CN110558435A - Astaxanthin nano liposome and preparation method and application thereof - Google Patents
Astaxanthin nano liposome and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an astaxanthin nanoliposome and a preparation method and application thereof. The preparation method comprises the following steps: s1, dissolving astaxanthin, cholesterol and phospholipid in a mixed solution of dichloromethane and trichloromethane, oscillating and fully mixing, decompressing and evaporating to remove an organic solvent so as to form a layer of uniform film, and then drying in vacuum; s2, adding a buffer solution into the film, eluting the film, and performing rotary dissolution and uniform dispersion to obtain an astaxanthin liposome suspension; s3, sealing the suspension in the dark at 20-30 ℃ for 6-24 h, and then, using a liposome extrusion instrument to filter the suspension to obtain the liposome. The astaxanthin nano liposome has the characteristics of excellent characterization characteristics, high encapsulation efficiency, uniform particle size, good dispersibility, good stability and strong antioxidant activity, obviously improves the specific growth rate and survival rate of prawns, the total antioxidant capacity and the immunity, and has the advantages of high synthesis rate, simple process and easy industrialization.
Description
Technical Field
the invention belongs to the technical field of biological materials. More particularly, relates to an astaxanthin nanoliposome and a preparation method and application thereof.
Background
Astaxanthin (Astaxannthin), the molecule of which is C40H52O4The lutein derivative has a melting point of about 224 ℃, is insoluble in water, has fat solubility, is easily soluble in most organic solvents such as chloroform, acetone, ethanol, ether and the like, belongs to a lutein beta-carotene family, and has wide distribution range in the natural world. The natural sources of natural astaxanthin mainly come from organisms such as algae, yeast, salmon, trout, shrimp, lobster and krill. Among them, Haematococcus pluvialis (Haematococcus pluvialis) and Phaffia rhodozyma (Phaffia rhodozyma) are the main sources of natural astaxanthin. The astaxanthin has physiological effects of resisting oxidation and aging, enhancing the disease resistance and coloring of organisms and the like, and has wide application prospect in the industries of livestock raising, aquatic products, food and medicine.
Astaxanthin demand is also increasing with global water production increasing at a 24% per year rate. However, the molecular structure of free astaxanthin is unstable, and the free astaxanthin is extremely sensitive to environmental factors such as light, heat, oxygen and the like, so that isomerization or degradation is easily caused, and the biological effect cannot be normally exerted. The litopenaeus vannamei has the characteristics of high growth speed, strong stress resistance, high feed conversion rate, high meat processing rate and the like, and becomes one of the most main varieties for prawn culture in China. However, as the cultivation density increases, the cultivation environment deteriorates continuously, which leads to increased disease of the prawns and reduced quality of the prawn products. Therefore, the exploration of the astaxanthin nanoliposome with good physicochemical characteristics, strong antioxidant activity and good stability is a problem to be solved urgently.
Liposome (Liposome) is an artificially prepared biological membrane, and the inner core formed by amphiphilic substances such as phospholipid molecules is a double-layer closed vesicle with aqueous phase and a cell membrane-like structure. The traditional liposome preparation methods include a thin film hydration method, an ether/ethanol injection method, a reverse evaporation method, high-pressure homogenization, ultrasound, extrusion and the like used in post-molding processing in recent years. Among them, the ether/ethanol injection method mainly allows phospholipid to be completely dissolved in organic solution ethanol/ether, and then the mixed solution is rapidly injected into a large amount of PBS buffer solution by sucking the mixed solution with a fine needle of a syringe, thereby forming liposomes. The method is simple in operation, quick in aging and mild, so that the influence on substances wrapped by the liposome is small, and the method is widely applied to preparation of the liposome. However, organic solutions such as ethanol and ether are not easy to remove, and due to solubility, the liposome prepared by the method has a low encapsulation rate. The reverse evaporation method is mainly to dissolve the raw materials for preparing liposome such as phospholipid in organic solvent, then inject the aqueous solution containing embedding substance into the organic solvent, and then perform ultrasonic disintegration for a period of time to form stable W/O water-in-oil emulsion, and finally perform decompression rotary evaporation to remove the organic solvent, thus forming liposome with good properties and low toxicity. The particle size of the liposomes prepared by this method is mainly limited by the lipid material dissolved and the kind of organic solvent used.
