CN117721023A - Production method of schizochytrium limacinum microcapsule rich in DHA algae oil - Google Patents
Production method of schizochytrium limacinum microcapsule rich in DHA algae oil Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering, and discloses a production method of schizochytrium microcapsule rich in DHA algae oil, which comprises the steps of activating schizochytrium with the seed age of 24 hours and the preservation number of CGMCC No.23203 to obtain schizochytrium seed liquid, performing primary seed tank expansion culture and secondary seed tank expansion culture on the schizochytrium seed liquid, and performing tertiary fermentation to obtain schizochytrium fermentation liquid; collecting schizochytrium fermentation liquor, carrying out solid-liquid separation, cleaning and primary filtration to obtain schizochytrium crude liquor, and carrying out flocculation and secondary filtration to obtain pure schizochytrium liquor; mixing schizochytrium liquid with embedding wall material, homogenizing, spray drying, premixing to produce schizochytrium microcapsule powder with embedding rate up to 90%, DHA content up to 5-16%, obvious nutritious effect and wide application range.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a production method of schizochytrium limacinum microcapsules rich in DHA algae oil.
Background
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. Currently, the main source of obtaining docosahexaenoic acid (DHA) by microbiological methods is the high-density fermentation culture of schizochytrium, which can accumulate a large amount of fat in vivo, and 70% exists in the form of triglycerides, wherein DHA can account for 45-55% of the total fatty acids.
DHA is a linear fatty acid containing 22 carbon atoms and 6 double bonds, belonging to the omega-3 series of unsaturated fatty acids. 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 algae oil is mainly applied to the fields of infant formula milk powder, health food, animal nutrition, food beverage and the like, and researches show that the intake of DHA algae oil is critical to the formation and development of infant brain cells and visual organs, and has the functional characteristics of preventing postpartum depression, enhancing human body resistance, preventing and reducing atherosclerosis, coronary heart disease and the like.
The microcapsule technology is an operation of embedding micro core substances such as solid, liquid, gas, etc. in semipermeable or airtight microcapsules by using natural or synthetic polymer substances, and has been widely used in the fields of medicine, daily chemical industry, biology, etc. The microcapsule is mainly characterized in that the microcapsule can protect the embedded food ingredients, isolate the embedded food ingredients from the external environment and prevent the embedded food ingredients from generating adverse quality changes, so that the original color, aroma, taste, nutrition ingredients and certain bioactive substances of the food are effectively maintained, the microcapsule also has a good masking effect on some food ingredients with unpleasant smell, and the microcapsule can be converted into a stable solid form by microcapsule technology for some materials which are difficult to store or process and store, so that the nutritional value and the processing performance of the processed materials are greatly improved.
In the production process of DHA algae oil, the wall breaking of thalli, decoloring, deodorizing, antioxidation and other treatments are required, and the DHA algae oil is difficult to preserve. The optimized schizochytrium strain and microcapsule technology are utilized to produce the microcapsule rich in DHA algae oil, so that the defects of complex production process, easy oxidation and the like of DHA algae oil can be effectively overcome, the problems of bitter and astringent schizochytrium thallus, multiple attachments and the like are well solved, the application range of schizochytrium is greatly improved, and more nutritional and healthy products are developed.
Disclosure of Invention
The invention aims to provide a production method of schizochytrium limacinum microcapsules rich in DHA algae oil, which solves the problems of complex production process and easy oxidation of DHA algae oil.
The aim of the invention can be achieved by the following technical scheme:
a method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
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 separating to obtain fermentation solid precipitate, and performing aftertreatment on the fermentation solid precipitate to obtain pure schizochytrium wet bodies;
step four: processing schizochytrium wet body to obtain schizochytrium microcapsule.
Further, in the first step, the schizochytrium is named as schizochytrium (schizochytrium sp.) TKD-1, which was deposited in China general microbiological culture collection center (cgmcc No. 23203) at the date of 08, 2021.
Further, in the first step, the activation method specifically includes: 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.
Further, the components of the activation medium include: glucose, yeast extract, corn steep liquor dry powder, KCl, trace elements and phosphate buffer with pH value of 6.8.
