CN116555042A - Schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil - Google Patents
Schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil Download PDFInfo
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- CN116555042A CN116555042A CN202310220412.5A CN202310220412A CN116555042A CN 116555042 A CN116555042 A CN 116555042A CN 202310220412 A CN202310220412 A CN 202310220412A CN 116555042 A CN116555042 A CN 116555042A
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- Prior art keywords
- algae oil
- schizochytrium
- schizochytrium limacinum
- seed
- seed tank
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- 241000195493 Cryptophyta Species 0.000 title claims abstract description 99
- 238000000855 fermentation Methods 0.000 title claims abstract description 31
- 230000004151 fermentation Effects 0.000 title claims abstract description 31
- 241000003595 Aurantiochytrium limacinum Species 0.000 title claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/02—Refining fats or fatty oils by chemical reaction
- C11B3/04—Refining fats or fatty oils by chemical reaction with acids
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/10—Refining fats or fatty oils by adsorption
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B5/00—Preserving by using additives, e.g. anti-oxidants
- C11B5/0021—Preserving by using additives, e.g. anti-oxidants containing oxygen
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B5/00—Preserving by using additives, e.g. anti-oxidants
- C11B5/0085—Substances of natural origin of unknown constitution, f.i. plant extracts
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B5/00—Preserving by using additives, e.g. anti-oxidants
- C11B5/0092—Mixtures
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6434—Docosahexenoic acids [DHA]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Emergency Medicine (AREA)
- Fats And Perfumes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil, belonging to the technical field of bioengineering, wherein the schizochytrium limacinum has a preservation number of CGMCC No.23203; activating schizochytrium limacinum with 24 hours of seed age to obtain schizochytrium limacinum seed liquid, performing tertiary fermentation on the schizochytrium limacinum seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium limacinum fermentation liquid, collecting the schizochytrium limacinum fermentation liquid, centrifugally collecting thalli, drying, crushing, extracting to obtain crude algae oil, filling nitrogen, degumming, decoloring and deodorizing the crude algae oil to obtain refined algae oil, adding 4-6wt% of composite antioxidant into the refined algae oil, and uniformly mixing to obtain double-low DHA algae oil with low peroxide value and low anisidine value; the produced DHA algae oil has peroxide value less than or equal to 1mmol, anisole value less than 2, strong oxidation resistance, good quality and easy storage.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil.
Background
Docosahexaenoic acid is also called DHA, and has a molecular formula of C 22 H 32 O 2 The molecular weight is 328.49, and the fatty acid belongs to omega-3 series unsaturated fatty acid, and is a linear fatty acid containing 22 carbon atoms and 6 double bonds. DHA is colorless and odorless, insoluble in water, soluble in ethanol, and miscible with organic solvents such as chloroform, diethyl ether and petroleum ether, and has fat solubility. DHA is relatively active in chemical property and is extremely easy to be subjected to oxygen, illumination, overheating and metal ions (such as Fe 2+ ，Cu 2+ And free radicals) and undergo chemical reactions such as oxidation, polymerization, rancidity, double bond conjugation, etc., to form certain volatile and non-volatile species such as aldehydes, acids, ketols, carbonyl compounds, etc.
DHA is mainly distributed in the human body at the parts of brain grey matter, nervous system, retina system, cardiac muscle, eosinophil white blood cells, breast milk and the like, and has important physiological functions. Since the human body cannot synthesize DHA by itself, it is necessary to take sufficient DHA from the outside to maintain health. At present, DHA mainly comes from fish oil, algae oil, fungi and the like, wherein DHA fish oil is low in price but is caused by objective factors such as pollution and the like, and fish oil safety problems have attracted attention of scientists and bioengineering management institutions in various countries.
Schizochytrium (Schizochytrium) is also known as Schizochytrium, belonging to the order of Rhizoctonia, thraustochytriales a marine fungus of the family thraustochytriaceae, because of the characteristics of high growth speed, easy culture and the like, the method is widely applied to scientific research and commercial production. At present, the main source of DHA obtained by the microbiological method is the high-density fermentation culture of schizochytrium, a large amount of fat can be accumulated in the body, and 70 percent of DHA exists in the form of triglyceride, wherein the DHA can account for 45-55 percent of the total fatty acid.
