CN106616015B - Method for preparing slow-release sea cucumber bait by using fish leftovers and immobilized enzyme - Google Patents
Method for preparing slow-release sea cucumber bait by using fish leftovers and immobilized enzyme Download PDFInfo
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- CN106616015B CN106616015B CN201611081141.6A CN201611081141A CN106616015B CN 106616015 B CN106616015 B CN 106616015B CN 201611081141 A CN201611081141 A CN 201611081141A CN 106616015 B CN106616015 B CN 106616015B
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C—CHEMISTRY; METALLURGY
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
Abstract
The invention discloses a method for preparing slow-release sea cucumber bait by using fish leftovers and immobilized enzymes. Which solves the problem that the existing fish leftovers can not be digested and utilized by directly feeding the sea cucumbers. Inoculating a protease-producing strain obtained by separating and screening in a fish intestinal tract in a culture medium, and culturing a seed bacterial liquid; putting the fish leftover meat paste into seed bacteria liquid for fermentation to form fermentation liquid; injecting the chitosan solution into a mixed solution of sodium hydroxide and methanol, and condensing into a hollow spherical chitosan carrier; activating the carrier in glutaraldehyde water solution; wrapping enzyme in a hollow spherical chitosan carrier to form immobilized enzyme; then the immobilized enzyme and the fermentation liquor are put into the sea cucumber culture water area. The method has reasonable process and feasible operation. The immobilized enzyme has high stability and long half-life cycle, slowly performs enzymolysis on fish leftover fermentation liquor to release sea cucumber bait, and simultaneously performs degradation on deposited residual bait, thereby reducing bait casting times and labor cost.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for preparing slow-release sea cucumber bait by using fish leftovers and immobilized enzymes.
Background
The sea cucumber is known to have high nutritional value and medicinal value and is regarded as an important seafood. In recent years, the annual increase of the sea cucumber consumption demand leads to the over-development of natural sea cucumber resources and the rapid decrease of population quantity, so that the artificial culture of the sea cucumber is developed. The bait is a material basis for the growth and development of the sea cucumbers, and natural bait cannot meet the requirements of the sea cucumber culture along with the development of large-scale sea cucumber culture, so that the bait needs to be fed manually. At present, the problems of high labor cost, low utilization rate of the fed baits, difficulty in mastering the amount and time of manually fed baits and the like exist in the manual feeding of the baits.
The fish is a rich marine resource in coastal areas of China, the leftovers (fish minced meat and viscera) after the fish processing have low cost and high content of protein and taurine, and are ideal natural baits for the sea cucumber, but the leftovers of the fish which are not processed have the defect that the sea cucumber cannot be digested and utilized.
Disclosure of Invention
In order to overcome the defect that the fish leftovers can not be digested and utilized by directly feeding the sea cucumbers in the prior art, the invention provides the method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme, which has reasonable process and high intestinal utilization rate of the sea cucumber bait.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme is characterized by comprising the following steps of: which comprises the following steps:
(a) fermentation treatment of fish leftovers
(a1) Culture of zymocyte liquid
Inoculating a W2 strain into a DAE culture medium, controlling the temperature to be 20-25 ℃, and culturing for 2-3 days under the condition of 150-180 r/min to obtain a seed bacterial liquid, wherein the OD600 of the seed bacterial liquid is 1.0-1.2; wherein, the formula of the DAE culture medium is as follows: 0.4-0.8 g of peptone, 0.05-0.2 g of yeast extract, 0.01-0.02 g of iron phosphate and 90-100 ml of seawater; the strain W2 is a protease-producing strain separated and screened from a fish intestinal tract, and the inoculation ratio of the strain W2 to the DAE culture medium is 1: 300-500;
(a2) preparation of fish leftover fermentation liquor
Selecting fish leftovers as raw materials, and grinding the fish leftovers into a meat paste; weighing minced fish leftovers, putting the minced fish leftovers into the seed bacterial liquid prepared in the step (a1), controlling the temperature to be 20-25 ℃, culturing for 2-3 d under the condition of 150-180 r/min, and taking fermentation liquid to obtain fish leftovers fermentation liquid; wherein the feed-liquid ratio of the minced fish leftovers to the seed bacterial liquid is 1: 20-25;
(b) preparation of immobilized enzymes
(b1) Preparation of the support
Weighing chitosan, and dissolving the chitosan in a hydrochloric acid solution with the concentration of 20-30% to prepare a chitosan solution; then injecting the prepared chitosan solution into a mixed solution containing 15% of sodium hydroxide and 30% of methanol, and condensing into hollow spherical chitosan; wherein the material-liquid ratio of the chitosan to the hydrochloric acid solution is 1: 3-5, and the deacetylation degree of the chitosan is 80-85%;
(b2) activation of the support