Further, the trace elements are FeSO with a mass ratio of 3:3:3:2:2:0.04:0.04 4 ·7H 2 O、MnCl 2 ·4H 2 O、ZnSO 4 ·7H 2 O、NiSO 4 ·6H 2 O、CuSO 4 ·5H 2 O、CoCl 2 ·6H 2 O、Na 2 MoO 4 ·2H 2 O is dissolved in purified water to prepare the water-soluble catalyst.
Further, in the second step, the method for enlarging and culturing the first-stage seed tank specifically comprises the following steps:
inoculating schizochytrium limacinum seed liquid into a primary seed tank expansion culture medium according to an inoculum size of 10%, setting a culture temperature of 28 ℃, and culturing for 36h at a rotating speed of 220r/min to obtain primary seed tank seed liquid with a concentration of 34-40g/L;
the method for the expansion culture of the secondary seed tank comprises the following steps:
inoculating the first-stage tank fungus seed liquid into a second-stage tank fungus seed liquid according to an inoculum size of 8%, setting a culture temperature of 28 ℃, and culturing for 24 hours at a rotating speed of 220r/min to obtain a second-stage tank fungus seed liquid with a concentration of 68-75 g/L;
the three-stage fermentation method specifically comprises the following steps:
inoculating the second-stage tank fungus seed solution into a fermentation culture medium according to an inoculum size of 8%, setting a culture temperature of 28 ℃ and a rotation speed of 300r/min, and culturing for 100 h.
Further, in the third step, the post-processing includes the following steps:
the first step: cleaning
Mixing purified water and fermentation solid sediment with the mass ratio of 3-5:1, stirring to form uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifuging, collecting sediment, repeating for 2-3 times, and collecting schizochytrium limacinum coarse sediment which is centrifugally separated for the last time;
and a second step of: flocculation
Mixing purified water and schizochytrium coarse precipitate in a mass ratio of 4-6:1 under stirring to form a uniform dispersion, adding a composite flocculant into the dispersion, stirring for 20-30min at a stirring rate of 300r/min, and standing for 15-20min to form schizochytrium bacterial precipitate;
and a third step of: filtration
Filtering the schizochytrium bacterial deposit by using a plate-and-frame filter press under the pressure of 0.3-0.6MPa, and collecting filter-pressed solid matters.
Further, in the second step, the composite flocculant comprises chitosan with the dosage of 0.5-1mg/L and modified kaolin with the dosage of 30-40 mg/L; the preparation method of the modified kaolin specifically comprises the following steps: placing kaolin into a muffle furnace, raising the temperature to 750-800 ℃, activating for 1-3h, taking out, mixing with sodium hydroxide solution with the concentration of 3mol/L, raising the temperature to 85-95 ℃, carrying out alkali treatment for 1-3h, centrifuging, separating, and drying and crushing the product.
Further, in the fourth step, the processing includes the following steps:
step S1: liquefaction process
Mixing purified water and schizochytrium in a volume ratio of 2-5:1 to obtain a pure schizochytrium solution;
step S2: mixing
Placing schizochytrium solution, arabic gum, modified corn starch and whey protein powder in a mass ratio of 30:2:1:1 into a stirring kettle, stirring and mixing for 20-30min at a rotating speed of 2000-3000rpm to obtain a mixed solution;
step S3: homogenizing
Heating the mixed solution to 50-60deg.C, homogenizing under 25-35MPa for 2-3 times to obtain homogenized solution
Step S4: spray drying the homogeneous solution with a spray dryer, setting the air inlet temperature at 165-200deg.C, the air outlet temperature at 70-95deg.C, and the feeding speed at 5-20L/h to obtain schizochytrium microcapsule powder;
s5, a step of S5; premixing
Adding anticaking agent into schizochytrium microcapsule powder, and stirring and mixing uniformly.
Further, in the step S5, the anti-caking agent consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1:1, and the mass of the anti-caking agent added is 1-2% of that of schizochytrium microcapsule powder.
The invention has the beneficial effects that:
the schizochytrium microcapsule produced by the invention has the embedding rate of more than 90 percent and DHA content of 5-16 percent, and effectively solves the problems of complex processes and storage and transportation such as decoloration, deodorization, antioxidation and the like in DHA algae oil production, and the problems of bitter and astringent schizochytrium strain, more attachments and the like by embedding DHA, and can be directly used as food materials for producing foods and health care nutrition products.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing analysis of schizochytrium gene KEGG;
FIG. 2 is a microscopic view of schizochytrium.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but 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.