The algae oil is a pure plant DHA, and the DHA algae oil is extracted from microalgae without being transmitted by food chains, and has very low eicosapentaenoic acid (EPA) content and is relatively safer. At present, DHA algae oil is mainly applied to the fields of infant formula milk powder, health food, animal nutrition, food and beverage and the like. The research shows that the intake of DHA algae oil is critical to the formation and development of brain cells and visual organs of infants, and has the functional characteristics of preventing postpartum depression, enhancing human body resistance, preventing and reducing atherosclerosis, coronary heart disease and the like. Along with the improvement of the living standard of people, the demand of people for DHA is larger and the quality requirement is higher, so that the DHA algae oil meets the demands of the masses.
The peroxide value is an index for indicating the oxidation degree of grease, fatty acid and the like, the anisole value indicates the quantity of secondary products such as aldehyde, ketone, quinone and the like in the grease, and the anisole value can be used for evaluating the oxidation stability of the grease and respectively measuring the primary oxidation products and the secondary oxidation products of the grease after oxidation. After DHA is oxidized, oxidation rancidity of DHA causes reduced shelf life, reduced nutrition components of beneficial accompanies such as vitamins, sterols, etc., and color and viscosity change of oil, thereby losing safety. The hydroperoxide generated in the oxidation and rancidity process of the grease has potential carcinogenicity, can be combined with macromolecular substances (DNA and protein) in a human body to react, so that the chains of the macromolecules are broken, the DNA and the protein are denatured, an enzyme system of the human body is destroyed, the normal metabolism of the human body is influenced, and various abnormal physiological conditions of the human body are finally induced. For example, the patent of the invention with publication number of CN102864111B discloses a schizochytrium limacinum strain with DHA yield up to 23g/L, and the strain with higher yield is obtained by screening, but the prior art cannot effectively reduce the problems of easy oxidation, high peroxide value and high anisidine value of DHA algae oil, so that the DHA algae oil has the problems of easy deterioration, heavy fishy smell, short shelf life and the like, therefore, how to improve the oxidation stability and reduce the peroxide value and the anisidine value is the key for ensuring the quality of the DHA algae oil.
Disclosure of Invention
The invention aims to provide schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil, so as to solve the problems in the background technology.
The aim of the invention can be achieved by the following technical scheme:
schizochytrium sp TKD-1, deposited at the China general microbiological culture Collection center, accession number: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is CGMCC No.23203.
Schizochytrium colony and cell morphology: schizochytrium sp TKD-1 is cultured on seawater culture medium at 25deg.C for 7 days, with colony diameter of 3-5mm, white color and light brown color at later stage; the cell wall is thin, spherical, transparent, and the cell diameter is 6.0-18.2 μm.
Schizochytrium rRNA gene sequence determination (18S rRNA sequence fragment):
Part 1：
5'-AGCCATGCATGTGTAAGTATAAGCGATTGTACTGTGAGACTGCGAACGGCTCATTATATCAGTAATAATTTCTTCGGTAGTTTCTTTTATATGGATACCTGCAGTAATTCTGGAAATAATACATGCTGTAAGAGCCCTGTATGGGGCTGCACTTATTAGATTGAAGCCGATTTTATTGGTGAATCATGATAATTGAGCAGATTGACT-3'
Part 2：
5'-TTAATTTTTATTTATGGAATTGAGTGCTTGGTCGGAAGGCCTGGCTAATCCTTGGAACGCTCATCGTGCTGGGGCTAGATTTTTGCAATTATTAATCTCCAACGAGGAATTCCTAGTAAACGCAAGTCATCAGCTTGCATTGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCACCTACCGATTGAACGGTCCGATGAAACCATGGGATGTTTCTGTTTGGATTAATTT-3'
schizochytrium limacinum and application thereof in fermentation production of double-low DHA algae oil, comprising the following steps:
step one, obtaining seed liquid: screening out strains, namely Schizochytrium sp TKD-1, and activating the strains with 24 hours of age to obtain Schizochytrium strain seed liquid;
step two, fermentation: determining that the first-level seed tank bacteria concentration of schizochytrium is 34+/-2 g/L and the second-level seed tank bacteria concentration of schizochytrium is 68+/-2 g/L; performing three-stage fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid;
step three, extracting: collecting schizochytrium fermentation liquor, centrifugally collecting thalli, drying, crushing and extracting to obtain crude algae oil;
step four, refining: degumming, decolorizing and deodorizing crude algae oil to obtain refined algae oil;
step five: adding 4-6wt% of compound antioxidant into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
Further, the method for screening the strains in the first step comprises the following steps: the schizochytrium limacinum is obtained from natural environment, is subjected to directional domestication after full gene sequencing, is screened out schizochytrium limacinum strains, is sealed by glycerol, and is stored at the temperature of minus 80 ℃.