The Chinese patent with the application number of 201410578096.X discloses an astaxanthin solid lipid nanoparticle and a preparation method thereof. The astaxanthin solid lipid nanoparticle is prepared from a lipid material and a water phase according to the mass ratio of 1: 11, preparing; wherein, by mass fraction, the lipid material comprises: 1-15% of solid fat, 85-98.9% of vegetable oil and 0.1% of astaxanthin; in the aqueous phase: 1-10% of surfactant and 90-99% of deionized water. However, the liposome prepared by the method has the defects of low encapsulation efficiency (the encapsulation efficiency is only 65-76%), poor dispersion stability (easy agglomeration and precipitation), easy capture by a monocyte phagocytic system, weak targeting property and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of astaxanthin nanoliposome which has good stability, high encapsulation efficiency, uniform particle size, good dispersibility and easy effective absorption by organisms.
the invention also aims to provide the astaxanthin nano liposome prepared by the method. The astaxanthin nano liposome has the characteristics of excellent characterization characteristics (high entrapment rate, uniform particle size and good dispersity), good stability and strong antioxidant activity.
The invention further aims to provide application of the astaxanthin nano-liposome prepared by the preparation method in preparation of shrimp feed. The astaxanthin nano liposome obviously improves the specific growth rate and survival rate of prawns, obviously improves alkaline phosphatase, superoxide dismutase, total antioxidant capacity, polyphenol oxidase and lysozym activity in liver and pancreas in the blood serum of prawns, and obviously reduces the content of the blood serum and the liver and pancreas malondialdehyde of the prawns; the astaxanthin nanoliposome can reduce the relative abundance of Proteobacteria (Proteobacteria) in intestinal tracts of prawns, and reduce the percentage of Vibrio (Vibrio) and Shewanella (Shewanella).
The above purpose of the invention is realized by the following technical scheme:
a preparation method of astaxanthin nano-liposome comprises the following steps:
S1, dissolving astaxanthin, cholesterol and phospholipid in a mixed solution of dichloromethane and trichloromethane, oscillating and fully mixing, decompressing and evaporating to remove an organic solvent so as to form a layer of uniform film, and then drying in vacuum;
S2, adding a buffer solution into the film, eluting the film, dissolving and dispersing uniformly to obtain an astaxanthin liposome suspension;
S3, sealing the suspension in the dark at 20-30 ℃ for 6-24 h, and then, using a liposome extrusion instrument to filter the suspension to obtain the liposome.
In a preferred embodiment of the present invention, the mass ratio of cholesterol to phospholipid in S1 is 1: 4-8, preferably 1: 5-7, more preferably 1: 6.
In a preferred embodiment of the present invention, the ratio of the mass of astaxanthin in S1 to the total mass of cholesterol and phospholipids is 0.5 to 2.5: 210, preferably 1: 210.
In a preferred embodiment of the present invention, the buffer is a phosphate buffer; the pH value of the buffer solution is controlled to be 6.0-8.0, and the pH value of the buffer solution is preferably 7.0-7.5.
in a preferred embodiment of the present invention, the concentration of the phosphate buffer is 0.01 to 0.02 mM.
In a preferred embodiment of the invention, the phospholipid is soy lecithin.
In a preferred embodiment of the invention, after the astaxanthin liposome suspension is obtained in S2, the suspension is further subjected to ultrasonic treatment.
In a preferred embodiment of the present invention, the ultrasonic conditions are: ultrasonic power is 50 ~ 70KHZAnd the ultrasonic temperature is 25-30 ℃, and the ultrasonic time is 20-40 min.
in the preferred embodiment of the present invention, the conditions for controlling the reduced pressure evaporation in S1 are as follows: s1 the conditions for controlling the reduced pressure evaporation are as follows: the vacuum degree is 0.08-0.1 MPa, and the temperature is-10 to-13 ℃.
in the preferred embodiment of the present invention, the conditions for controlling the dissolution and dispersion uniformity in S2 are as follows: the temperature is 37-40 ℃, the rotating speed is 100-500 r/min, and the time is 1-2 h.
In a preferred embodiment of the present invention, the volume ratio of the mixed solution of dichloromethane and chloroform to the buffer solution is 1: 4 to 7.
In a preferred embodiment of the present invention, the volume ratio of dichloromethane to chloroform in the mixed solution of dichloromethane and chloroform is 1: 4.
In a preferred embodiment of the present invention, the vacuum drying conditions in S1 are: the vacuum degree is 10-11 Pa, and the temperature is-50 to-60 ℃.
