CN114271412A

CN114271412A - Mixed feed for improving water mildew resistance of tilapia and preparation method thereof

Info

Publication number: CN114271412A
Application number: CN202210017735.XA
Authority: CN
Inventors: 张晨; 魏汉; 刘派
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-01-08
Filing date: 2022-01-08
Publication date: 2022-04-05
Anticipated expiration: 2042-01-08
Also published as: CN114271412B

Abstract

The invention belongs to the technical field of feeds, and particularly provides a mixed feed for improving the water mildew resistance of tilapia and a preparation method thereof. The invention obtains the low-fiber tea-residue protein containing high-stability polyphenol by drying and crushing the tea-residue, extracting and recovering the protein in the tea-residue, and compounding the tea-residue protein, soybean oil, vitamins, mineral substances, fish meal, flour and leftovers of gramineous crops into the mixed feed. The mixed feed prepared by the invention has strong inhibition effect on mould, can obviously reduce the mould of the feed, and can improve the nonspecific immunity of tilapia and the resistance of tilapia to saprolegniasis.

Description

Mixed feed for improving water mildew resistance of tilapia and preparation method thereof

Technical Field

The invention belongs to the technical field of feeds, and particularly relates to a mixed feed for improving the resistance of tilapia to saprolegniasis and a preparation method thereof.

Background

Tilapia mossambica is a main economic fish in aquaculture industry, and has the advantages of short culture period, strong environmental adaptability and the like. However, the aquaculture industry in China is moving towards intensification and scale, the aquaculture range and density are gradually expanded, and the demand for protein resources is also increasing. On one hand, due to a high-density culture mode, the immunity of tilapia is obviously reduced, and the phenomena of outbreak of fish diseases, slow growth rate of fish bodies and the like occur. On the other hand, protein resources for aquatic feed in China are in short supply, and the utilization rate of non-grain protein resources is extremely low, so that research on development and effective utilization of raw materials of aquatic feed protein is urgently needed. The disease resistance and the feeding value of non-grain protein resources are deeply researched, the current situations of fish immunity reduction and feed protein resource shortage can be relieved, and a sustainable development green aquatic product technology industry is constructed.

The feed protein resource is a basic resource for the development of aquaculture industry, and the aquatic feed protein resource in China has a great demand gap. The tea residue is used as a non-grain plant protein resource, contains 25-30% of protein, and contains polyphenol antioxidant, and can enhance fish immunity. Taking the tea resources in Fujian province as an example, only two Fujian province tea processing enterprises produce about 10 ten thousand tons of tea leaves and about 2.5 ten thousand tons of protein. The tea residue has the problems of poor palatability, low digestibility and the like, and is difficult to be used as a protein resource for feeding. However, the tea residue protein is a high-quality and palatable easily-digestible plant source protein, and can be used as a protein source feed or an anti-microbial feed for aquaculture.

Tea leaf protein as oneThe high-quality low-fiber vegetable protein can be extracted by an alkaline method to enhance the bioavailability. When the tea residue protein is extracted by an alkaline method, the protein in the tea residue is released by damaging cell walls by alkali liquor at high temperature, and simultaneously, polyphenols are released, and the protein and the Ca are mixed under the specific condition²⁺The combination forms a complex, and ensures that the polyphenols are difficult to degrade or destroy. The compound is compounded with soybean oil, vitamins, mineral substances, fish meal, flour and other raw materials to prepare a mixed feed, so that the daily nutritional requirements of tilapia are guaranteed, the construction of the immunity of tilapia can be enhanced, and the infection rate of saprolegniasis is reduced.

Disclosure of Invention

The invention aims to provide a mixed feed for improving the resistance of tilapia to saprolegniasis and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention firstly provides a preparation method of a mixed feed for improving the resistance of tilapia to saprolegniasis, which specifically comprises the following steps:

1) pretreatment of the tea leaves: drying the collected tea leaves, and crushing to obtain tea leaf powder;

2) extracting tea leaf protein: uniformly mixing the tea residue powder with alkali liquor, reacting for 0.5-6h at 80-100 ℃, centrifuging the mixture, and collecting supernatant to obtain tea residue protein extract;

