CN115836707A - Defatted microalgae powder, preparation method and application thereof, and marine fish compound feed - Google Patents

Defatted microalgae powder, preparation method and application thereof, and marine fish compound feed Download PDF

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CN115836707A
CN115836707A CN202211529464.2A CN202211529464A CN115836707A CN 115836707 A CN115836707 A CN 115836707A CN 202211529464 A CN202211529464 A CN 202211529464A CN 115836707 A CN115836707 A CN 115836707A
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powder
parts
microalgae powder
microalgae
fish
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王成强
李宝山
曹体宏
孙永智
黄炳山
王际英
宋志东
郝甜甜
王晓艳
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Shandong Marine Resource and Environment Research Institute
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Shandong Marine Resource and Environment Research Institute
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    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

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Abstract

The invention relates to the technical field of aquatic feed processing, in particular to defatted microalgae powder, a preparation method and application thereof and a compound feed for marine fishes. The invention provides a preparation method of degreased microalgae powder, which comprises the following steps: (1.1) mixing the microalgae powder with ethanol, reacting for 6-8 h, centrifuging, and removing supernatant to obtain microalgae powder precipitate; (1.2) mixing the microalgae powder precipitate with water and a surfactant, and carrying out ultrasonic oscillation for 5-10 min to obtain a microalgae powder precipitate mixed solution; (1.3) mixing the microalgae powder precipitate mixed solution with a complex enzyme, performing enzymolysis for 4-6 h at 40-60 ℃, centrifuging, and removing upper clear oil to obtain a degreased microalgae powder mixed solution; and (1.4) drying the degreased microalgae powder mixed solution to obtain degreased microalgae powder. The defatted microalgae powder prepared by the method is more suitable for preparing a compound feed which is beneficial to the growth of the marine fish and improves the immunity of the marine fish.

Description

Defatted microalgae powder, preparation method and application thereof, and marine fish compound feed
Technical Field
The invention relates to the technical field of aquatic feed processing, in particular to defatted microalgae powder, a preparation method and application thereof and a compound feed for marine fishes.
Background
Microalgae are primary producers of marine food chains, are various and widely distributed, and are concerned in the development of novel feed raw materials at present because of the fact that microalgae contain rich protein and lipid nutrients, particularly essential amino acids and highly unsaturated fatty acids. In particular, in recent years, the production scale of microalgae is developed, the yield is continuously increased, the cost is further reduced, the microalgae is widely applied to the feed manufacturing industry, many persons in the industry introduce the microalgae as a novel protein raw material into a feed formula, and the microalgae replaces part of fish meal to relieve the situation of the deficiency of fish meal resources at present, but the utilization rate of the microalgae powder directly added into the feed is low, and the microalgae powder is particularly used for aquaculture feed and mainly has the following problems. Firstly, the nutrient composition of single microalgal flour is unbalanced and lacks essential amino acids; secondly, if the microalgae powder is used for replacing part of the fish meal, the feed has no food calling performance to animals due to the reduction of the using amount of the fish meal; thirdly, due to the existence of microalgae cell walls, the nutrient is low in digestion and utilization rate. Therefore, the wall-broken microalgae is more favorable for digestion and absorption of cultured animals, and the oil and fat contained in the microalgae are high-value raw materials in the food industry and the pharmaceutical industry, so that the wall-broken microalgae is used as a feed raw material after the algae oil is extracted, and the diversity high-value utilization of the microalgae powder can be better realized. However, the current technology for extracting algae oil by breaking walls of microalgae powder has the problems of higher cost, complex process and the like, and the application and high-value utilization of the microalgae powder in the aspect of aquatic feeds are limited.
Disclosure of Invention
The invention aims to provide a method for wall breaking and degreasing after proportioning different microalgae flour according to a proportion to obtain degreased microalgae flour with rich nutrition, and the degreased microalgae flour is matched with other feed raw materials and additives to be applied to a feed formula for marine fish. The feed produced by the method meets the requirements of essential amino acids of seawater culture varieties of lateolabrax japonicus, sebastes schlegeli, grouper and the like, is added with other nutrients to form a more scientific and balanced formula of the mixed feed, realizes the use of a novel protein raw material in aquatic feed, reduces the using amount of a grain protein source, and can realize healthy growth and ecological culture of cultured fishes.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of degreased microalgae powder, which comprises the following steps:
(1.1) mixing the microalgae powder with ethanol, reacting for 6-8 h, centrifuging, and removing supernatant to obtain microalgae powder precipitate;
(1.2) mixing the microalgae powder precipitate with water and a surfactant, and carrying out ultrasonic oscillation for 5-10 min to obtain a microalgae powder precipitate mixed solution;
(1.3) mixing the microalgae powder precipitate mixed solution with a complex enzyme, performing enzymolysis for 4-6 h at 40-60 ℃, centrifuging, and removing upper clear oil to obtain a degreased microalgae powder mixed solution;
(1.4) drying the degreased microalgae powder mixed solution to obtain degreased microalgae powder;
the mixing mass ratio of the microalgal powder to the ethanol is 1;
the compound enzyme is a mixture of neutral protease, cellulase and papain;
the neutral protease is: cellulase: the mass ratio of the papain is 2-4;
the activity of the neutral protease is more than or equal to 100U/mg;
the activity of the cellulase is more than or equal to 3000U/g;
the activity of the papain is more than or equal to 50U/mg.