Schizochytrium employed in the examples below were obtained from the natural environment, sequenced, subjected to directional domestication after sequencing, screened for schizochytrium (schizochytrium sp.) TKD-1 species, and stored in glycerol at-80 ℃.
The schizochytrium sp TKD-1 has been deposited in the China general microbiological culture collection center (cgmccno.23203) at the date 08 and 09 of 2021.
The sequence of the rRNA gene of Schizochytrium (Schizochytriumsp.) TKD-1 (18 SrRNA sequence fragment) is shown below:
Part1：
5'-AGCCATGCATGTGTAAGTATAAGCGATTGTACTGTGAGACTGCGAACGGCT CATTATATCAGTAATAATTTCTTCGGTAGTTTCTTTTATATGGATACCTGCAGTAATT CTGGAAATAATACATGCTGTAAGAGCCCTGTATGGGGCTGCACTTATTAGATTGAAGC CGATTTTATTGGTGAATCATGATAATTGAGCAGATTGACT-3'
Part2：
5'-TTAATTTTTATTTATGGAATTGAGTGCTTGGTCGGAAGGCCTGGCTAATCC TTGGAACGCTCATCGTGCTGGGGCTAGATTTTTGCAATTATTAATCTCCAACGAGGAATTCCTAGTAAACGCAAGTCATCAGCTTGCATTGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCACCTACCGATTGAACGGTCCGATGAAACCATGGGATGTTTCTGTTTGGATTAATTT-3'
FIG. 1 is a graph of a schizochytrium sp (Schizochytrium sp.) genome KEGG analysis, and it is understood from FIG. 1 that schizochytrium TKD-1 comprises 9 major metabolic pathways, respectively carbohydrate metabolism, amino acid metabolism, other amino acid metabolism, lipid metabolism, energy metabolism, cofactors and vitamin metabolism, nucleotide metabolism, transport reactions, crossover metabolism and other metabolism. Lipid metabolism is the highest in all pathways.
FIG. 2 is a microscopic view of Schizochytrium (Schizochytrium sp.) TKD-1 after 7 days of culture at 25℃on seawater medium, as seen in FIG. 2, the schizochytrium colony diameter is 3-5mm, white, and light brown in the late stage; the cell wall is thin, spherical, transparent, and the cell diameter is 6.0-18.2 μm.
The preparation method of the culture medium used in the following examples was:
the first step: 10mL of Na with concentration of 0.05mol/L 2 HPO 4 10mL of the solution had KH concentration of 0.05mol/L 2 PO 4 Stirring and mixing the solution to prepare a phosphate buffer solution with the pH value of 6.8;
and a second step of: 0.3g of FeSO 4 ·7H 2 O, 0.3g MnCl 2 ·4H 2 O, 0.3g ZnSO 4 ·7H 2 O, 0.2g NiSO 4 ·6H 2 O, 0.2g of CuSO 4 ·5H 2 O, 0.004g of CoCl 2 ·6H 2 O and 0.004g of Na 2 MoO 4 ·2H 2 O is dissolved in 10mL of purified water, stirred and mixed uniformly to form trace elements;
and a third step of: adding 2.9g glucose, 0.052g yeast extract, 0.104g corn steep liquor dry powder, 0.149g trace element and 0.016g trace element into the phosphate buffer solution prepared in the first step, and stirring and mixing uniformly.
The modified kaolin used in the following examples was prepared by the following method:
and (3) placing the kaolin in a muffle furnace, raising the temperature to 800 ℃, activating for 2 hours, taking out, mixing 10g of activated kaolin with 100mL of sodium hydroxide solution with the concentration of 3mol/L, raising the temperature to 90 ℃, conducting alkali treatment for 2 hours, centrifuging, separating, and drying and crushing the product.