3) and (3) recovering tea residue protein: adding acid solution into the tea residue protein extractive solution to adjust pH of the tea residue protein extractive solution to 5-8, and adding Ca into the tea residue protein extractive solution²⁺Uniformly mixing the solution, centrifuging, freeze-drying the centrifuged precipitate, and crushing to obtain tea residue protein;

4) compounding feeds: uniformly mixing tea residue protein, soybean oil, vitamin and mineral salt premix, fish meal, flour and grass family crop leftovers, crushing, performing steam conditioning treatment by using a conditioner, introducing saturated steam, wherein the conditioning temperature is 85-95 ℃, and stopping conditioning when the water content of powder is 23-28%; then the mixture enters an expansion cavity for extrusion expansion, the expansion pressure is 3.0-10.5 MPa, the expansion temperature is 130-160 ℃, and the time is 5-30 s; and finally drying at 90-100 ℃ until the water content is lower than 10%, thus obtaining the finished feed.

Further, in the step 1) of the preparation method, the tea leaves are dried at 40-60 ℃ until the water content is lower than 10%.

Further, in the step 2) of the preparation method, the feed-liquid ratio of the tea residue powder to the alkali liquor is 1:10-40 w/v; the alkali liquor is 0.1-0.4M sodium hydroxide solution.

Further, in the step 3) of the preparation method, the acid solution is 0.5-4.0M hydrochloric acid solution, and the Ca is²⁺The solution is 0.1-2.0M calcium chloride solution; the tea residue protein extracting solution contains Ca²⁺The volume ratio of the solution was 1: 1.

Further, in the step 4) of the preparation method, the weight ratio of the tea residue protein, the soybean oil, the vitamin and mineral salt premix, the fish meal, the flour and the leftovers of the gramineous crops is 3-10: 1-5: 5-25: 10-30: 30-80.

Further, every 100 weight parts of vitamin and mineral salt premix comprises 6 weight parts of vitamin A and vitamin B₁8 parts by weight of vitamin B₂4 parts of vitamin D, 1 part of folic acid, 20 parts of calcium hydrophosphate, 1 part of ferrous sulfate, 35 parts of zinc sulfate, 5 parts of sodium selenite and 10 parts of calcium lactate.

Further, the leftovers of the gramineous crops comprise bean pulp, wheat bran and rice bran, wherein the weight ratio of the bean pulp to the wheat bran to the rice bran is 10-40: 5-30.

The invention also provides a mixed feed prepared by the preparation method.

The invention also provides application of the mixed feed in tilapia culture.

In the technical scheme, the tea residue powder is added into alkali liquor and reacts for 30 min-6 h at 80-100 ℃, so that the tea residue protein can be released from cell membranes through broken cell walls, and the protein in the tea residue can be released at high temperatureBinding to polyphenol under alkaline conditions to form a complex. Ca for recovery of tea leaf protein²⁺The solution can not only make the tea residue protein combined with polyphenols settle rapidly, but also make Ca precipitate rapidly²⁺Chelating with polyphenols through coordination bonds, protecting polyphenols from being oxidized, and improving stability of polyphenols. Meanwhile, the protein in the tea leaves is extracted by a high-temperature alkaline method, so that the low-fiber vegetable protein with good bioavailability can be obtained. In the feed compounding, the tea residue protein is added to enhance the preservation period of the mixed feed and mineral Ca²⁺The content of (a), and the resistance to saprolegniasis of tilapia; the addition of soybean oil, vitamins and minerals can ensure the normal nutrient intake of fatty acid, vitamins and minerals of tilapia, thereby ensuring the normal physiological function and metabolism operation of tilapia; the addition of the fish meal and the flour can ensure the normal nutrient intake of protein and carbohydrate of the tilapia. The invention has the following remarkable advantages:

the mixed feed for resisting the saprolegniasis of tilapia provided by the invention is not added with any antibiotic medicine. According to the invention, on the basis of daily ingredient nutrition balance, a proper amount of tea residue protein is added, so that the self-immunity of tilapia is improved, and thus, the occurrence of tilapia saprolegniasis is reduced. The embodiment proves that the mixed feed for resisting the tilapia saprolegniasis and the preparation method thereof can not only increase the capability of the feed for resisting the tilapia saprolegniasis, but also improve the resistance of the tilapia saprolegniasis, thereby reducing the application of preservatives in the feed and reducing the use of antibiotic drugs in the feed for resisting the tilapia saprolegniasis.