Preferably, the microalgae powder is one or more of spirulina powder, chlorella powder and chlorella powder.
Preferably, when the microalgal flour is a mixed flour of spirulina flour, chlorella flour and parachloropsis, the mass ratio of the spirulina flour to the chlorella flour is as follows: chlorella powder: the pseudo-micro-green algae powder is 5-12.
Preferably, the concentration of the ethanol in the step (1.1) is 75 to 80vt%;
the reaction temperature in the step (1.1) is 30-40 ℃;
the rotating speed of the centrifugation in the step (1.1) is 3000-5000 rpm;
the centrifugation time in the step (1.1) is 5-10 min.
Preferably, the mass ratio of the microalgae powder precipitate mixed with water in the step (1.2) is 1;
the surfactant in the step (1.2) is sodium dodecyl sulfate;
mixing the microalgae powder precipitate and the surfactant according to the mass ratio of 100:0.1 to 0.2;
the power of the ultrasound in the step (1.2) is 100-150W.
Preferably, the mass ratio of the mixed solution of the microalgae powder precipitate in the step (1.3) to the complex enzyme is 100;
the pH value during enzymolysis in the step (1.3) is 6.0-7.5;
the rotation speed in the enzymolysis in the step (1.3) is 40-60 rpm;
the rotating speed of the centrifugation in the step (1.3) is 3000-5000 rpm;
the centrifugation time in the step (1.3) is 5-10 min;
the drying in the step (1.4) is freeze drying;
the freeze drying method comprises the following steps: after being completely frozen at the temperature of minus 20 ℃, the mixture is dried for 36 to 48 hours in vacuum at the temperature of minus 55 to minus 60 ℃.
The invention also provides the defatted microalgae powder prepared by the preparation method.
The invention also provides application of the defatted microalgae powder in preparation of the compound feed for the marine fish.
The invention also provides a marine fish compound feed containing the defatted microalgae powder, which comprises the following components in parts by weight:
20 to 30 parts of fish meal, 8 to 15 parts of casein, 8 to 12 parts of soybean protein concentrate, 15 to 30 parts of degreased microalgal flour, 6 to 10 parts of wheat gluten, 8 to 10 parts of wheat flour, 3 to 5 parts of fish oil, 4 to 5 parts of soybean oil, 1 to 1.5 parts of soybean lecithin, 0.6 to 1.0 part of phagostimulant, 0.4 to 0.6 part of taurine, 0.8 to 1.5 parts of vitamin premix, 1.0 to 2.0 parts of mineral premix, 0.1 to 0.3 part of ethoxyquinoline, 0.4 to 0.6 part of monocalcium phosphate, 0.3 to 0.7 part of vitamin C and 1.0 to 4.0 parts of sodium carboxymethylcellulose;
the phagostimulant is prepared by mixing 1.5-2.5 weight percent of betaine, 1.5-2.5 weight percent of glycine and 1 weight percent of hypoxanthine nucleotide.
The invention also provides a preparation method of the compound feed for marine fishes, which comprises the following steps:
(10.1) mixing fish meal, casein, soybean protein concentrate, defatted microalgae powder, wheat gluten, wheat flour, and sodium carboxymethylcellulose, and pulverizing to particle size of less than 150 μm to obtain intermediate 1;
(10.2) mixing the intermediate 1 with vitamin premix, mineral premix, taurine, monocalcium phosphate, vitamin C, ethoxyquin, phagostimulant, soybean lecithin, soybean oil and fish oil, curing, granulating and drying to obtain the compound feed for the marine fish;
the drying temperature is 65-75 ℃;
the water content of the marine fish compound feed is less than 12%.
The invention provides defatted microalgae powder, a preparation method and application thereof and a compound feed for marine fishes. The invention carries out wall breaking and degreasing treatment on the microalgae powder by utilizing a biological catalysis-aqueous enzymatic method, has easily obtained raw materials, high work efficiency and easy popularization, applies the obtained degreased microalgae powder to the compound feed for the marine fish, strengthens the nutrition balance of the compound feed by properly proportioning with other feed raw materials, and enhances the growth performance of fish bodies and can also reduce the nitrogen and phosphorus discharge amount of the fish bodies by adding nutritional factors such as phagostimulant and the like. The feed prepared from the components of the invention meets the growth performance of the marine fish.
The microalgae powder degreasing process adopts a biocatalysis-aqueous enzymatic method, the method is a mild oil extraction method, the method is easy to implement, the operation is simple and convenient, the extraction time is short, the energy consumption is low, and the yield of microalgae oil is high.