Accordingly, the astaxanthin nanoliposome prepared by the preparation method is also within the protection scope of the invention. The encapsulation rate of the liposome is 83-95 percent; the particle size is 100-188 nm; the dispersion index is 0.02-0.04; the Zeta potential is-55.78 +/-1.63 mv.
The invention also relates to application of the astaxanthin nano liposome prepared by the preparation method in preparation of shrimp feed. Experiments show that the astaxanthin nano liposome is greatly improved in antioxidant activity, water solubility and stability, can be effectively absorbed by organisms easily, obviously improves the specific growth rate and survival rate of the prawns, obviously improves the alkaline phosphatase, superoxide dismutase, total antioxidant capacity, polyphenol oxidase and lysozyme activity in hepatopancreas in blood serum of the prawns, and obviously reduces the content of the blood serum of the prawns and the content of hepatopancreas malondialdehyde; the astaxanthin nanoliposome can reduce the relative abundance of Proteobacteria (Proteobacteria) in intestinal tracts of prawns, and reduce the percentage of Vibrio (Vibrio) and Shewanella (Shewanella).
Compared with the prior art, the invention has the following beneficial effects:
(1) The prepared astaxanthin nano liposome encapsulates astaxanthin in liposome, reduces the sensitivity of the liposome to external environment, enhances the stability of the liposome, has high antioxidant activity and good water solubility, is easy to be absorbed by organisms, and has higher bioavailability.
(2) The prepared astaxanthin nano liposome has high entrapment rate, uniform particle size, good dispersibility, good in-vitro slow release effect and high stability.
(3) The prepared astaxanthin nano liposome obviously improves the specific growth rate and survival rate of prawns, and obviously improves the total oxidation resistance and immunocompetence of the prawns.
(4) The astaxanthin nanoliposome has high synthesis rate and simple process and is easy to industrialize.
(5) The reaction condition is mild, safe and environment-friendly.
Drawings
FIG. 1 is a high performance liquid chromatography chromatogram for astaxanthin detection.
FIG. 2 shows a standard curve of astaxanthin.
FIG. 3 shows the appearance and dispersion state of astaxanthin nanoliposome; wherein the left bottle is astaxanthin nano liposome solution; the middle bottle is astaxanthin nano liposome solution diluted by 10 times; the right bottle is natural astaxanthin).
FIG. 4 is a transmission electron microscope image of astaxanthin nanoliposome.
Fig. 5 shows the particle size distribution of astaxanthin nanoliposomes.
Fig. 6 is a potential distribution diagram of astaxanthin nanoliposome.
Fig. 7 shows the results of comparing the clearance rates of astaxanthin nanoliposome (ASTL) and Astaxanthin (AST) with respect to DPPH and ABTS.
fig. 8 shows the variation of the encapsulation efficiency of astaxanthin nanoliposomes (ASTL) with time.
FIG. 9 shows the effect of astaxanthin addition on the encapsulation efficiency.
FIG. 10 is a graph showing the effect of cholesterol to soy lecithin ratio on encapsulation efficiency.
FIG. 11 is a graph showing the effect of buffer pH on encapsulation efficiency.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 an astaxanthin nanoliposome
1. The preparation method of the astaxanthin nano liposome comprises the following steps:
(1) Accurately weighing 1.0mg of astaxanthin, 210mg of cholesterol and soybean lecithin (the mass ratio of the two is 1: 6); completely dissolving the mixture at normal temperature by using 5mL of mixed solution of dichloromethane and trichloromethane, carrying out vortex oscillation until no insoluble substances exist, removing the organic solvent by using a rotary evaporator in vacuum at low temperature until a layer of uniform orange thin film is obtained on the bottle wall of a round-bottom flask of the rotary evaporator, and then putting the round-bottom flask into a vacuum drying oven for vacuum drying for 1 h;
(2) Slowly pouring 20mL of prepared phosphate buffer solution with the pH value of 7.5 into a round-bottom flask, and rotating the round-bottom flask at a proper speed so as to uniformly dissolve substances on the inner wall of the flask in the buffer solution; if phospholipid precipitates, carrying out ultrasonic oscillation to obtain astaxanthin liposome suspension;
(3) Sealing the suspension in dark, and standing at 25 deg.C for 6 h; then, a liposome extrusion instrument is used for film coating, so that the astaxanthin nano liposome is obtained, and the astaxanthin nano liposome is stored in a refrigerator at the temperature of 4 ℃.