Drawings

FIG. 1: stability of polyphenols of tea residue proteins under different recovery conditions.

FIG. 2: crude fiber content in tea residue protein.

FIG. 3: the product feed A, B, C has mould inhibiting effect.

FIG. 4: influence of the finished feed A, B, C on the nonspecific immunity of tilapia. And A, plasma lysozyme activity. And B, plasma SOD enzyme activity.

FIG. 5: the effect of the finished feed A, B, C on incidence rate of saprolegniasis infection and mortality rate after infection of tilapia. And A, incidence rate. And B, mortality.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

The vitamin and mineral salt premixes used in the following examples contain 6 parts by weight of vitamin A and 6 parts by weight of vitamin B per 100 parts by weight of the vitamin and mineral salt premixes₁8 parts by weight of vitamin B₂4 parts of vitamin D, 1 part of folic acid, 20 parts of calcium hydrophosphate, 1 part of ferrous sulfate, 35 parts of zinc sulfate, 5 parts of sodium selenite and 10 parts of calcium lactate.

Example 1

This example determined the polyphenol stability of tea leaf protein under different recovery conditions.

Drying the collected tea leaves at 60 ℃ until the water content is lower than 10%, and then crushing to obtain tea leaf powder. Mixing 1kg tea residue powder with 20L 0.2M sodium hydroxide solution, reacting at 95 deg.C for 4 hr, centrifuging at 4000g for 10min, and collecting supernatant to obtain tea residue protein extractive solution.

Recovery conditions 1: adjusting pH of the tea residue protein extractive solution to 5.0 with 1M HCl solution, adding 0.1M CaCl equal to the volume of the tea residue protein extractive solution₂The solution was mixed well and then centrifuged at 4000g for 10 min.

Recovery conditions 2: adjusting pH of the tea residue protein extract to 5.0 with 1M HCl solution, mixing well, and centrifuging at 4000g for 10 min.

Recovery conditions 3: adjusting pH of the tea residue protein extractive solution to 6.0 with 1M HCl solution, adding 0.1M CaCl equal to the volume of the tea residue protein extractive solution₂The solution was mixed well and then centrifuged at 4000g for 10 min.

Recovery conditions 1: adjusting pH of the tea residue protein extract to 6.0 with 1M HCl solution, mixing well, and centrifuging at 4000g for 10 min.

Placing the centrifuged precipitate into a 3kDa dialysis bag (the external liquid is distilled water, the system is exposed in the air, the room temperature is 25 ℃), and finally determining the content of the polyphenol bound in the internal liquid of the dialysis bag by using a ferrous tartrate method. And (3) expressing the polyphenol stability by the content of the combined polyphenol, namely the polyphenol stability = the content of the combined polyphenol of the liquid in the dialysis bag/the content of the polyphenol of the tea residue protein extracting solution. Significance level P<0.05. As shown in FIG. 1, Ca²⁺The stability of polyphenol can be effectively increased by adopting the method.

Example 2

In this example, the crude fiber content of the tea residue protein was measured.

Drying the collected tea leaves at 60 ℃ until the water content is lower than 10%, and then crushing to obtain tea leaf powder. Mixing 1kg tea residue powder with 20L 0.2M sodium hydroxide solution, reacting at 95 deg.C for 4 hr, centrifuging at 4000g for 10min, and collecting supernatant to obtain tea residue protein extractive solution. Adjusting pH of the tea residue protein extract to 5.0 with 1M hydrochloric acid solution, adding 0.1M calcium chloride solution with the same volume as the tea residue protein extract, mixing, centrifuging at 4000g for 10min, freeze drying the centrifuged precipitate, and pulverizing to particle size of less than 0.4mm to obtain tea residue protein. The content of crude fiber in the tea residue protein is measured by a method for measuring crude fiber in GB/T5009.10-2003 food, and the content of the crude fiber in the tea residue protein is calculated by W (crude fiber)/W (dry weight of the tea residue protein). As can be seen from FIG. 2, the content of crude fiber in the tea residue protein was extremely low.