The activation method comprises the following steps: the screened schizochytrium was inoculated in an inoculum size of 10% in an activation medium, cultured at 28℃for 3 days, and then cultured at 26℃for 2 days.
The components of the activation medium include: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
Further, the specific steps of degumming, decoloring and deodorizing in the fourth step are as follows:
1. adding crude algae oil into a hydration tank, heating to 65+ -5deg.C, adding 10wt% of phosphoric acid water solution of 65+ -5deg.C, stirring for 15min at 30r/min, standing for 3 hr, removing colloid and water, and degumming;
2. heating the degummed crude algae oil to 80+/-5 ℃, adding 1.5wt% of silicon dioxide into the crude algae oil, stirring for 30min, cooling to below 60 ℃, and filtering to obtain decolorized algae oil;
3. putting decolorized algae oil into deodorizing tower, and deodorizing under 1500-1700Pa vacuum degree to obtain refined algae oil.
Further, in the fifth step, the compound antioxidant is prepared from diglyceride, a leopard camphor flavonoid extract and quinoa saponin according to the following weight ratio of 3-5:1:3 mass ratio.
Further, the third step is carried out under the condition of nitrogen filling and oxygen isolation, and the nitrogen filling procedure is optimized: 15+ -5 vt percent of nitrogen is filled into the nitrogen-filled and oxygen-discharged workbench, 15+ -5 vt percent of air is pumped out, and the repeated operation ensures that the concentration of the nitrogen is more than or equal to 98 percent. And in the refining process of the step four, the nitrogen concentration of the device is more than or equal to 98 percent.
The invention has the beneficial effects that:
in the invention, the dry weight of schizochytrium limacinum cells reaches 160.64 g.L -1 DHA yield reaches 86.61 g.L -1 The yield is higher, and the DHA algae oil yield is improved. DHA algae oil content also reaches more than 50%.
The DHA algae oil produced by the fermentation culture and optimized refining process of schizochytrium limacinum is short in production time, high in yield, strong in antioxidant capacity and low in raw material cost; the phosphoric acid aqueous solution adopted in the refining process can convert the phospholipid and the phospholipid metal complex into the hydrated phospholipid, thereby effectively reducing the content of colloid and trace metals in the crude algae oil; the crude algae oil contains a large amount of chlorophyll, and the silicon dioxide has strong adsorption capacity to pigment, so that the algae oil is prevented from oxidative deterioration caused by the chlorophyll; the deodorizing step can remove the odor substances in the crude algae oil, and can reduce the oxidation strength and the anisidine value; the related operation is carried out under the condition of nitrogen protection, and oxygen can be effectively isolated so as to prevent the oxidation of algae oil. The components in the composite antioxidant are mixed for use to play a role in synergy, wherein the diglyceride has good high temperature resistance and oxidation resistance; the camphor-flavonoids extract of leopard skin can effectively remove free radicals in grease, so that the oxidation resistance of DHA algae oil is improved, and quinoa saponin is composed of a hydrophilic carbohydrate frame and a lipophilic triterpene or steroid structure, and has high oxidation resistance. The produced DHA algae oil has peroxide value less than or equal to 1mmol, anisole value less than 2, strong oxidation resistance, good quality and easy storage and application.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a microscopic view of schizochytrium in example 1 of the present invention;
FIG. 2 is a diagram of the whole genome sequencing of schizochytrium in example 1 of the present invention;
FIG. 3 is a line graph of the effect of the mono-and complex antioxidants of the invention on the antioxidant activity of DHA algae oil.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Schizochytrium sp is obtained from natural environment, subjected to whole-gene sequencing (the whole-genome sequencing result is shown in figure 2), subjected to directional domestication, screened out Schizochytrium sp TKD-1 strain (shown in figure 1), sealed with glycerol, and stored at-80 ℃.
