
when the 72 hour fermentation was completed and a sample was taken, the Intracellular pH at this time point was measured using an Intracellular pH test kit (purchased from abcam, Intracellular pH Assay kit ab 228552), and the pH at the inner wall of the cell membrane at this time point was 5.5. Since the pH of the fermenter was kept constant at 7.2 at all times, it was calculated that the pH gradient Δ pH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-1.7, i.e., the pH difference between the pH inside the Phaffia yeast cells and the pH outside the cells during fermentation was kept at-1.7;
cell dry weight determination was performed after the entire 72 hour fermentation was completed: weighing the fermentation liquor, centrifuging at the rotating speed of 4000rpm for 5 minutes, washing the obtained precipitate with water once, centrifuging at the rotating speed of 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃, weighing, and measuring the dry weight concentration of cells in the fermentation liquor to be 34 g/L.
1g of strain sample after 72 hours of fermentation is taken, after centrifugal washing, 3mol/L hydrochloric acid aqueous solution with the same volume is added into the strain sample, and the strain sample is heated in boiling water bath for 5 minutes to break cell walls. And extracting the cell after wall breaking by using acetone to obtain an acetone solution of astaxanthin. All acetone residues were removed by distillation at 45 ℃ in the absence of light. Then using High Performance Liquid Chromatography (HPLC), the astaxanthin content was determined to be about 11.8mg/g cell dry weight; the astaxanthin content 11.8mg/g obtained by the measurement is multiplied by the cell dry weight concentration 34g/L obtained by the fermentation liquid, and the concentration of the astaxanthin in the fermentation batch is 401.2 mg/L.
Example four
The fermentation method can be applied to batch fermentation occasions and also can be applied to continuous fermentation occasions. This example illustrates the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermentor.
Firstly, 400mg of nano titanium dioxide (Allatin, CAS 13463/67/7, SKU # T104936) is weighed, and the solid particle size of the titanium dioxide is 60 nanometers; in addition, 10mL of nano alumina water dispersion (Aladdin, CAS 1344/28/1, SKU # A119404) is prepared, wherein the nano alumina water dispersion contains 20wt% of nano alumina particles, and the particle size of the nano alumina particles is 5-10 nanometers; mixing 400mg of the nano titanium dioxide into 10mL of nano alumina water dispersion liquid, and uniformly stirring to obtain 10mL of nano TiO containing 400mg of nano titanium dioxide2/Al2O3Mother liquor; (since the amount of 400mg is small, the volume is hardly increased by adding 10ml of nano-alumina aqueous dispersion, so that nano-TiO is obtained2/Al2O3Mother liquor was also 10 mL);
phaffia rhodozyma (ATCC 24202) is inoculated into 100ml of seed culture medium for fermentation to prepare a Phaffia seed solution; the seed culture medium is usually a yeast extract peptone glucose medium, which generally comprises the following components: 2% of glucose, 1% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of carbon-source-free sterile fermentation medium autoclaved at 121 ℃ is placed in a 5L fermenter for 30 minutes using 5mol/L NaOH aqueous solutionAnd 2mol/L hydrochloric acid aqueous solution is used for controlling and adjusting the pH value of the fermentation medium to 7.2; the composition of the sterile fermentation medium described in this example includes: 80g/L glucose, 1g/L yeast extract, 1g/L magnesium sulfate, 6g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L dipotassium phosphate and 0.2g/L calcium chloride; in particular, 10ml of prepared medium containing 400mg of nano TiO is added2Of nano TiO2/Al2O3Mother liquor, thereby adding 200mg/L of nano TiO into the fermentation liquor2；
Inoculating the obtained Phaffia rhodozyma seed liquid into a sterile fermentation culture medium with the pH value constant at 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled to be 15-25 ℃, the initial stirring rotating speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation volume in the fermentation tank is 0.5-2 vvm, and the stirring rotating speed and the ventilation volume are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process;
when the 72 hour fermentation was completed and a sample was taken, the Intracellular pH at this time point was measured using an Intracellular pH test kit (purchased from abcam, Intracellular pH Assay kit ab 228552), and the pH at the inner wall of the cell membrane at this time point was 5.2. Since the pH of the fermenter was kept constant at 7.2 at all times, it was calculated that the pH gradient Δ pH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-2.0, i.e., the pH difference between the pH inside the Phaffia yeast cells and the pH outside the cells during fermentation was kept at-2.0;
cell dry weight determination was performed after the entire 72 hour fermentation was completed: weighing the fermentation liquor, centrifuging at the rotating speed of 4000rpm for 5 minutes, washing the obtained precipitate with water once, centrifuging at the rotating speed of 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃, weighing, and measuring the dry weight concentration of cells in the fermentation liquor to be 35 g/L.
1g of strain sample after 72 hours of fermentation is taken, after centrifugal washing, 3mol/L hydrochloric acid aqueous solution with the same volume is added into the strain sample, and the strain sample is heated in boiling water bath for 5 minutes to break cell walls. And extracting the cell after wall breaking by using acetone to obtain an acetone solution of astaxanthin. All acetone residues were removed by distillation at 45 ℃ in the absence of light. Then measuring the astaxanthin content by High Performance Liquid Chromatography (HPLC) to be about 16.8mg/g cell dry weight; the astaxanthin content 16.8mg/g obtained by the measurement is multiplied by the cell dry weight concentration 35g/L obtained by the fermentation liquid, and the concentration of the astaxanthin obtained by the fermentation of the batch is 588 mg/L.