Example 1
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: inoculating schizochytrium limacinum with 24h of seed age into an activation culture medium according to 10% of inoculum size, culturing for 3 days at 28 ℃, and culturing for 2 days at 26 ℃ to obtain schizochytrium limacinum seed liquid;
step two: inoculating schizochytrium limacinum seed liquid into a primary seed tank expansion culture medium according to an inoculum size of 10%, setting a culture temperature of 28 ℃, and culturing for 36h at a rotating speed of 220r/min to obtain a primary seed tank seed liquid with a concentration of 36 g/L; inoculating the first-stage tank fungus seed liquid into a second-stage tank fungus seed liquid according to an inoculum size of 8%, setting a culture temperature of 28 ℃, and culturing for 24 hours at a rotating speed of 220r/min to obtain a second-stage tank fungus seed liquid with a concentration of 72 g/L; inoculating the second-stage pot fungus seed solution into a fermentation medium according to an inoculum size of 8%, setting a culture temperature to be 28 ℃, and culturing for 100 hours at a rotating speed of 300r/min to obtain schizochytrium fermentation liquor;
step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, stirring and mixing 3000mL of purified water and 100g of fermentation solid precipitate to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the process for 2 times, collecting the schizochytrium crude precipitate obtained by the last centrifugal separation, stirring and mixing 200mL of purified water and 50g of schizochytrium crude precipitate to form a uniform dispersion, adding a composite flocculant consisting of 0.1g of chitosan and 6g of modified kaolin into the dispersion, stirring for 20min at a stirring rate of 300r/min, standing for 15min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate by using a plate-and-frame filter press under the pressure of 0.3MPa, and collecting the solid to obtain pure filter-pressed schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder into a stirring kettle, stirring and mixing for 20min at a rotating speed of 2000rpm to obtain a mixed solution, heating the mixed solution to 50 ℃, homogenizing the mixed solution for 2 times under a pressure of 25MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to 165 ℃, the air outlet temperature to 80 ℃ and the feeding speed to 5L/h, and uniformly stirring and mixing 20g of schizochytrium microcapsule powder and 0.2g of an anti-caking agent to obtain the schizochytrium microcapsule, wherein the anti-caking agent consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1:1.
Example 2
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: the same as in example 1;
step two: the same as in example 1;
step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, mixing 3500mL of purified water and 100g of fermentation solid precipitate with stirring to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the process for 2 times, collecting the schizochytrium crude precipitate obtained by the last centrifugal separation, mixing 240mL of purified water and 50g of schizochytrium crude precipitate with stirring to form a uniform dispersion, adding a composite flocculant consisting of 0.14g of chitosan and 7.68g of modified kaolin into the dispersion, stirring for 25min at a stirring rate of 300r/min, standing for 15min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate by using a plate-and-frame filter press under the pressure of 0.4MPa, and collecting filter-pressed solids to obtain pure schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder in a stirring kettle, stirring and mixing for 25min at a rotating speed of 2500rpm to obtain a mixed solution, heating the mixed solution to 55 ℃, homogenizing the mixed solution for 3 times under a pressure of 30MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to be 170 ℃, the air outlet temperature to be 85 ℃ and the feeding speed to be 10L/h, and uniformly stirring and mixing 20g of schizochytrium microcapsule powder with 0.3g of an anticaking agent to obtain the schizochytrium microcapsule, wherein the schizochytrium microcapsule consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1.
Example 3
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: the same as in example 1;
step two: the same as in example 1;
step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, mixing 3800mL of purified water and 100g of fermentation solid precipitate with stirring to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the steps for 3 times, collecting the schizochytrium crude precipitate centrifugally separated in the last time, mixing 260mL of purified water and 50g of schizochytrium crude precipitate with stirring to form a uniform dispersion, adding a composite flocculant consisting of 0.2g of chitosan and 9.1g of modified kaolin into the dispersion, stirring for 25min at a stirring rate of 300r/min, standing for 15min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate by using a plate-and-frame filter press under the pressure of 0.4MPa, and collecting filter-pressed solids to obtain pure schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder in a stirring kettle, stirring and mixing for 25min at a rotating speed of 2500rpm to obtain a mixed solution, heating the mixed solution to 55 ℃, homogenizing the mixed solution for 3 times under a pressure of 30MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to 175 ℃, the air outlet temperature to 90 ℃ and the feeding speed to 12L/h, and uniformly stirring and mixing 20g of schizochytrium microcapsule powder and 0.3g of an anti-caking agent to obtain the schizochytrium microcapsule, wherein the schizochytrium microcapsule consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1.