2. The physicochemical properties of the astaxanthin nanoliposome obtained in example 1 were investigated, including the measurement of appearance, particle size, potential, encapsulation efficiency, antioxidant activity, and the like.
(1) The chromatogram and standard curve of astaxanthin high performance liquid phase detection are shown in figures 1 and 2.
As can be seen from fig. 1, the retention time of astaxanthin is 4.182min, the peak pattern is good, no tailing, no impurity peaks, indicating that no degradation or isomerization of astaxanthin occurs, and astaxanthin can be stably present in the chromatographic solvent.
And measuring peak area values of astaxanthin standard substance solutions with different concentrations at a wavelength of 475nm, repeating for 3 times, and taking an average value. As can be seen from fig. 2, the linear regression equation of astaxanthin obtained by linear regression of the peak area (Y) with concentration (X) based on the data obtained is: 165.4057X +4.3059, R20.9991, astaxanthin therefore has a good linear relationship with peak area in the concentration range of 1.0-5.0. mu.g/mL.
(2) Appearance and dispersion state observation of astaxanthin nanoliposome
Adding buffer solution with pH of 7.5 into the prepared astaxanthin nanoliposome dispersion system, diluting ten times, shaking uniformly, standing for 15min, simultaneously placing astaxanthin standard substance with the same amount as the astaxanthin nanoliposome into buffer solution with the same amount, using astaxanthin nanoliposome solution as blank control, observing the appearance of the dispersion system with naked eyes, and observing the formation of insoluble substances and the dispersion condition.
As shown in fig. 3, the appearance of the astaxanthin nanoliposome dispersion sample solution is a uniform, orange translucent emulsion; diluting with buffer solution 10 times, shaking gently, standing for 15min, and dispersing completely and uniformly without solid suspended substance or precipitate; meanwhile, natural astaxanthin with the same mass is placed in the buffer solution with the same volume, is gently shaken and stands for the same time, and is difficult to dissolve.
(3) Transmission electron microscope observation of astaxanthin nanoliposome
and (3) dripping a trace amount of astaxanthin nanoliposome onto a glass slide, adding 2% phosphotungstic acid on a copper net for negative dyeing for 5min, volatilizing for 15-20min under natural conditions, observing the form under a transmission electron microscope and taking a photo.
As shown in fig. 4, the electron microscope image showed that the morphology of the astaxanthin nanoliposome particles was spherical or ellipsoidal, the dispersibility was good, and the particle diameters were almost uniform. Observation is carried out in a large visual field, and no obvious impurity interference exists in the visual field, which indicates that the synthesis rate of the astaxanthin nanoliposome is high. With further increase of the magnification, the surface of the astaxanthin nanoliposome particles can be seen to be smooth.
(4) Determination of particle size and dispersion index of astaxanthin nano-liposome
taking 10 mu L of the prepared astaxanthin nanoliposome sample solution, uniformly mixing with 990 mu L of ultrapure water, placing into a polystyrene-styrene cuvette, removing bubbles, shaking up, and measuring the particle size and the particle size distribution of the astaxanthin nanoliposome by using a Nano-ZS90 laser particle size measuring instrument of Malvern company.
As shown in FIG. 5, the particle size of the astaxanthin nanoliposome was 188.6. + -. 2.69nm, and the dispersion index (PDI) was 0.02. + -. 0.02.
(5) Zeta potential determination of astaxanthin nanoliposome
1.0mL of the above test sample solution was added to a clean polystyrene-propylene U-tube, and the Zeta potential of the astaxanthin nanoliposome was measured using a Nano-ZS90 laser particle size analyzer from Malvern.
As can be seen from FIG. 6, the Zeta potential of the astaxanthin nano-liposome is-55.78 + -1.63 mv, and the astaxanthin nano-liposome is electronegative.
(6) Antioxidant activity test of astaxanthin nanoliposome
1) DPPH radical scavenging Capacity determination
As can be seen from fig. 7, the astaxanthin nanoliposome dispersion solution lost 41.36% of astaxanthin while the astaxanthin methanol solution consumed 83.49% of the total amount of astaxanthin by scavenging an equal amount of DPPH radicals; the difference of the astaxanthin nanoliposome and the astaxanthin methanol solution on DPPH free radical scavenging rate is obvious (P is less than 0.05).