Putting the hollow spherical chitosan prepared in the step (b 1) into a glutaraldehyde aqueous solution with the concentration of 4-8%, stirring and reacting for 4-6 h at room temperature, standing overnight at 4 ℃, removing supernatant, washing for 2-3 times with water, and removing residual glutaraldehyde to obtain glutaraldehyde-activated hollow spherical chitosan; wherein the material-liquid ratio of the hollow spherical chitosan to the glutaraldehyde aqueous solution is 1: 2-3;
(b3) encapsulation of enzymes
Adding the glutaraldehyde-activated hollow spherical chitosan prepared in the step (b 2) into neutral protease, alkaline protease and acid protease preparations prepared from buffers with different pH values respectively, stirring and reacting for 2-3 h at room temperature, standing overnight at 4 ℃, and washing with water to remove free enzymes to obtain immobilized neutral protease, alkaline protease and acid protease; wherein the pH value of the buffer solution of the neutral protease is 6.8-7.5; the pH value of the buffer solution of the alkaline protease is 8.5-10.5; the pH value of the buffer solution of the acid protease is 4.0-4.5;
(c) throw in
And (c) putting the immobilized neutral protease, the immobilized alkaline protease and the immobilized acidic protease prepared in the step (b 3) and the fish leftover fermentation liquor prepared in the step (a 2) into the sea cucumber culture water area according to the feed-liquid ratio of 1: 50-100.
The invention inoculates protease-producing strain separated and screened from fish intestinal tract in culture medium, and cultures seed bacterial liquid; putting the fish leftover meat paste into seed bacteria liquid for fermentation to form fermentation liquid rich in protein; injecting the chitosan solution into a mixed solution of sodium hydroxide and methanol, and condensing into a hollow spherical chitosan carrier; activating the carrier in glutaraldehyde water solution; wrapping enzyme in a hollow spherical chitosan carrier by using an immobilized enzyme technology to form an immobilized enzyme; and putting the immobilized enzyme and the fermentation liquor into a sea cucumber culture water area together. The immobilized enzyme has high stability and long half-decay period, the fish leftover fermentation liquor and the fish leftover fermentation liquor are put into a sea cucumber feeding water area together, and a substrate can be subjected to contact reaction with the enzyme through a pore passage to carry out enzymolysis on protein in the fish leftover waste liquor, so that the intestinal utilization rate of sea cucumber bait is improved, the discharge of metabolites is reduced, the growth rate and the feed conversion rate of the sea cucumber are improved, a certain degradation effect on residual bait deposited on the seabed or the bottom of a pool can be realized, the discharge of nitrogen and phosphorus in the breeding industry is reduced, and the breeding ecological environment is protected. The invention has reasonable process, wherein the adopted protease is acidic protease, neutral protease and alkaline protease simulating the front intestine to the back intestine in the sea cucumber body; the chitosan can also play a role in purifying water quality after being put into a sea cucumber raising water area. Because the action substrates of the enzyme are limited, the fish leftover fermentation liquor which is put once cannot be completely digested immediately, and the fish leftover fermentation liquor and the immobilized protease are slowly and continuously matched to supply nutrients for the sea cucumbers. The bait throwing device and the bait throwing method can reduce the times of throwing the bait and the labor cost by throwing the bait in one step. The invention fully utilizes marine wastes, improves the utilization rate of marine resources, reduces environmental pollution, increases the variety of the holothurian culture baits, and has wide market prospect.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
The method for preparing the slow-release sea cucumber bait by utilizing the fish leftovers and the immobilized enzyme comprises the following process steps:
(a) fermentation treatment of fish leftovers
(a1) Culture of zymocyte liquid
Inoculating a W2 strain into a DAE culture medium, controlling the temperature to be 22 ℃, and culturing for 3d under the condition of 160r/min to obtain a seed bacterial liquid, wherein the OD600 of the seed bacterial liquid is 1.1; wherein, the formula of the DAE culture medium is as follows: 0.5g of peptone, 0.1g of yeast extract, 0.015g of iron phosphate and 95ml of seawater; the strain W2 is a protease-producing strain separated and screened from the fish intestinal tract, and the inoculation ratio of the strain W2 to the DAE culture medium is 1: 400;
(a2) preparation of fish leftover fermentation liquor
Selecting fish leftovers as raw materials, and feeding the fish leftovers into a grinder to grind the fish leftovers into a meat paste; weighing meat paste fish leftovers into the seed bacterial liquid prepared in the step (a1), wherein the feed liquid ratio of the meat paste fish leftovers to the seed bacterial liquid is 1: 22; controlling the temperature at 23 ℃, culturing for 3d under the condition of 160r/min, and taking fermentation liquor to obtain fish leftover fermentation liquor;
(b) preparation of immobilized enzymes
(b1) Preparation of the support
Weighing chitosan with deacetylation degree of 82% and dissolving in 25% hydrochloric acid solution, wherein the material-to-liquid ratio of the chitosan to the hydrochloric acid solution is 1:4, and preparing into chitosan solution; then injecting the prepared chitosan solution into a mixed solution containing 15% of sodium hydroxide and 30% of methanol, and condensing into hollow spherical chitosan;