Example 4
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: the same as in example 1;
step two: the same as in example 1;
step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, stirring 4000mL of purified water and 100g of fermentation solid precipitate to mix to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the process for 3 times, collecting the schizochytrium crude precipitate obtained by the last centrifugal separation, stirring and mixing 280mL of purified water and 50g of schizochytrium crude precipitate to form a uniform dispersion, adding a composite flocculant consisting of 0.22g of chitosan and 10g of modified kaolin into the dispersion, stirring for 30min at a stirring rate of 300r/min, standing for 20min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate by using a plate-and-frame filter press under the pressure of 0.4MPa, and collecting the solid to obtain pure schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder in a stirring kettle, stirring and mixing for 25min at a rotating speed of 2600rpm to obtain a mixed solution, heating the mixed solution to 55 ℃, homogenizing the mixed solution for 3 times at a pressure of 32MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to be 180 ℃, the air outlet temperature to be 90 ℃ and the feeding speed to be 15L/h, uniformly stirring and mixing 20g of schizochytrium microcapsule powder with 0.32g of an anticaking agent to obtain the schizochytrium microcapsule, wherein the schizochytrium microcapsule consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1.
Example 5
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: the same as in example 1;
step two: the same as in example 1;
step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, stirring 4000mL of purified water and 100g of fermentation solid precipitate to mix to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the process for 3 times, collecting the schizochytrium crude precipitate obtained by the last centrifugal separation, stirring and mixing 280mL of purified water and 50g of schizochytrium crude precipitate to form a uniform dispersion, adding a composite flocculant consisting of 0.25g of chitosan and 11g of modified kaolin into the dispersion, stirring for 30min at a stirring rate of 300r/min, standing for 20min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate by using a plate-and-frame filter press under the pressure of 0.5MPa, and collecting the solid to obtain pure schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder in a stirring kettle, stirring and mixing for 25min at the rotation speed of 2800rpm to obtain a mixed solution, heating the mixed solution to 55 ℃, homogenizing the mixed solution for 3 times under the pressure of 32MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to 195 ℃, the air outlet temperature to 90 ℃, and the feeding speed to 18L/h, obtaining schizochytrium microcapsule powder, uniformly stirring and mixing 20g of schizochytrium microcapsule powder with 0.36g of an anti-caking agent to obtain the schizochytrium microcapsule, wherein the schizochytrium microcapsule consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate with the mass ratio of 2:2:1:1.
Example 6
A method for producing schizochytrium limacinum microcapsules rich in DHA algae oil, which comprises the following steps:
step one: the same as in example 1;
step two: the same as in example 1; step three: collecting schizochytrium fermentation liquor, centrifugally separating to obtain a fermentation solid precipitate, mixing 5000mL of purified water and 100g of fermentation solid precipitate with stirring to form a uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifugally collecting the precipitate, repeating the steps for 3 times, collecting the schizochytrium crude precipitate obtained by the last centrifugal separation, mixing 300mL of purified water and 50g of schizochytrium crude precipitate with stirring to form a uniform dispersion, adding a composite flocculant consisting of 0.3g of chitosan and 12g of modified kaolin into the dispersion, stirring for 30min at a stirring rate of 300r/min, standing for 20min to form a schizochytrium bacterial precipitate, filtering the schizochytrium bacterial precipitate with a plate-and-frame filter press under the pressure of 0.6MPa, and collecting the solid to obtain pure schizochytrium wet bodies;
step four: mixing 60mL of purified water and 30g of schizochytrium wet body to obtain a pure schizochytrium solution, placing the schizochytrium solution, 6g of Arabic gum, 3g of modified corn starch and 3g of whey protein powder into a stirring kettle, stirring and mixing for 30min at a rotating speed of 3000rpm to obtain a mixed solution, heating the mixed solution to 60 ℃, homogenizing the mixed solution for 3 times under a pressure of 35MPa to form a homogenized solution, using a spray dryer to spray-dry the homogenized solution, setting the air inlet temperature to be 200 ℃, the air outlet temperature to be 95 ℃ and the feeding speed to be 20L/h, uniformly stirring and mixing 20g of schizochytrium microcapsule powder with 0.4g of an anticaking agent to obtain the schizochytrium microcapsule, wherein the schizochytrium microcapsule consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1.