Data for astaxanthin production in examples one to four are compared as shown in the following table
Figure DEST_PATH_IMAGE001
The above table summarizes the addition of different nano TiO2/Al2O3 Influence on the fermentation production of astaxanthin by Phaffia rhodozyma. In the example where 500mg/L of conventional micron-sized titanium dioxide was added, the astaxanthin content was 3.2mg/g of cell dry weight and the total astaxanthin concentration was about 124.8 mg/L. While adding 100mg/L and 200mg/L nano TiO2/Al2O3 In the embodiment, the synthesis of astaxanthin is as high as 442.9mg/L and 588mg/L, and a good effect of stimulating the synthesis of astaxanthin is achieved.
The invention has the advantages that: the invention uses nano-alumina water dispersion as a carrier to process and dissolve nano-titanium dioxide into a fermentation culture medium, so that the nano-titanium dioxide can be efficiently delivered into yeast cells during fermentation, the pH value in the cells can be rapidly regulated, the pH value imbalance between the inside and the outside of the phaffia yeast cells is kept during fermentation, namely the pH environment inside and outside the phaffia yeast cell membranes is changed, the pH value gradient inside and outside the phaffia yeast cell membranes is caused, the stress reaction of the phaffia yeast is further excited, the metabolic pathway inside the phaffia yeast is carried out towards the direction of producing astaxanthin, and the content of the astaxanthin in the phaffia yeast is effectively increased.

Claims (8)

1.TiO2/Al2O3The method for promoting the high yield of astaxanthin by phaffia rhodozyma is characterized by comprising the following steps: theThe method comprises the following steps: inoculating Phaffia yeast seed liquid with nano TiO2/Al2O3Carrying out fermentation culture in a sterile fermentation culture medium of mother liquor, wherein the nano TiO is2/Al2O3The mother liquor is a nano titanium dioxide mixed liquor taking a nano alumina water dispersion liquid as a carrier, and the pH value inside and outside the phaffia rhodozyma cells is kept unbalanced during fermentation, so that the stress reaction of the phaffia rhodozyma is excited, and the metabolic pathway in the phaffia rhodozyma is oriented to the direction of producing astaxanthin.
2. The method for promoting high yield of astaxanthin by phaffia according to claim 1 by TiO2/Al2O3, wherein: adding 100-200 mg of nano TiO into each liter of sterile fermentation medium2/Al2O3The pH value of the sterile fermentation medium of the mother liquor is kept between 5.2 and 7.8, the pH value in the Phaffia rhodozyma cells during fermentation is lower than the pH value outside the cells, and the pH difference between the pH value in the Phaffia rhodozyma cells and the pH value outside the cells during fermentation is kept between-2.0 and-1.4.
3. The method for promoting high yield of astaxanthin from Phaffia rhodozyma by TiO2/Al2O3 as claimed in claim 1 or 2, wherein: nano TiO22/Al2O3The preparation method of the mother liquor comprises the following steps: mixing nano titanium dioxide into nano alumina water dispersion liquid, and uniformly stirring to obtain nano TiO2/Al2O3The mother liquor of the nano TiO2/Al2O3 contains 18-22 wt% of nano alumina particles, 1-2 wt% of nano titanium dioxide and 76-81 wt% of water, wherein the size of the solid nano titanium dioxide particles is 40-80 nanometers, and the particle size of the nano alumina particles is 5-10 nanometers.
4. The method for promoting high yield of astaxanthin by phaffia according to claim 3 by TiO2/Al2O3, wherein: the pH value of the sterile fermentation medium is controlled and adjusted by using 2-5 mol/L NaOH aqueous solution and 1-2 mol/L hydrochloric acid aqueous solution.
5. The method for promoting high yield of astaxanthin by phaffia according to claim 1 by TiO2/Al2O3, wherein: the preparation method of the Phaffia yeast seed liquid comprises the following steps: the method comprises the following steps of inoculating Phaffia rhodozyma into a seed culture medium for fermentation to prepare a Phaffia rhodozyma seed solution, wherein the seed culture medium usually adopts a yeast extract peptone glucose culture medium, and the seed culture medium comprises the following components: 1-3% of glucose, 1-3% of yeast extract and 1-2% of peptone.
6. The method for promoting high yield of astaxanthin by phaffia according to claim 5 by TiO2/Al2O3, wherein: the seed fermentation process is carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the phaffia rhodozyma seed liquid is controlled at 15-25 ℃.
7. The method for promoting high yield of astaxanthin by phaffia according to claim 1 by TiO2/Al2O3, wherein: the sterile fermentation medium comprises the following components: 10-100 g/L of glucose, 0.5-1 g/L of yeast extract, 0.5-1 g/L of magnesium sulfate, 2-6 g/L of ammonium sulfate, 1-3 g/L of monopotassium phosphate, 1-3 g/L of dipotassium phosphate and 0.1-0.2 g/L of calcium chloride.
8. The method for promoting high yield of astaxanthin by phaffia according to claim 7 by TiO2/Al2O3, wherein: inoculating the phaffia yeast seed liquid into an aseptic fermentation culture medium, and then performing fermentation culture in a fermentation tank, wherein the fermentation culture period is 48-72 hours; wherein the temperature of the fermentation liquor in the fermentation tank is controlled at 20 ℃, the initial stirring speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial aeration in the fermentation tank is 0.5-2 vvm, and the stirring speed and the aeration are set to ensure that the dissolved oxygen in the fermentation liquor is 20-60% in the whole fermentation culture process.
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