The defatted microalgae powder selects three kinds of microalgae for simultaneous wall breaking and degreasing, so that essential amino acids can be supplemented with each other, and the defatted microalgae powder is applied to the marine fish compound feed as a novel protein source, thereby not only providing the protein source for fish, but also additionally providing nutrients such as polysaccharide, mineral substances, vitamins and the like. Meanwhile, the discharge of nitrogen and phosphorus is reduced, the pollution of the feed to water quality is reduced, and the growth of the fish body is not influenced.
The sea water fish compound feed added with the degreased microalgae powder has scientific formula, reasonable process and balanced nutrition composition, improves the proportion of the 'non-grain' protein source microalgae powder in the compound feed, reduces the using amount of grain protein, and controls the content of fish meal to be below 30 percent. And by properly supplementing the phagostimulant and other additives, the food intake and the conversion rate of the fish to the compound feed are improved, and the absorption and the utilization of nutrition are promoted.
The nutrient components in the feed prepared by the invention are stable in water, the loss of nutrient substances in the feed is reduced, and the feed has a proper sedimentation speed in water and accords with the feeding habit of seawater fishes.
The sea fish compound feed taking the defatted microalgae powder as the protein raw material is applicable to fish with a monomer specification of more than 50g, in particular to yellow perch, sebastes schlegeli or grouper. Compared with the feed prepared by the traditional formula, the feed increases the utilization of non-grain protein sources, reduces the using amount of fish meal, increases the disease resistance and quality of the fish body under the condition of ensuring the normal growth of the fish body, reduces the discharge amount of nitrogen and phosphorus, and is more ecological and environment-friendly in culture.
Detailed Description
The invention provides a preparation method of degreased microalgae powder, which comprises the following steps:
(1.1) mixing the microalgae powder with ethanol, reacting for 6-8 h, centrifuging, and removing supernatant to obtain microalgae powder precipitate;
(1.2) mixing the microalgae powder precipitate with water and a surfactant, and carrying out ultrasonic oscillation for 5-10 min to obtain a microalgae powder precipitate mixed solution;
(1.3) mixing the microalgae powder precipitate mixed solution with a complex enzyme, performing enzymolysis for 4-6 h at 40-60 ℃, centrifuging, and removing upper clear oil to obtain a degreased microalgae powder mixed solution;
(1.4) drying the degreased microalgae powder mixed solution to obtain degreased microalgae powder;
in the invention, the microalgae powder is one or more of spirulina powder, chlorella powder and chlorella powder, preferably mixed powder of the spirulina powder, the chlorella powder and the chlorella powder; the mass ratio of the spirulina powder to the chlorella powder to the pseudo-chlorella powder is 5-12. In the present invention, the mixing ratio of the microalgal flour to ethanol is 1. In the invention, the concentration of the ethanol is 75-80 vt%, and is preferably 77.5vt%; the reaction temperature in the step (1.1) is 30-40 ℃, and preferably 35 ℃; the reaction time of the step (1.1) is preferably 7h; the rotating speed of the centrifugation in the step (1.1) is 3000-5000 rpm, preferably 4000rpm; the centrifugation time in the step (1.1) is 5-10 min, preferably 7.5min.
In the present invention, the mass ratio of the microalgal flour precipitate of step (1.2) to water is 1; the surfactant in the step (1.2) is sodium dodecyl sulfate; mixing the microalgae powder precipitate and the surfactant according to the mass ratio of 100:0.1 to 0.2, preferably 100:0.15; the power of the ultrasound in the step (1.2) is 100-150W, preferably 125W; the time of the ultrasonic oscillation in the step (1.2) is preferably 7.5min.
In the invention, the complex enzyme in the step (1.3) is a mixture of neutral protease, cellulase and papain; the neutral protease is: cellulase: the mass ratio of the papain is 2-4; the activity of the neutral protease is more than or equal to 100U/mg; the activity of the cellulase is more than or equal to 3000U/g; the activity of the papain is more than or equal to 50U/mg. The mass ratio of the mixed solution of the microalgae powder precipitate and the complex enzyme in the step (1.3) is 100; the temperature of the enzymolysis in the step (1.3) is preferably 50 ℃; the pH value during enzymolysis in the step (1.3) is 6.0-7.5, and is preferably 6.75; the rotation speed in the enzymolysis in the step (1.3) is 40-60 rpm, preferably 50rpm; the enzymolysis time in the step (1.3) is preferably 5 hours; the rotating speed of the centrifugation in the step (1.3) is 3000-5000 rpm, preferably 4000rpm; the centrifugation time in the step (1.3) is 5-10 min, preferably 7.5min; the drying in the step (1.4) is freeze drying; the freeze drying method comprises the following steps: after being completely frozen at the temperature of minus 20 ℃, the mixture is dried for 36 to 48 hours in vacuum at the temperature of minus 55 to minus 60 ℃. The freeze drying method in the step (1.4) is preferably as follows: completely freezing at-20 deg.C, and vacuum drying at-57.5 deg.C for 42h.