The following tests were performed on schizochytrium microcapsules prepared in examples 1-6 of the present invention and on commercially available capsules (wuhan paletto biotechnology limited):
(1) DHA content determination
Accurately weighing microcapsule 0.1g, adding 5ml KOH/methanol solution with concentration of 0.4mol/l, ultrasonic crushing for 15min, and adding 5ml BF during water bath at 60deg.C for 1 hr 3 The methanol solution (14%) was heated in a water bath for 1h. Extraction was performed using n-hexane (1:1 solution: n-hexane) and 1mL of the supernatant was chromatographed in a brown bottle.
The chromatographic method comprises the following steps: the chromatographic analysis selects a gas chromatograph matched with a capillary column, the sample injection amount is 2 mu L, the sample injection temperature is 250 ℃, and the column temperature condition is as follows: the temperature is maintained at 80 ℃ for 2min, then the temperature is increased to 120 ℃ at a speed of 10 ℃/min, then the temperature is increased to 180 ℃ at 5 ℃/min, the temperature is maintained for 2min, then the temperature is increased to 230 ℃ at 2 ℃/min, and the temperature is maintained at 230 ℃ for 5min. Helium is used as a carrier gas. DHA content in the microcapsules was qualitatively and quantitatively calculated as retention time and peak area of C19:0 standard, respectively.
(2) And (3) embedding rate measurement: the embedding rate is the percentage of the DHA content of the microcapsule after embedding and the DHA content in the pure schizochytrium limacinum solution before embedding.
(3) Microencapsulation efficiency assay: microencapsulation efficiency= (total DHA content of microcapsule particles-surface DHA content of microcapsule particles)/total DHA content of microcapsule particles.
The method for measuring the total DHA content of the microcapsule particles comprises the following steps: accurately weighing 0.1g schizochytrium microcapsule, adding 5ml KOH/methanol solution (0.4 mol/L) for ultrasonic crushing (15 min), and standing at 60deg.CWater bath for 1h, during which 5ml of BF was added 3 The methanol solution (14%) was heated in a water bath for 1h. Extraction was performed by n-hexane (1:1 solution: n-hexane), and 1mL of the supernatant was taken and chromatographed in a brown flask.
The method for measuring DHA content on the surface of microcapsule particles comprises the following steps: accurately weighing 0.1g schizochytrium microcapsule, adding 10mL of normal hexane, stirring, filtering, collecting filtrate, and repeating for 3 times to obtain DHA extract. Rotary evaporating DHA extractive solution, adding 5ml KOH/methanol solution (0.4 mol/L) at 60deg.C in water bath for 1 hr, adding 5ml BF 3 Methanol solution (14%), heating in water bath at 60deg.C for 1 hr, adding 20mL saturated sodium chloride solution, adding 5mL n-hexane, shaking, and collecting supernatant to determine DHA content.
(4) Peroxide value measurement: refer to GB5009.227-2016.
The test results are shown in the following table:
as shown by the test results of the table, the DHA content of the schizochytrium microcapsules prepared in the examples 1-6 is 5% -16%, the microcapsule embedding rate is over 99.5%, the microcapsule embedding efficiency is over 99.11%, and the peroxide value is lower than 0.09, wherein the schizochytrium microcapsules in the example 5 have the optimal comprehensive quality, and the schizochytrium microcapsules in the example 1 have the worst comprehensive quality, but are better than those of the commercially available capsules.
The nutritional ingredients of schizochytrium microcapsules prepared in example 5 were tested and the results are shown in the following table:
| composition of the components | Content ratio (%) |
| Total protein | 20.89 |
| Total fat | 18.98 |
| Total sugar | 6.38 |
| Ash content | 4.85 |
| Total amino acids | 7.38 |
The foregoing is merely illustrative and explanatory of the principles of the invention, as various modifications and additions may be made to the specific embodiments described, or similar thereto, by those skilled in the art, without departing from the principles of the invention or beyond the scope of the appended claims.
Claims (10)
1. A method for producing schizochytrium microcapsules rich in DHA algae oil, which is characterized by comprising the following steps:
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 separating to obtain fermentation solid precipitate, and performing aftertreatment on the fermentation solid precipitate to obtain pure schizochytrium wet bodies;
step four: processing schizochytrium wet body to obtain schizochytrium microcapsule.
2. The method of claim 1, wherein in the first step, the Schizochytrium is classified as Schizochytrium sp TKD-1, which was deposited in the China general microbiological culture Collection center (cccc) under the accession number of 23203 at the 09 th year 2021.