The Schizochytrium sp TKD-1 has been deposited in China general microbiological culture Collection center (CGMCC) under the accession number CGMCC No.23203 at the date 08 and 09 of 2021.
The Schizochytrium sp TKD-1 is cultured on a seawater culture medium at 25 ℃ for 7 days, the colony diameter is 3-5mm, the colony is white, and the later period is light brown; the cell wall is thin, spherical, transparent, and the cell diameter is 6.0-18.2 μm.
The sequence of the rRNA gene of Schizochytrium sp TKD-1 (18S rRNA sequence fragment) is shown below:
Part 1：
5'-AGCCATGCATGTGTAAGTATAAGCGATTGTACTGTGAGACTGCGAACGGCTCATTATATCAGTAATAATTTCTTCGGTAGTTTCTTTTATATGGATACCTGCAGTAATTCTGGAAATAATACATGCTGTAAGAGCCCTGTATGGGGCTGCACTTATTAGATTGAAGCCGATTTTATTGGTGAATCATGATAATTGAGCAGATTGACT-3'
Part 2：
5'-TTAATTTTTATTTATGGAATTGAGTGCTTGGTCGGAAGGCCTGGCTAATCCTTGGAACGCTCATCGTGCTGGGGCTAGATTTTTGCAATTATTAATCTCCAACGAGGAATTCCTAGTAAACGCAAGTCATCAGCTTGCATTGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCACCTACCGATTGAACGGTCCGATGAAACCATGGGATGTTTCTGTTTGGATTAATTT-3'
example 2
The double-low DHA algae oil is produced by fermenting the strain in the embodiment 1, which comprises the following steps:
step one: inoculating a Schizochytrium sp TKD-1 strain with the age of 24 hours into an activation culture medium according to an inoculum size of 10%, transferring into a constant-temperature shaking table with the shaking speed of 110r/min, culturing for 3 days at 28 ℃, and culturing for 2 days at 26 ℃ to obtain Schizochytrium strain seed liquid;
wherein the components of the activation medium include: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
Step two: determining that the first-level seed tank bacteria concentration of schizochytrium is 34+/-2 g/L and the second-level seed tank bacteria concentration of schizochytrium is 68+/-2 g/L; and performing tertiary fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid.
Step three: collecting schizochytrium fermentation broth, centrifuging for 10min at 4000r/min, washing schizochytrium with distilled water for 3 times, and vacuum drying at 55deg.C; chloroform and methanol were mixed in 2mL using a nitrogen-filled oxygen-removal table with a nitrogen concentration of 99%: mixing 1mL of the mixture to obtain a chloroform-methanol mixed solution, and then crushing schizochytrium limacinum cells and mixing the schizochytrium limacinum cells with the chloroform-methanol mixed solution according to the ratio of 1g: mixing 3mL, extracting at 50deg.C under 200W microwave assistance for 60min, centrifuging at 4000r/min for 10min, collecting chloroform-methanol mixed liquid layer, and drying under nitrogen protection to obtain crude algae oil.
Step four: adding crude algae oil into a hydration tank, heating to 65+/-5 ℃, adding 10wt% of phosphoric acid aqueous solution of the crude algae oil while stirring, after the addition is completed within 10min, stirring for 15min under the condition of 30r/min after the addition is completed, standing for 3h, removing colloid and water at the bottom layer, and finishing degumming treatment; heating the degummed crude algae oil to 80+/-5 ℃, adding 1.5wt% of silicon dioxide into the crude algae oil, stirring for 30min, cooling to below 60 ℃, and filtering to obtain decolorized algae oil; putting the decolorized algae oil into a deodorizing tower, and deodorizing under 1500Pa vacuum degree to obtain refined algae oil; the device adopted in the refining process is provided with a nitrogen charging port and a waste gas discharging port, so that the nitrogen concentration in the device is ensured to be 99%, and the purpose of isolating oxygen is achieved.