The invention also provides the defatted microalgae powder prepared by the preparation method.
The invention also provides application of the defatted microalgae powder in preparation of the compound feed for the marine fish.
The invention also provides a marine fish compound feed containing the defatted microalgae powder, which comprises the following components in parts by weight:
20-30 parts of fish meal, preferably 22-28 parts of fish meal, and further preferably 25 parts of fish meal;
8-15 parts of casein, preferably 10-13 parts of casein, and more preferably 11.5 parts of casein;
8-12 parts of soybean protein concentrate, preferably 9-11 parts, and more preferably 10 parts;
15-30 parts of degreased microalgal flour, preferably 19-26 parts of degreased microalgal flour, and more preferably 22.5 parts of degreased microalgal flour;
6-10 parts of wheat gluten, preferably 7-9 parts of wheat gluten, and more preferably 8 parts of wheat gluten;
8 to 10 parts of wheat flour, preferably 8.5 to 9.5 parts, and more preferably 9 parts;
3-5 parts of fish oil, preferably 3.5-4.5 parts, and more preferably 4 parts;
4-5 parts of soybean oil, preferably 4.5 parts;
1-1.5 parts of soybean lecithin, preferably 1.25 parts;
0.6-1.0 part of phagostimulant, preferably 0.7-0.9 part, and more preferably 0.8 part;
0.4 to 0.6 part of taurine, preferably 0.45 to 0.55 part of taurine, and more preferably 0.5 part of taurine;
0.8 to 1.5 parts of vitamin premix, preferably 1.0 to 1.3 parts, and more preferably 1.15 parts;
1.0-2.0 parts of mineral premix, preferably 1.2-1.8 parts, and more preferably 1.5 parts;
0.1 to 0.3 part of ethoxyquinoline, preferably 0.15 to 0.25 part, and more preferably 0.2 part;
0.4 to 0.6 part of calcium dihydrogen phosphate, preferably 0.45 to 0.55 part, and more preferably 0.5 part;
0.3 to 0.7 part of vitamin C, preferably 0.4 to 0.6 part of vitamin C, and more preferably 0.5 part of vitamin C;
1.0-4.0 parts of sodium carboxymethylcellulose, preferably 2-3 parts of sodium carboxymethylcellulose, and more preferably 2.5 parts of sodium carboxymethylcellulose;
the phagostimulant is prepared by mixing betaine, glycine and hypoxanthine nucleotide according to the weight ratio of 1.5-2.5.
The invention also provides a preparation method of the compound feed for marine fishes, which comprises the following steps:
(10.1) mixing fish meal, casein, soybean protein concentrate, defatted microalgae powder, gluten powder, wheat flour and sodium carboxymethylcellulose, and pulverizing to particle size of less than 150 μm to obtain intermediate 1;
(10.2) mixing the intermediate 1 with vitamin premix, mineral premix, taurine, calcium dihydrogen phosphate, vitamin C, ethoxyquin, phagostimulant, soybean lecithin, soybean oil and fish oil, curing, granulating and drying to obtain the compound feed for the marine fish;
the curing is carried out in a modulator, the feeding speed is adjusted to be 68-78 Hz, the temperature of the modulator is 92-95 ℃, and the optimal temperature is 93.5 ℃; the temperature of the main cylinder is 92-95 ℃, and preferably 93.5 ℃; the host frequency is 42 to 52%, preferably 47%. The drying temperature is 65-75 ℃, and preferably 70 ℃; the water content of the compound feed for marine fish<12％ 。
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The compositions of essential amino acids in the fish meal, soy protein concentrate, spirulina powder, chlorella powder and nannochloropsis powder selected in the examples of the present invention are shown in table 1.
TABLE 1 comparison of essential amino acid compositions (g/100 g) of several major feedstocks
Figure BDA0003971680020000071
Example 1
Uniformly mixing the spirulina powder, the chlorella powder and the nannochloropsis sp powder according to the mass ratio of 7.5.
Mixing microalgal powder and 75vt% ethanol according to a mass ratio of 1:8, reacting at 30 ℃ for 8 hours, centrifuging at 3000rpm for 10min to obtain ethanol supernatant and microalgae powder precipitate, and discarding the ethanol supernatant.
And adding water into the microalgae powder precipitate according to the material-liquid ratio of 1.
Mixing 3 parts of neutral protease, 2 parts of papain and 1 part of cellulase uniformly according to the parts by weight to obtain the complex enzyme required by enzymolysis. Wherein, the neutral protease, the papain and the cellulase are all commercial products, the activity of the neutral protease is more than or equal to 100U/mg, the activity of the papain is more than or equal to 50U/mg, and the activity of the cellulase is more than or equal to 3000U/g.