(b2) activation of the support
Putting the hollow spherical chitosan prepared in the step (b 1) into a glutaraldehyde aqueous solution with the concentration of 6%, wherein the material-liquid ratio of the hollow spherical chitosan to the glutaraldehyde aqueous solution is 1: 2.5; stirring and reacting for 5h at room temperature, standing overnight at 4 ℃, removing supernatant, washing with water for 3 times, and removing residual glutaraldehyde to obtain hollow spherical chitosan activated by glutaraldehyde;
(b3) encapsulation of enzymes
Adding the glutaraldehyde-activated hollow spherical chitosan prepared in the step (b 2) into a neutral protease preparation prepared by a buffer solution with the pH value of 7.0, an alkaline protease preparation prepared by a buffer solution with the pH value of 9.0 and an acidic protease preparation prepared by a buffer solution with the pH value of 4.2 respectively, stirring and reacting for 3 hours at room temperature, standing overnight at 4 ℃, and washing with water to remove free enzymes to obtain immobilized neutral protease, alkaline protease and acidic protease;
(c) throw in
And (c) putting the immobilized neutral protease, the immobilized alkaline protease and the immobilized acidic protease prepared in the step (b 3) and the fish leftover fermentation liquor prepared in the step (a 2) into the sea cucumber aquaculture water area according to the feed-liquid ratio of 1: 80.
The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme provided by the embodiment has the advantages of reasonable process and reliable operation. The immobilized enzyme has high stability and long half-life cycle, slowly carries out enzymolysis on fish leftover fermentation liquor, releases sea cucumber bait, simultaneously plays a role in degrading residual bait deposited on the sea bottom or the pool bottom, reduces the discharge of nitrogen and phosphorus in the aquaculture industry, and adopts chitosan which also plays a role in purifying water quality and protects the aquaculture ecological environment. It reduces the times of throwing baits and the labor cost. The invention makes full use of marine wastes, improves the utilization rate of marine resources and reduces environmental pollution.
Example 2
The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme is characterized by comprising the following steps of: which comprises the following steps:
(a) fermentation treatment of fish leftovers
(a1) Culture of zymocyte liquid
Inoculating a W2 strain into a DAE culture medium, controlling the temperature to be 25 ℃, and culturing for 2d under the condition of 150r/min to obtain a seed bacterial liquid; the OD600 of the seed bacterial liquid is 1.2; wherein, the formula of the DAE culture medium is as follows: 0.8g of peptone, 0.05g of yeast extract, 0.02g of iron phosphate and 100ml of seawater; the strain W2 is a protease-producing strain separated and screened from the fish intestinal tract, and the inoculation ratio of the strain W2 to the DAE culture medium is 1: 500;
(a2) preparation of fish leftover fermentation liquor
Selecting fish leftovers as raw materials, and feeding the fish leftovers into a grinder to grind the fish leftovers into a meat paste; weighing meat paste fish leftovers into the seed bacterial liquid prepared in the step (a1), wherein the feed-liquid ratio of the meat paste fish leftovers to the seed bacterial liquid is 1: 25; controlling the temperature to be 20 ℃, culturing for 2d under the condition of 180r/min, and taking fermentation liquor to obtain fish leftover fermentation liquor;
(b) preparation of immobilized enzymes
(b1) Preparation of the support
Weighing chitosan with deacetylation degree of 85 percent, dissolving the chitosan in hydrochloric acid solution with concentration of 20 percent, and preparing chitosan solution with the material-to-liquid ratio of the chitosan to the hydrochloric acid solution being 1: 5; then injecting the prepared chitosan solution into a mixed solution containing 15% of sodium hydroxide and 30% of methanol, and condensing into hollow spherical chitosan;
(b2) activation of the support
Putting the hollow spherical chitosan prepared in the step (b 1) into a glutaraldehyde aqueous solution with the concentration of 8%, wherein the material-liquid ratio of the hollow spherical chitosan to the glutaraldehyde aqueous solution is 1: 2; stirring and reacting for 4h at room temperature, standing overnight at 4 ℃, removing supernatant, washing with water for 3 times, and removing residual glutaraldehyde to obtain hollow spherical chitosan activated by glutaraldehyde;
(b3) encapsulation of enzymes
Adding the glutaraldehyde-activated hollow spherical chitosan prepared in the step (b 2) into a neutral protease preparation prepared by a buffer solution with the pH value of 6.8, an alkaline protease preparation prepared by a buffer solution with the pH value of 10.5 and an acidic protease preparation prepared by a buffer solution with the pH value of 4.0 respectively, stirring and reacting for 2.5h at room temperature, standing overnight at 4 ℃, and washing with water to remove free enzymes to obtain immobilized neutral protease, alkaline protease and acidic protease;
(c) throw in
And (c) putting the immobilized neutral protease, the immobilized alkaline protease and the immobilized acidic protease prepared in the step (b 3) and the fish leftover fermentation liquor prepared in the step (a 2) into a sea cucumber culture water area according to the feed-liquid ratio of 1: 50.