Step five: adding 4wt% of compound antioxidant (diglyceride: flavonoids extract of leopard skin camphor, quinoa saponin=3:1:3) into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
Example 3
The double-low DHA algae oil is produced by fermenting the strain in the embodiment 1, which comprises the following steps:
step one: inoculating a Schizochytrium sp TKD-1 strain with the age of 24 hours into an activation culture medium according to an inoculum size of 10%, transferring into a constant-temperature shaking table with the shaking speed of 110r/min, culturing for 3 days at 28 ℃, and culturing for 2 days at 26 ℃ to obtain Schizochytrium strain seed liquid;
wherein the components of the activation medium include: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
Step two: determining that the first-level seed tank bacteria concentration of schizochytrium is 34+/-2 g/L and the second-level seed tank bacteria concentration of schizochytrium is 68+/-2 g/L; and performing tertiary fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid.
Step three: collecting schizochytrium fermentation broth, centrifuging for 10min at 4000r/min, washing schizochytrium with distilled water for 3 times, and vacuum drying at 55deg.C; chloroform and methanol were mixed in 2mL using a nitrogen-filled oxygen-removal table with a nitrogen concentration of 99%: mixing 1mL of the mixture to obtain a chloroform-methanol mixed solution, and then crushing schizochytrium limacinum cells and mixing the schizochytrium limacinum cells with the chloroform-methanol mixed solution according to the ratio of 1g: mixing 3mL, extracting at 50deg.C under 200W microwave assistance for 60min, centrifuging at 4000r/min for 10min, collecting chloroform-methanol mixed liquid layer, and drying under nitrogen protection to obtain crude algae oil.
Step four: adding crude algae oil into a hydration tank, heating to 65+/-5 ℃, adding 10wt% of phosphoric acid aqueous solution of the crude algae oil while stirring, after the addition is completed within 10min, stirring for 15min under the condition of 30r/min after the addition is completed, standing for 3h, removing colloid and water at the bottom layer, and finishing degumming treatment; heating the degummed crude algae oil to 80+/-5 ℃, adding 1.5wt% of silicon dioxide into the crude algae oil, stirring for 30min, cooling to below 60 ℃, and filtering to obtain decolorized algae oil; putting the decolorized algae oil into a deodorizing tower, and deodorizing under 1550Pa vacuum degree to obtain refined algae oil; the device adopted in the refining process is provided with a nitrogen charging port and a waste gas discharging port, so that the nitrogen concentration in the device is ensured to be 99%, and the purpose of isolating oxygen is achieved.
Step five: adding 5wt% of compound antioxidant (diglyceride: flavonoids extract of leopard skin camphor, quinoa saponin=4:1:3) into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
Example 4
The double-low DHA algae oil is produced by fermenting the strain in the embodiment 1, which comprises the following steps:
step one: inoculating a Schizochytrium sp TKD-1 strain with the age of 24 hours into an activation culture medium according to an inoculum size of 10%, transferring into a constant-temperature shaking table with the shaking speed of 110r/min, culturing for 3 days at 28 ℃, and culturing for 2 days at 26 ℃ to obtain Schizochytrium strain seed liquid;
wherein the components of the activation medium include: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
Step two: determining that the first-level seed tank bacteria concentration of schizochytrium is 34+/-2 g/L and the second-level seed tank bacteria concentration of schizochytrium is 68+/-2 g/L; and performing tertiary fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid.
Step three: collecting schizochytrium fermentation broth, centrifuging for 10min at 4000r/min, washing schizochytrium with distilled water for 3 times, and vacuum drying at 55deg.C; chloroform and methanol were mixed in 2mL using a nitrogen-filled oxygen-removal table with a nitrogen concentration of 98%: mixing 1mL of the mixture to obtain a chloroform-methanol mixed solution, and then crushing schizochytrium limacinum cells and mixing the schizochytrium limacinum cells with the chloroform-methanol mixed solution according to the ratio of 1g: mixing 3mL, extracting at 50deg.C under 200W microwave assistance for 60min, centrifuging at 4000r/min for 10min, collecting chloroform-methanol mixed liquid layer, and drying under nitrogen protection to obtain crude algae oil.