Adding 3wt% of complex enzyme into the mixed solution of microalgae powder precipitate, setting stirring speed at 45rpm under the conditions of pH 6.5 and temperature of 45 ℃, performing enzymolysis for 5h, centrifuging at 3500rpm for 8min to obtain supernatant clear oil, which is microalgae powder free oil, and processing to obtain microalgae oil used in food, medicine, etc. In the test, the residual mixed solution after the upper layer of clear oil is discarded is stirred and uniformly mixed, and then is completely frozen at the temperature of minus 20 ℃, and is frozen and vacuum-dried for 40 hours at the temperature of minus 55 ℃, so that the defatted microalgae powder with the water content of less than 5 percent is obtained.
The prepared degreased microalgae powder is applied to a marine fish compound feed as a protein raw material, and the compound feed comprises the following raw materials in parts by mass:
30 parts of fish meal, 10 parts of casein, 9 parts of soybean protein concentrate, 19 parts of defatted microalgae powder, 7 parts of wheat gluten, 8 parts of wheat flour, 3.6 parts of fish oil, 4.4 parts of soybean oil, 1 part of soybean lecithin, 0.6 part of a phagostimulant, 0.5 part of taurine, 1.0 part of a vitamin premix, 1.0 part of a mineral premix, 0.2 part of ethoxyquin, 0.5 part of monocalcium phosphate, 0.4 part of vitamin C and 3.7 parts of sodium carboxymethylcellulose.
The phagostimulant comprises the following components in parts by weight: betaine 0.24 parts, glycine 0.24 parts, hypoxanthine nucleotide 0.12 parts
The preparation method of the compound feed for the marine fishes comprises the following steps:
preparing materials: weighing the raw materials according to the weight parts of the components.
Crushing: mixing the above weighed fish meal, casein, soybean protein concentrate, defatted microalgae powder, wheat gluten, wheat flour, and sodium carboxymethylcellulose for the first time to obtain coarse powder with fineness of
Figure BDA0003971680020000081
Screening a screen; then, the mixture is pulverized for the second time by an ultrafine pulverizer until the particle size is 145 μm, thereby obtaining an intermediate 1.
And (3) secondary mixing: and mixing the intermediate 1 with the weighed vitamin premix, mineral premix, taurine, monocalcium phosphate, ethoxyquin, phagostimulant, vitamin C, soybean lecithin, soybean oil and fish oil, and stirring for 120s.
Preparing and granulating: heating and curing the secondarily mixed material through a modulator, controlling the feeding speed to be 70Hz, controlling the temperature of the modulator to be 95 ℃, the temperature of a main machine cylinder to be 95 ℃ and the frequency of the main machine to be 45%, and selecting a proper template for granulating according to the particle size. Drying at 65 ℃ until the water content of the material is less than 12% to obtain the compound feed for the marine fish. And then packaging to obtain a finished product.
The content of essential amino acid in the marine fish compound feed is detected, and the result is as follows: valine 2.35%, lysine 3.07%, isoleucine 2.07%, leucine 4.04%, phenylalanine 2.37%, methionine 1.05%, threonine 2.17%, histidine 1.55%, arginine 3.33%. The crude protein content was 52.47%, the crude fat content was 10.94%.
The specific method for detecting the relevant indexes in the marine fish formula feed comprises the following steps of measuring the crude protein content by a Kjeldahl method (GB/T6432-2018), measuring the crude fat content by a Soxhlet extraction method (GB/T6433-2006), and measuring the amino acid content by an acid hydrolysis method (GB/T18246-2019) by using a full-automatic amino acid measuring instrument (Hitachi L-8900, japan).
Example 2
Uniformly mixing the spirulina powder, the chlorella powder and the nannochloropsis sp powder according to the mass ratio of 11.
Mixing microalgae powder and 80vt% ethanol according to a mass ratio of 1:9, reacting at 35 ℃ for 7 hours, centrifuging at 4000rpm for 8min to obtain ethanol supernatant and microalgae powder precipitate, and discarding the ethanol supernatant.
Adding water into the microalgae powder precipitate according to the material-liquid ratio of 1.
Mixing 2 parts of neutral protease, 2 parts of papain and 1 part of cellulase uniformly according to the parts by weight to obtain the complex enzyme required by enzymolysis. Wherein, the neutral protease, the papain and the cellulase are all commercial products, the activity of the neutral protease is more than or equal to 100U/mg, the activity of the papain is more than or equal to 50U/mg, and the activity of the cellulase is more than or equal to 3000U/g.
Adding 2wt% of complex enzyme into the microalgae powder precipitation mixed solution, setting the stirring speed to 50rpm under the conditions of pH 7.0 and temperature of 50 ℃, performing enzymolysis for 4h, centrifuging for 5min under the condition of 4000rpm, and treating the supernatant clear oil to obtain the microalgae powder free oil which can be used in the aspects of food, medicine and the like. In the test, the residual mixed solution after the upper layer of clear oil is discarded is stirred and uniformly mixed, and then is completely frozen at the temperature of minus 20 ℃, and is frozen and vacuum-dried for 36 hours at the temperature of minus 60 ℃ to obtain the degreased microalgae powder with the water content of less than 5 percent.