Example 3

The embodiment provides a preparation method of a mixed feed for improving the resistance of tilapia to saprolegniasis, which specifically comprises the following steps:

(1) compounding feeds: uniformly mixing 2kg of soybean oil, 2kg of vitamin and mineral salt premix, 17kg of fish meal, 10kg of flour, 30kg of soybean meal, 8kg of wheat bran and 10kg of rice bran, and crushing the mixture until the granularity is less than 0.4 mm; carrying out steam tempering treatment by using a tempering device, introducing saturated steam, wherein the tempering temperature is 90 ℃, and stopping tempering when the water content of the powder is 25%; then the mixture enters a puffing cavity for extrusion and puffing, wherein the puffing pressure is 5.0MPa, the puffing temperature is 140 ℃, and the time is 10 s; and finally drying at 95 ℃ until the water content is lower than 10 percent to obtain the finished feed A.

Example 4

(1) pretreatment of the tea leaves: drying the collected tea leaves at 60 ℃ until the water content is lower than 10%, and then crushing to obtain tea leaf powder.

(2) Extracting tea leaf protein: and (2) uniformly mixing 1kg of the tea residue powder obtained in the step (1) with 20L of 0.2M sodium hydroxide solution, reacting for 4h at 95 ℃, centrifuging for 10min at 4000g, and collecting supernatant to obtain a tea residue protein extracting solution.

(3) And (3) recovering tea residue protein: and (3) adjusting the pH value of the tea residue protein extracting solution obtained in the step (2) to 5.0 by using 1M hydrochloric acid solution, adding 0.1M calcium chloride solution which is equal to the tea residue protein extracting solution in volume, uniformly mixing, centrifuging for 10min at 4000g, freeze-drying the centrifuged precipitate, and crushing to obtain tea residue protein with the granularity of less than 0.4 mm.

(4) Compounding feeds: uniformly mixing 4kg of the tea residue protein obtained in the step (3) with 2kg of soybean oil, 2kg of vitamin and mineral salt premix, 17kg of fish meal, 10kg of flour, 30kg of soybean meal, 8kg of wheat bran and 10kg of rice bran, and crushing the mixture until the granularity is less than 0.4 mm; carrying out steam tempering treatment by using a tempering device, introducing saturated steam, wherein the tempering temperature is 90 ℃, and stopping tempering when the water content of the powder is 25%; then the mixture enters a puffing cavity for extrusion and puffing, wherein the puffing pressure is 5.0MPa, the puffing temperature is 140 ℃, and the time is 10 s; and finally drying at 95 ℃ until the water content is lower than 10 percent to obtain the finished feed B.

Example 5

(4) Compounding feeds: uniformly mixing 8kg of the tea residue protein obtained in the step (3) with 2kg of soybean oil, 2kg of vitamin and mineral salt premix, 17kg of fish meal, 10kg of flour, 30kg of soybean meal, 8kg of wheat bran and 10kg of rice bran, and crushing the mixture to the granularity of less than 0.4 mm; carrying out steam tempering treatment by using a tempering device, introducing saturated steam, wherein the tempering temperature is 90 ℃, and stopping tempering when the water content of the powder is 25%; then the mixture enters a puffing cavity for extrusion and puffing, wherein the puffing pressure is 5.0MPa, the puffing temperature is 140 ℃, and the time is 10 s; and finally, drying at 95 ℃ until the water content is lower than 10 percent to obtain the finished feed C.

Application examples

The finished feed A, B, C prepared in examples 3-5 was used for tilapia (gift tilapia).

FIG. 3 is a graph showing the effect of finished feed A, B, C on mold colonies. 1g of the A, B, C powder was dissolved in 50mL of distilled water for 30min with 100w of ultrasonic power, and then 0.2mL of the supernatant was injected into the mold-stained wells of the medium and cultured in an incubator (25 ℃, 200rpm/min, 12 h). As can be seen from FIG. 3, the finished feed B, C shows a significant black zone of inhibition, and thus the mixed feed with tea residue protein has a significant effect of resisting mold.

FIG. 4 shows the effect of the finished feed A, B, C on the tilapia plasma lysozyme activity and SOD enzyme activity. The day of formal culture of tilapia is 60 days, each group is repeated three times, and each time is 20 fish in a fish tank. The experiment adopts the feeding of the full feed, the feeding is carried out for 2 times (9: 00, 17: 00) every day, the aeration and oxygenation are carried out for 24 hours in the culture period, the water temperature is 22 +/-2 ℃, the pH value is 6.9-7.4, and the content of ammonia nitrogen is lower than 0.03 mg/L. The significance of the difference of each group was analyzed by one-way ANOVA and Duncan's method multiple comparisons, and the significance level P was less than 0.05. As can be seen from FIG. 4, the mixed feed added with tea residue protein can obviously enhance the activity of tilapia mossambica plasma lysozyme and the activity of plasma SOD enzyme.