Step four: adding crude algae oil into a hydration tank, heating to 65+/-5 ℃, adding 10wt% of phosphoric acid aqueous solution of the crude algae oil while stirring, after the addition is completed within 10min, stirring for 15min under the condition of 30r/min after the addition is completed, standing for 3h, removing colloid and water at the bottom layer, and finishing degumming treatment; heating the degummed crude algae oil to 80+/-5 ℃, adding 1.5wt% of silicon dioxide into the crude algae oil, stirring for 30min, cooling to below 60 ℃, and filtering to obtain decolorized algae oil; putting the decolorized algae oil into a deodorizing tower, and deodorizing under 1600Pa vacuum degree to obtain refined algae oil; the device adopted in the refining process is provided with a nitrogen charging port and a waste gas discharging port, so that the nitrogen concentration in the device is guaranteed to be 98%, and the purpose of isolating oxygen is achieved.
Step five: adding 5wt% of compound antioxidant (diglyceride: flavonoids extract of leopard skin camphor, quinoa saponin=5:1:3) into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
Example 5
The double-low DHA algae oil is produced by fermenting the strain in the embodiment 1, which comprises the following steps:
step one: inoculating a Schizochytrium sp TKD-1 strain with the age of 24 hours into an activation culture medium according to an inoculum size of 10%, transferring into a constant-temperature shaking table with the shaking speed of 110r/min, culturing for 3 days at 28 ℃, and culturing for 2 days at 26 ℃ to obtain Schizochytrium strain seed liquid;
wherein the components of the activation medium include: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
Step two: determining that the first-level seed tank bacteria concentration of schizochytrium is 34+/-2 g/L and the second-level seed tank bacteria concentration of schizochytrium is 68+/-2 g/L; and performing tertiary fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid.
Step three: collecting schizochytrium fermentation broth, centrifuging for 10min at 4000r/min, washing schizochytrium with distilled water for 3 times, and vacuum drying at 55deg.C; chloroform and methanol were mixed in 2mL using a nitrogen-filled oxygen-removal table with a nitrogen concentration of 99%: mixing 1mL of the mixture to obtain a chloroform-methanol mixed solution, and then crushing schizochytrium limacinum cells and mixing the schizochytrium limacinum cells with the chloroform-methanol mixed solution according to the ratio of 1g: mixing 3mL, extracting at 50deg.C under 200W microwave assistance for 60min, centrifuging at 4000r/min for 10min, collecting chloroform-methanol mixed liquid layer, and drying under nitrogen protection to obtain crude algae oil.
Step four: adding crude algae oil into a hydration tank, heating to 65+/-5 ℃, adding 10wt% of phosphoric acid aqueous solution of the crude algae oil while stirring, after the addition is completed within 10min, stirring for 15min under the condition of 30r/min after the addition is completed, standing for 3h, removing colloid and water at the bottom layer, and finishing degumming treatment; heating the degummed crude algae oil to 80+/-5 ℃, adding 1.5wt% of silicon dioxide into the crude algae oil, stirring for 30min, cooling to below 60 ℃, and filtering to obtain decolorized algae oil; putting the decolorized algae oil into a deodorizing tower, and deodorizing under 1700Pa vacuum degree to obtain refined algae oil; the device adopted in the refining process is provided with a nitrogen charging port and a waste gas discharging port, so that the nitrogen concentration in the device is ensured to be 99%, and the purpose of isolating oxygen is achieved.
Step five: adding 6wt% of compound antioxidant (diglyceride: flavonoids extract of leopard skin camphor, quinoa saponin=3:1:3) into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
Comparative example 1: based on the embodiment 5, the concentration of nitrogen adopted in the third step and the fourth step is controlled to be 99%, and the DHA algae oil is prepared without adding a compound antioxidant.
Comparative example 2: based on the embodiment 5, nitrogen protection is not adopted in the process of the third step and the fourth step, and the DHA algae oil is prepared without adding a compound antioxidant.