The prepared degreased microalgae powder is applied to a marine fish compound feed as a protein raw material, and the compound feed comprises the following raw materials in parts by weight:
25 parts of fish meal, 11 parts of casein, 10 parts of soybean protein concentrate, 22 parts of defatted microalgae powder, 7.2 parts of wheat gluten, 8 parts of wheat flour, 3.8 parts of fish oil, 4.4 parts of soybean oil, 1.1 parts of soybean lecithin, 0.8 part of phagostimulant, 0.5 part of taurine, 1.0 part of vitamin premix, 1.5 parts of mineral premix, 0.2 part of ethoxyquin, 0.5 part of monocalcium phosphate, 0.6 part of vitamin C and 2.4 parts of sodium carboxymethylcellulose.
The phagostimulant comprises the following components in parts by weight: 0.32 part of betaine, 0.32 part of glycine and 0.16 part of hypoxanthine nucleotide.
A marine fish compound feed was prepared according to the preparation method of example 1.
The content of essential amino acid in the marine fish compound feed is detected, and the result is as follows: valine 2.39%, lysine 3.03%, isoleucine 2.12%, leucine 4.10%, phenylalanine 2.42%, methionine 1.03%, threonine 2.18%, histidine 1.60%, arginine 3.37%. The crude protein content was 52.48%, the crude fat content was 10.92%.
The specific method for detecting the relevant indexes in the marine fish formula feed is the same as that described in 'example 1'.
Example 3
Uniformly mixing the spirulina powder, the chlorella powder and the parachlorococcus sp powder according to the mass ratio of 9.
Mixing microalgae powder and 78vt% ethanol according to a mass ratio of 1:8.5, reacting at 40 deg.C for 6h, centrifuging at 3500rpm for 10min to obtain ethanol supernatant and microalgae powder precipitate, and removing ethanol supernatant.
And adding water into the microalgae powder precipitate according to the material-liquid ratio of 1.5, uniformly mixing, adding sodium dodecyl sulfate accounting for 0.2% of the mass of the microalgae powder precipitate, and oscillating in 100W ultrasonic waves for 10min to obtain the microalgae powder precipitate mixed solution.
Mixing 4 parts of neutral protease, 3 parts of papain and 2 parts of cellulase uniformly according to the parts by weight to obtain the complex enzyme required by enzymolysis. Wherein, the neutral protease, the papain and the cellulase are all commercial products, the activity of the neutral protease is more than or equal to 100U/mg, the activity of the papain is more than or equal to 50U/mg, and the activity of the cellulase is more than or equal to 3000U/g.
Adding 2wt% of complex enzyme into the microalgae powder precipitation mixed solution, setting the stirring speed to 40rpm under the conditions of pH 7.5 and temperature of 55 ℃, performing enzymolysis for 6h, centrifuging for 10min under the condition of 3000rpm, and treating the supernatant clear oil to obtain the microalgae powder free oil which can be used in the aspects of food, medicine and the like. In the test, the residual mixed solution after the upper layer of clear oil is discarded is stirred and uniformly mixed, and then is completely frozen at the temperature of minus 20 ℃, and is frozen and vacuum-dried for 48 hours at the temperature of minus 55 ℃ to obtain the degreased microalgae powder with the water content of less than 3 percent.
The prepared degreased microalgae powder is applied to a marine fish compound feed as a protein raw material, and the compound feed comprises the following raw materials in parts by weight:
20 parts of fish meal, 12 parts of casein, 11 parts of soybean protein concentrate, 25 parts of defatted microalgae powder, 8 parts of wheat gluten, 8.5 parts of wheat flour, 4 parts of fish oil, 4.5 parts of soybean oil, 1.2 parts of soybean lecithin, 1.0 part of phagostimulant, 0.6 part of taurine, 0.8 part of vitamin premix, 1.2 parts of mineral premix, 0.1 part of ethoxyquin, 0.6 part of monocalcium phosphate, 0.5 part of vitamin C and 1 part of sodium carboxymethylcellulose.
The phagostimulant comprises the following components in parts by weight: 0.4 part of betaine, 0.4 part of glycine and 0.2 part of hypoxanthine nucleotide.
A marine fish compound feed was prepared according to the preparation method of example 1.
The content of essential amino acid in the marine fish compound feed is detected, and the result is as follows: valine 2.38%, lysine 3.05%, isoleucine 2.10%, leucine 4.10%, phenylalanine 2.44%, methionine 1.02%, threonine 2.15%, histidine 1.65%, arginine 3.35%. The crude protein content was 52.39%, the crude fat content was 10.99%.
The specific method for detecting the relevant indexes in the marine fish formula feed is the same as that described in 'example 1'.
Application example 1
Test time: day 1 in 2019, month 7 to day 25 in 2019, month 8.
And (3) test management: selecting the pearl giant grouper with the initial weight of 50.75 +/-2.75 g.