2) ABTS free radical scavenging Capacity assay
As can be seen from the data and significance analysis in fig. 7, the decrease in astaxanthin in the astaxanthin nanoliposome dispersion was 30.16% of the total amount of astaxanthin, and the decrease in astaxanthin in the astaxanthin methanol solution was 65.61% of the total amount of astaxanthin, with the same amount of ABTS radicals scavenging, which was significantly different (P < 0.05).
(7) Storage stability test of astaxanthin nanoliposome
The astaxanthin nanoliposome prepared as above was stored in a dark standard atmosphere at 4 ℃ for 50 days, and the particle size, dispersion index, potential and Encapsulation Efficiency (EE) of the astaxanthin nanoliposome were measured at 0d, 10d, 20d, 30d, 40d and 50d of the storage, and the results are shown in Table 1 and FIG. 8.
Wherein Wt is the astaxanthin content embedded by the liposome in each determination time period; w0 is the content of embedded astaxanthin in the day of preparation of the astaxanthin nanoliposome.
TABLE 1 changes over time in particle size, dispersion index, Zeta potential
As can be seen from Table 1, in terms of particle size, the astaxanthin nanoliposome tends to become smaller in particle size during storage for as long as 50d, the particle size does not change significantly at 30d before storage (P > 0.05), and the particle size begins to become smaller and has a significant difference after 30d (P < 0.05); in terms of dispersion index, there was a significant increase (P < 0.05) over a period after 30d, but much less than 0.3; in the Zeta potential, the significant increase occurs at 10d (P < 0.05), and no significant change occurs any more (P > 0.05) in the period before 40d after 10d, and by the time of 50d, the charge quantity carried by the astaxanthin nanoliposome particles is significantly reduced once more (P < 0.05) compared with that of the first 30d, but the absolute value of the surface charge quantity of the particles in the study period is more than 30 mv.
meanwhile, as can be seen from fig. 8, from 10d, the encapsulation efficiency of the astaxanthin nanoliposome is significantly reduced (P < 0.05), and is reduced from 95.95% to 93.38%; the encapsulation efficiency in 10d to 20d has not changed significantly (P > 0.05); in the time period from 30d to 50d, the encapsulation efficiency of the astaxanthin nano liposome is in a remarkable reduction trend (P < 0.05), and at the time of 50d, the encapsulation efficiency is reduced to 87.16%.
In terms of retention, the change in retention (RR) was measured at 10d of storage to describe the storage stability of the astaxanthin nanoliposome under the environmental conditions. Experiments show that after the astaxanthin nanoliposome is preserved for 10 days, the residual astaxanthin still accounts for 84.0 percent of the original content.
Example 2 optimization of astaxanthin nanoliposome preparation
(1) Effect of astaxanthin dosage on Liposome encapsulation efficiency
Astaxanthin nanoliposomes were prepared in the same manner as in example 1, except that the conditions were not changed, except that the amount of astaxanthin added was 0.5, 1.0, 1.5, 2.0, and 2.5mg as one-factor variables, the total amount of cholesterol and soybean lecithin was 210mg (mass ratio: 1: 6), the pH of the buffer was 7.5, and the effect of the amount of astaxanthin added on the nanoliposome encapsulation efficiency was examined.
As can be seen from FIG. 9, when the addition amount of astaxanthin is 1.0mg, the encapsulation efficiency is the highest, reaching 94.12%; but when the adding amount is less than 1.0mg, the encapsulation efficiency is lower; when the added amount of astaxanthin exceeds 1.0mg, the encapsulation efficiency tends to be low.
(2) Effect of the Mass ratio of Cholesterol to Soy lecithin on the encapsulation efficiency of liposomes
Based on example 1, the mass ratio of cholesterol to soybean lecithin is 1: 4. 1: 5. 1: 6. 1: 7. 1: 8 is a single-factor variable, under the conditions that the added amount of astaxanthin is 1.0mg, the total amount of cholesterol and soybean lecithin is 210mg, and the pH of the buffer solution is 7.5, the astaxanthin nanoliposome is prepared by the method of the above example 1, the encapsulation efficiency is measured, and the influence of the mass ratio of cholesterol and soybean lecithin on the encapsulation efficiency of the liposome is studied.