The appearance of the nitrogen-charging and oxygen-discharging workbench adopted in the embodiment 2-embodiment 5 and the comparative example 1 is similar to that of a sterile super-clean workbench, and the workbench is different in that the workbench has tightness, and is provided with an air inlet and an air outlet, wherein the air inlet is connected with a nitrogen-charging tank, the air outlet is connected with a gas collecting tank for gas recovery, the workbench is also provided with an operation port with a rubber sleeve, and when an operator stretches into the workbench, the rubber sleeve is attached to an arm to prevent external air from entering; when in use, 15+ -5 vt percent of nitrogen is firstly filled into the workbench, then 15+ -5 vt percent of air is pumped out, and the operation is repeated until the concentration of the nitrogen reaches a preset value.
The DHA algae oils prepared in example 2-example 5 and comparative example 1-comparative example 2 were tested, 20 samples were taken for each group, and the peroxide value, the anisidine value and the yield of each group of DHA algae oil were measured, and the results are shown in Table 1:
TABLE 1
As can be seen from Table 1, the DHA algae oil produced in example 2-example 5 has peroxide value less than or equal to 1mmol, anisole value less than 2, and is obviously superior to the existing national standard (GB 26400-2011 food additive docosahexaenoic acid oil (fermentation method)).
The antioxidant effect of the three natural antioxidants added alone and compounded on DHA algae oil at 5% antioxidant addition was compared using Schaal oven method (see FIG. 3).
As can be seen from FIG. 3, at the same addition amount, the antioxidant activity of the diglyceride > quinoa saponin > the flavonoids extract of Litsea coreana. Further researches show that the three compound antioxidant effects are superior to the antioxidant effects of the two compound additives, and the effect of the three antioxidants when added independently is not obvious. This is because two or more antioxidants can be synergistically combined, and the main reason is that some substances can assist the antioxidants to act, such as metal ion chelators, or reduce the free radicals of the antioxidants generated by the antioxidants to play an antioxidant role, thereby regenerating the antioxidants.
It should be noted that in this document, terms such as "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. Schizochytrium sp TKD-1, which is classified and named as Schizochytrium sp, is preserved in China general microbiological culture Collection center (CGMCC No. 23203) on the 09 th year of 2021.
2. Use of schizochytrium in accordance with claim 1 for the fermentative production of double-low DHA algae oil.
3. The use according to claim 2, characterized by the steps of:
step one: activating schizochytrium limacinum with 24 hours of seed age to obtain schizochytrium limacinum seed liquid;
step two: performing three-stage fermentation on schizochytrium seed liquid after primary seed tank expansion culture and secondary seed tank expansion culture to obtain schizochytrium fermentation liquid;
step three: collecting schizochytrium fermentation liquor, centrifugally collecting thalli, drying, crushing and extracting to obtain crude algae oil;
step four: degumming, decolorizing and deodorizing crude algae oil to obtain refined algae oil;
step five: adding 4-6wt% of compound antioxidant into refined algae oil, and mixing uniformly to obtain double-low DHA algae oil.
4. The use according to claim 2, wherein the method of activation in step one is: the schizochytrium was inoculated in an inoculum size of 10% in an activation medium, cultured at 28℃for 3 days, and then cultured at 26℃for 2 days.
5. The use according to claim 4, wherein the components of the activation medium comprise: 145g/L glucose, 2.6g/L yeast extract, 5.2g/L corn steep liquor dry powder and K + 10mM, trace element 0.8g/L, phosphate buffer with pH value of 6.8.
6. The use according to claim 2, wherein the first seed tank for the first seed tank expansion culture has a schizochytrium first seed tank bacterial concentration of 34.+ -.2 g/L and the second seed tank for the second seed tank expansion culture has a schizochytrium second seed tank bacterial concentration of 68.+ -.2 g/L.
7. The use according to claim 2, wherein in step five the complex antioxidant is prepared from diglycerides, flavonoids extract of leopard camphor and quinoa saponins according to 3-5:1:3 mass ratio.
8. The method according to claim 2, wherein the operation in step three and step four is performed at a nitrogen concentration of 98% or more.
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