The juvenile fishes are used as test fishes, after temporary culture is carried out for 15 days, the test fishes are randomly placed in 6 culture buckets (the diameter is 75cm, the depth is 80 cm), 30 juvenile fishes are placed in each bucket and divided into two groups, the three groups are repeated, and the culture mode is circulating water culture. During the test period, the feed was fed twice at 08. The environment during the test was: the water temperature is controlled at 27 +/-1 ℃, the dissolved oxygen is greater than 6.2mg/L, the salinity is 25, the pH is 7.8, and the contents of ammonia nitrogen and nitrite nitrogen are both less than 0.1mg/L. The test was 8 weeks, and the body weight of each group of fish, the final body weight at the end of the test and the initial body weight at the beginning of the test were measured during the test period; recording the death status of the fish, the remaining terminal mantissas at the end of the experiment and the initial mantissas at the beginning of the experiment; the amount of discharged residual baits; dry weight condition of ingested feed; calculating survival rate, weight gain rate, specific growth rate, feed coefficient and protein efficiency. The specific calculation formula is as follows:
survival (SR,%) = terminal mantissa/initial mantissa × 100%;
weight gain rate (WGR,%) = [ (final weight-initial weight)/initial weight ] × 100%;
specific growth rate (SGR,%/day) = { [ Ln (final body weight) -Ln (initial body weight) ]/day of experiment } × 100%;
feed Factor (FCR) = ingested feed dry weight/(final weight-initial weight);
protein efficiency (PER,%) = [ (final body weight-initial body weight)/(feed intake dry weight × content of protein in feed) ] × 100%.
Specific growth indices are shown in table 2.
TABLE 2 Effect of different feeds on the growth of grouper
Item Items Control group Test group
Initial body weight/g 50.67±2.56 50.83±2.37
Final body weight/g of Final body weight 119.45±5.21 121.50±4.19
Survival rate SR/%) 93.33±3.33 94.44±4.56
Weight gain WGR/%) 135.76±8.87 139.77±7.47
Specific growth rate SGR/% 1.23±0.03 1.25±0.05
Feed factor FCR 1.05±0.05 1.01±0.03
Protein efficiency PER/%) 1.90±0.10 1.98±0.06
Table 2 shows that the weight gain rate and the specific growth rate of the test group of the fishes are respectively increased by 11.05 percent and 7.77 percent compared with the control group, the feed coefficient is reduced by 4.08 percent compared with the control group, and the whole growth effect is better than that of the control group fed with commercial materials. In addition, the protein efficiency of the test group is 3.94% higher than that of the control group, which shows that the utilization rate of the protein in the feed of the test group by the test fish is higher, the N discharged into water is less, and the environmental pollution is reduced.
Water sample collection: after the feeding is finished, the circulating water system is closed, samples are taken at a position 25cm below the water surface after 6 hours, each barrel samples 250mL each time, and the ammonia nitrogen content and the soluble phosphate content in the water are measured.
As a result, the ammonia nitrogen excretion of the fish in the control group is 0.63mg/L, and the excretion of the soluble phosphate is 0.162mg/L; the ammonia nitrogen excretion of the test group fish is 0.52mg/L, and the excretion of soluble phosphate is 0.135mg/L. Compared with the control group, the ammonia nitrogen excretion and the soluble phosphate excretion of the test group fish are respectively reduced by 17.46 percent and 16.67 percent.
And (3) toxin counteracting test: after 8 weeks of culture test, 8 fish were taken out from each culture bucket for challenge test, and 50. Mu.L of Vibrio anguillarum bacterial liquid (5X 10) suspended in normal saline was intraperitoneally injected according to 50g of body weight 8 cfu/mL), then stocked in an indoor culture tank, continuously oxygenated in the tank, and the cumulative mortality rate after 72h of injection was recorded.
As a result: after 72 hours of toxin attacking, the mortality rate of the fish in the control group is 76.67%, the mortality rate of the fish in the test group is 63.33%, and compared with the control group, the cumulative mortality rate of the fish in the test group is reduced by 17.52%, which indicates that the fish in the test group has better disease resistance.
The test data result shows that compared with a control group, the test group fish has better growth performance and higher protein utilization rate; after the challenge test, the cumulative mortality of the tested fishes is lower than that of the control group, which shows that the disease resistance of the fish body is enhanced by the feed of the tested group; in addition, the nitrogen and phosphorus excretion amount in the culture water of the test group is lower than that of the control group, so that the pollution to the culture water environment is reduced. In conclusion, the feed product produced by the method has strong advantages and has certain positive significance for the healthy and efficient ecological development of the mariculture industry.