3. The method for producing schizochytrium limacinum microcapsules rich in DHA algae oil according to claim 1, wherein in the first step, the activating method is specifically as follows: 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.
4. A method of producing schizochytrium microcapsules enriched in DHA algae oil according to claim 3, characterized in that the components of the activation medium comprise: glucose, yeast extract, corn steep liquor dry powder, KCl, trace elements and phosphate buffer with pH value of 6.8.
5. The method for producing schizochytrium limacinum microcapsules rich in DHA algae oil according to claim 4, wherein the trace elements are FeSO with a mass ratio of 3:3:3:2:2:0.04:0.04 4 ·7H 2 O、MnCl 2 ·4H 2 O、ZnSO 4 ·7H 2 O、NiSO 4 ·6H 2 O、CuSO 4 ·5H 2 O、CoCl 2 ·6H 2 O、Na 2 MoO 4 ·2H 2 O is dissolved in purified water to prepare the water-soluble catalyst.
6. The method for producing schizochytrium microcapsules enriched in DHA algae oil according to claim 1, wherein in the second step, the first-stage seed tank concentration of schizochytrium in the first-stage seed tank expansion culture is 34-40g/L; the schizochytrium secondary seed tank strain concentration of the secondary seed tank expanded culture is 68-75g/L.
7. The method for producing schizochytrium microcapsules enriched in DHA algae oil according to claim 1, characterized in that in step three, the post-treatment comprises the following steps:
the first step: cleaning
Mixing purified water and fermentation solid sediment with the mass ratio of 3-5:1, stirring to form uniform dispersion, placing the uniform dispersion in a centrifugal machine, centrifuging, collecting sediment, repeating for 2-3 times, and collecting schizochytrium limacinum coarse sediment which is centrifugally separated for the last time;
and a second step of: flocculation
Mixing purified water and schizochytrium coarse precipitate in a mass ratio of 4-6:1 under stirring to form a uniform dispersion, adding a composite flocculant into the dispersion, stirring for 20-30min at a stirring rate of 300r/min, and standing for 15-20min to form schizochytrium bacterial precipitate;
and a third step of: filtration
Filtering the schizochytrium bacterial deposit by using a plate-and-frame filter press under the pressure of 0.3-0.6MPa, and collecting filter-pressed solid matters.
8. The method for producing schizochytrium limacinum microcapsules enriched in DHA algae oil according to claim 7, characterized in that in the second step, the composite flocculant comprises chitosan in an amount of 0.5-1mg/L and modified kaolin in an amount of 30-40 mg/L; the preparation method of the modified kaolin specifically comprises the following steps: placing kaolin into a muffle furnace, raising the temperature to 750-800 ℃, activating for 1-3h, taking out, mixing with sodium hydroxide solution with the concentration of 3mol/L, raising the temperature to 85-95 ℃, carrying out alkali treatment for 1-3h, centrifuging, separating, and drying and crushing the product.
9. The method for producing schizochytrium microcapsules enriched in DHA algae oil according to claim 1, wherein in step four, the processing treatment comprises the following steps:
step S1: liquefaction process
Mixing purified water and schizochytrium in a volume ratio of 2-5:1 to obtain a pure schizochytrium solution;
step S2: mixing
Placing schizochytrium solution, arabic gum, modified corn starch and whey protein powder in a mass ratio of 30:2:1:1 into a stirring kettle, stirring and mixing for 20-30min at a rotating speed of 2000-3000rpm to obtain a mixed solution;
step S3: homogenizing
Heating the mixed solution to 50-60deg.C, homogenizing under 25-35MPa for 2-3 times to obtain homogenized solution
Step S4: spray drying the homogeneous solution with a spray dryer, setting the air inlet temperature at 165-200deg.C, the air outlet temperature at 70-95deg.C, and the feeding speed at 5-20L/h to obtain schizochytrium microcapsule powder;
s5, a step of S5; premixing
Adding anticaking agent into schizochytrium microcapsule powder, and stirring and mixing uniformly.
10. The method for producing schizochytrium limacinum microcapsules enriched in DHA algae oil according to claim 9, wherein in step S5, the anticaking agent consists of calcium silicate, tricalcium phosphate, talcum powder and sodium hexametaphosphate in a mass ratio of 2:2:1:1, and the mass of the anticaking agent added is 1-2% of the schizochytrium limacinum microcapsule powder.
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