As can be seen from fig. 10, when the mass ratio of cholesterol to soybean lecithin was 1: when 6, the effect of embedding astaxanthin is the best. When the mass ratio is higher than 1: and 6, the encapsulation efficiency is increased along with the reduction of the ratio, and when the mass ratio is lower than 1: at 6, the encapsulation efficiency began to decrease as the content of soybean lecithin increased.
(3) effect of buffer pH on Liposome encapsulation efficiency
Astaxanthin nanoliposomes were prepared in the same manner as in example 1, with the buffer pH being 6.0, 6.5, 7.0, 7.5, 8.0, respectively, as single-factor variables, under the conditions that the added amount of astaxanthin was 1.0mg and the total amount of cholesterol and soybean lecithin was 210mg (mass ratio of 1: 6), and the encapsulation efficiency was measured to analyze the influence of the pH of the buffer on the encapsulation efficiency of the liposomes.
As can be seen from fig. 11, the encapsulation efficiency of the astaxanthin nanoliposome increases with the increase of the pH of the buffer solution, and is more obvious when the pH is in the range of 6.0 to 7.0, and the change of the encapsulation efficiency of the astaxanthin nanoliposome is downward when the pH exceeds 7.5.
(4) Quadrature test
On the basis of the single-factor test, 3 main influencing factors such as astaxanthin dosage, the mass ratio of cholesterol to soybean lecithin, buffer solution pH and the like are selected, and an orthogonal test for preparing the astaxanthin nanoliposome is carried out by taking the encapsulation efficiency as an index. The orthogonal experimental design is shown in table 2.
TABLE 2 orthogonal test factor horizon
As can be seen from Table 3, the influence factors on the encapsulation efficiency of astaxanthin nanoliposome are in the order of magnitude B (mass ratio of cholesterol to soybean lecithin)>C (buffer pH)>A (added amount of astaxanthin). The best combination is A1B1C1I.e. astaxanthin in an amount of 1.0mg, cholesterol: soybean lecithin ═ 1: 6 buffer pH 7.0, the encapsulation efficiency under this set of conditions was 92.18%, being the optimal test set. The optimum combination is A according to the value of k1B1C3. A is to be1B1C3The combination was tested in 3 parallel validation runs, resulting in an average encapsulation of 95.96% which is greater than that of orthogonal test A1B1C1Encapsulation efficiency under the combination conditions, Explanation A1B1C3The combination is superior to A1B1C1And (4) combining. Therefore, the use amount of astaxanthin is determined to be 1.0mg, and the mass ratio of cholesterol to soybean lecithin is 1: 6, the encapsulation efficiency of the astaxanthin nano liposome is the maximum under the condition that the pH value of the buffer solution is 7.5, and the preparation effect is the best.
TABLE 3 orthogonal test design and results
Example 3 Effect of astaxanthin nanoliposomes on growth and immunization of Litopenaeus vannamei
(1) Test grouping and processing
Selecting juvenile shrimps with normal appearance, strong constitution and 0.3 +/-0.01 g of initial weight, randomly distributing the juvenile shrimps into 12 250L culture barrels, and respectively adding 4 treatment groups, namely a blank control group (group B), an astaxanthin adding group (AST group), a blank liposome adding group (BL group) and an astaxanthin adding nanoliposome group (ASTL group) in example 1; each treatment group was set to 3 replicates, each of 35 shrimp, feeding period 43 d.
b, feeding the prawns to a basic feed, and feeding the prawns to the AST group, the BL group and the ASTL group to a basic feed containing astaxanthin, blank liposome and astaxanthin nano liposome respectively; respectively adding blank liposome, astaxanthin and astaxanthin nano liposome into the basic feed in a spraying and stirring manner; the astaxanthin content in AST group and ASTL group was 80mg astaxanthin per 1kg feed, and the blank liposome was added in the same amount as in ASTL group.
(2) growth performance of Litopenaeus vannamei of each feed group
As can be seen from Table 4, when four different feeds were fed, there was no significant difference in the weight gain and feed efficiency between the blank control group and each test group; in the aspect of specific growth rate of the prawns, both the AST group and the ASTL group are obviously higher than those of the B group and the BL group (P is less than 0.05), the difference between the AST feed group and the ASTL feed group is not significant (P is more than 0.05), and the difference between the B group and the BL group is not significant (P is more than 0.05); in the aspect of survival rate, the survival rate of the ASTL group is the highest and is 83.83 percent, and the survival rate is obviously higher than that of the B group, the AST group and the BL group; the survival rate of the litopenaeus vannamei is obviously improved by feeding the ASTL feed, and the survival rate of the litopenaeus vannamei has no obvious difference (P is more than 0.05) among other feed groups.