FIG. 5 is a graph showing the effect of finished feed A, B, C on incidence and mortality of Tilapia saprolegniasis infection. After the tilapia is formally cultured for 60 days, 0.2mL of 2.5 multiplied by 10 are respectively injected into the abdominal cavity⁷CFU/mL saprolegnia, and recording incidence and mortality of Tilapia saprolegniasis after each group injection is finished for 48 hours. The experiment adopts the feeding of the full feed, the feeding is carried out for 2 times (9: 00, 17: 00) every day, the aeration and oxygenation are carried out for 24 hours in the culture period, the water temperature is 22 +/-2 ℃, the pH value is 6.9-7.4, and the content of ammonia nitrogen is lower than 0.03 mg/L. The significance of the difference of each group is analyzed by adopting one-way ANOVA and Duncan's method multiple comparison, and the significance level P<0.05. As can be seen from FIG. 5, the mixed feed added with tea residue protein can obviously reduce incidence rate of tilapia saprolegniasis infection and mortality rate after infection.

According to the above embodiments and experimental results, the mixed feed added with tea residue protein provided by the invention does not contain antibiotic components, but can reduce feed mildew, improve nonspecific immunity of tilapia, and improve resistance of tilapia against saprolegniasis.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A preparation method of mixed feed for improving the resistance of tilapia to saprolegniasis is characterized by comprising the following steps: the method specifically comprises the following steps:

4) compounding feeds: mixing tea residue protein, soybean oil, vitamins, mineral salt premix, fish meal, flour and grass family crop leftovers, and pulverizing; performing steam tempering treatment by using a tempering device, introducing saturated steam, wherein the tempering temperature is 85-95 ℃, and stopping tempering when the water content of the powder is 23-28%; then the mixture enters an expansion cavity for extrusion expansion, the expansion pressure is 3.0-10.5 MPa, the expansion temperature is 130-160 ℃, and the time is 5-30 s; and finally drying at 90-100 ℃ until the water content is lower than 10%, thus obtaining the finished feed.

2. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 1, which is characterized by comprising the following steps: in the step 1), the tea leaves are dried at the temperature of 40-60 ℃ until the water content is lower than 10%.

3. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 1, which is characterized by comprising the following steps: in the step 2), the feed-liquid ratio of the tea residue powder to the alkali liquor is 1:10-40 w/v; the alkali liquor is 0.1-0.4M sodium hydroxide solution.

4. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 1, which is characterized by comprising the following steps: in the step 3), the acid solution is 0.5-4.0M hydrochloric acid solution, and the Ca is²⁺The solution is 0.1-2.0M calcium chloride solution; the tea residue protein extracting solution contains Ca²⁺The volume ratio of the solution was 1: 1.

5. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 1, which is characterized by comprising the following steps: in the step 4), the weight ratio of the tea residue protein, the soybean oil, the vitamin and mineral salt premix, the fish meal, the flour and the leftovers of the gramineous crops is 3-10: 1-5: 5-25: 10-30: 30-80.

6. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 5, wherein the preparation method comprises the following steps: every 100 weight parts of vitamin and mineral salt premix contains 6 weight parts of vitamin A and vitamin B₁8 parts by weight of vitamin B₂4 parts of vitamin D, 1 part of folic acid, 20 parts of calcium hydrophosphate, 1 part of ferrous sulfate, 35 parts of zinc sulfate, 5 parts of sodium selenite and 10 parts of calcium lactate.

7. The preparation method of the mixed feed for improving the resistance of tilapia mossambica to saprolegniasis according to claim 5, wherein the preparation method comprises the following steps: the leftovers of the gramineous crops comprise soybean meal, wheat bran and rice bran, wherein the weight ratio of the soybean meal to the wheat bran to the rice bran is 10-40: 5-30.

8. A mixed feed produced by the production method according to claim 1.

9. The use of the mixed feed of claim 8 in tilapia culture.