The embodiment can show that the invention provides defatted microalgae powder, a preparation method and application thereof and a compound feed for marine fishes. The defatted microalgae powder prepared by the method is more suitable for preparing compound feed capable of improving the growth and disease resistance of the marine fish.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method of degreased microalgae powder is characterized by comprising the following steps:
(1.1) mixing the microalgae powder with ethanol, reacting for 6-8 h, centrifuging, and removing supernatant to obtain microalgae powder precipitate;
(1.2) mixing the microalgae powder precipitate with water and a surfactant, and carrying out ultrasonic oscillation for 5-10 min to obtain a microalgae powder precipitate mixed solution;
(1.3) mixing the microalgae powder precipitate mixed solution with a complex enzyme, performing enzymolysis for 4-6 h at 40-60 ℃, centrifuging, and removing upper clear oil to obtain a degreased microalgae powder mixed solution;
(1.4) drying the degreased microalgae powder mixed solution to obtain degreased microalgae powder;
the mixing mass ratio of the microalgal powder to the ethanol is 1;
the complex enzyme is a mixture of neutral protease, cellulase and papain;
the neutral protease is: cellulase: the mass ratio of the papain is 2-4;
the activity of the neutral protease is more than or equal to 100U/mg;
the activity of the cellulase is more than or equal to 3000U/g;
the activity of the papain is more than or equal to 50U/mg.
2. The method according to claim 1, wherein the microalgal flour is one or more of spirulina powder, chlorella powder, and parachloropsis powder.
3. The preparation method according to claim 2, wherein when the microalgal flour is a mixed flour of spirulina powder, chlorella powder and parachloropsis powder, the mass ratio of the spirulina powder to the chlorella powder is: chlorella powder: the pseudo-micro-green algae powder is 5-12.
4. The method according to claim 2, wherein the concentration of ethanol in step (1.1) is 75 to 80vt%;
the reaction temperature in the step (1.1) is 30-40 ℃;
the rotating speed of the centrifugation in the step (1.1) is 3000-5000 rpm;
the centrifugation time in the step (1.1) is 5-10 min.
5. The preparation method according to claim 4, wherein the mass ratio of the microalgae powder precipitate in the step (1.2) to water is 1;
the surfactant in the step (1.2) is sodium dodecyl sulfate;
in the step (1.2), the mass ratio of the microalgae powder precipitate to the surfactant is 100:0.1 to 0.2;
the power of the ultrasound in the step (1.2) is 100-150W.
6. The preparation method according to claim 5, wherein the mass ratio of the mixed solution of the microalgae powder precipitate in the step (1.3) to the complex enzyme is 100;
the pH value during the enzymolysis in the step (1.3) is 6.0-7.5;
the rotation speed in the enzymolysis in the step (1.3) is 40-60 rpm;
the rotating speed of the centrifugation in the step (1.3) is 3000-5000 rpm;
the centrifugation time in the step (1.3) is 5-10 min;
the drying in the step (1.4) is freeze drying;
the freeze drying method comprises the following steps: after being completely frozen at the temperature of minus 20 ℃, the mixture is dried for 36 to 48 hours in vacuum at the temperature of minus 55 to minus 60 ℃.
7. The defatted microalgae powder prepared by the method according to any of claims 1 to 6.
8. Use of the defatted microalgae powder of claim 7 in preparing a compound feed for marine fish.
9. A marine fish compound feed containing the defatted microalgae powder of claim 7 is characterized by comprising the following components in parts by mass:
20 to 30 parts of fish meal, 8 to 15 parts of casein, 8 to 12 parts of soybean protein concentrate, 15 to 30 parts of degreased microalgal flour, 6 to 10 parts of wheat gluten, 8 to 10 parts of wheat flour, 3 to 5 parts of fish oil, 4 to 5 parts of soybean oil, 1 to 1.5 parts of soybean lecithin, 0.6 to 1.0 part of phagostimulant, 0.4 to 0.6 part of taurine, 0.8 to 1.5 parts of vitamin premix, 1.0 to 2.0 parts of mineral premix, 0.1 to 0.3 part of ethoxyquinoline, 0.4 to 0.6 part of monocalcium phosphate, 0.3 to 0.7 part of vitamin C and 1.0 to 4.0 parts of sodium carboxymethylcellulose;
the phagostimulant is prepared by mixing 1.5-2.5 weight percent of betaine, 1.5-2.5 weight percent of glycine and 1 weight percent of hypoxanthine nucleotide.
10. The method for preparing the compound feed for marine fish of claim 9, comprising the steps of:
(10.1) mixing fish meal, casein, soybean protein concentrate, defatted microalgae powder, wheat gluten, wheat flour, and sodium carboxymethylcellulose, and pulverizing to particle size of less than 150 μm to obtain intermediate 1;
(10.2) mixing the intermediate 1 with vitamin premix, mineral premix, taurine, monocalcium phosphate, vitamin C, ethoxyquin, phagostimulant, soybean lecithin, soybean oil and fish oil, curing, granulating and drying to obtain the compound feed for the marine fish;
the drying temperature is 65-75 ℃;
the water content of the marine fish compound feed is less than 12%.
CN202211529464.2A 2022-11-30 2022-11-30 Defatted microalgae powder, preparation method and application thereof, and marine fish compound feed Pending CN115836707A (en)

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