TABLE 4 growth Performance of Litopenaeus vannamei of each feed group
(3) Prawn serum immune index determination
The results of the shrimp serum immunological index measurements of each feed group are shown in Table 5. As can be seen from table 5, in the case of the activity of the serum immunoenzyme of the prawns, the activity of acid phosphatase (ACP) and the activity of Lysozyme (LZM) in the serum of each test group were not significantly different from those of group B (P > 0.05); the alkaline phosphatase (AKP) activity in the shrimp serum of the ASTL group is obviously higher than that of other feed groups (P is less than 0.05), the activity value is 17.65 (King units/100 mL), and the other test groups have no obvious difference (P is more than 0.05).
TABLE 5 serum immunity index of each feed group for shrimp
Experiments also find that the astaxanthin nano liposome obviously improves the specific growth rate and survival rate of prawns, obviously improves alkaline phosphatase, superoxide dismutase, total antioxidant capacity, polyphenol oxidase and lysozym activity in liver and pancreas in the blood serum of prawns, and obviously reduces the content of the blood serum and the liver and pancreas malondialdehyde of the prawns; the astaxanthin nanoliposome can reduce the relative abundance of Proteobacteria (Proteobacteria) in intestinal tracts of prawns, and reduce the percentage of Vibrio (Vibrio) and Shewanella (Shewanella).
The applicant declares that the above detailed description is a preferred embodiment described for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, i.e. it does not mean that the present invention must be implemented by means of the above embodiment. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. The preparation method of the astaxanthin nano liposome is characterized by comprising the following steps:
S1, dissolving astaxanthin, cholesterol and phospholipid in a mixed solution of dichloromethane and trichloromethane, oscillating and fully mixing, decompressing and evaporating to remove an organic solvent so as to form a layer of uniform film, and then drying in vacuum;
S2, adding a buffer solution into the film, eluting the film, dissolving and dispersing uniformly to obtain an astaxanthin liposome suspension;
S3, sealing the suspension in the dark at 20-30 ℃ for 6-24 h, and then, using a liposome extrusion instrument to filter the suspension to obtain the liposome.
2. The method according to claim 1, wherein the mass ratio of the cholesterol to the phospholipid of S1 is 1: 4-8, preferably 1: 5-7, more preferably 1: 6.
3. The method according to claim 2, wherein the ratio of the mass of astaxanthin S1 to the total mass of cholesterol and phospholipids is 0.5 to 2.5: 210, preferably 1: 210.
4. The method according to claim 1, wherein the buffer is a phosphate buffer; the pH value of the buffer solution is controlled to be 6.0-8.0, and the pH value of the buffer solution is preferably 7.0-7.5.
5. The method according to claim 1, wherein the phospholipid is soybean lecithin.
6. The method according to claim 1, wherein after the astaxanthin liposome suspension is obtained in S2, the suspension is further subjected to ultrasonic treatment.
7. The method according to claim 6, wherein the ultrasonic conditions are as follows: ultrasonic power is 50 ~ 70KHZand the ultrasonic temperature is 25-30 ℃, and the ultrasonic time is 20-40 min.
8. The method according to claim 1, wherein the conditions for controlling the reduced pressure evaporation in S1 are as follows: the vacuum degree is 0.08-0.1 MPa, and the temperature is-10 to-13 ℃; s2 the conditions for controlling the dissolution and the dispersion to be uniform are as follows: the temperature is 37-40 ℃, the rotating speed is 100-200 r/min, and the time is 1-2 h.
9. The astaxanthin nano-liposome prepared by the preparation method of any one of claims 1 to 8, wherein the encapsulation efficiency of the liposome is 83-95%; the particle size is 100-188 nm; the dispersion index is 0.02-0.04.
10. The use of the astaxanthin nanoliposome prepared by the preparation method according to any one of claims 1 to 8 in preparing shrimp feed.
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| CN115990236A (en) * | 2023-03-22 | 2023-04-21 | 广州研智化妆品有限公司 | Composition used after medical arts and preparation method thereof |
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| CN115990236A (en) * | 2023-03-22 | 2023-04-21 | 广州研智化妆品有限公司 | Composition used after medical arts and preparation method thereof |
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