The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme provided by the embodiment has the advantages of reasonable process and reliable operation. The immobilized enzyme has high stability and long half-life cycle, slowly performs enzymolysis on fish leftover fermentation liquor to release sea cucumber bait, and simultaneously performs degradation on residual bait deposited on the seabed or the pool bottom, thereby reducing the bait throwing times and labor cost. The invention makes full use of marine wastes, improves the utilization rate of marine resources and reduces environmental pollution.
Example 3
The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme is characterized by comprising the following steps of: which comprises the following steps:
(a) fermentation treatment of fish leftovers
(a1) Culture of zymocyte liquid
Inoculating a W2 strain into a DAE culture medium, controlling the temperature to be 20 ℃, and culturing for 3d under the condition of 180r/min to obtain a seed bacterial liquid; the OD600 of the seed bacterial liquid is 1.0; wherein, the formula of the DAE culture medium is as follows: 0.4g of peptone, 0.2g of yeast extract, 0.01g of ferric phosphate and 90ml of seawater; the strain W2 is a protease-producing strain separated and screened from the fish intestinal tract, and the inoculation ratio of the strain W2 to the DAE culture medium is 1: 300;
(a2) preparation of fish leftover fermentation liquor
Selecting fish leftovers as raw materials, and feeding the fish leftovers into a grinder to grind the fish leftovers into a meat paste; weighing meat paste fish leftovers into the seed bacterial liquid prepared in the step (a1), wherein the feed-liquid ratio of the meat paste fish leftovers to the seed bacterial liquid is 1: 20; controlling the temperature to be 25 ℃, culturing for 2.5d under the condition of 150r/min, and taking fermentation liquor to obtain fish leftover fermentation liquor;
(b) preparation of immobilized enzymes
(b1) Preparation of the support
Weighing chitosan with the deacetylation degree of 80 percent, dissolving the chitosan in a hydrochloric acid solution with the concentration of 30 percent, and preparing a chitosan solution, wherein the material-to-liquid ratio of the chitosan to the hydrochloric acid solution is 1: 3; then injecting the prepared chitosan solution into a mixed solution containing 15% of sodium hydroxide and 30% of methanol, and condensing into hollow spherical chitosan;
(b2) activation of the support
Putting the hollow spherical chitosan prepared in the step (b 1) into a glutaraldehyde aqueous solution with the concentration of 4%, wherein the material-liquid ratio of the hollow spherical chitosan to the glutaraldehyde aqueous solution is 1: 3; stirring and reacting for 6h at room temperature, standing overnight at 4 ℃, removing supernatant, washing with water for 2 times, and removing residual glutaraldehyde to obtain hollow spherical chitosan activated by glutaraldehyde;
(b3) encapsulation of enzymes
Adding the glutaraldehyde-activated hollow spherical chitosan prepared in the step (b 2) into a neutral protease preparation prepared by a buffer solution with the pH value of 7.5, an alkaline protease preparation prepared by a buffer solution with the pH value of 8.5 and an acidic protease preparation prepared by a buffer solution with the pH value of 4.5 respectively, stirring and reacting for 2 hours at room temperature, standing overnight at 4 ℃, and washing with water to remove free enzymes to obtain immobilized neutral protease, alkaline protease and acidic protease;
(c) throw in
And (c) putting the immobilized neutral protease, the immobilized alkaline protease and the immobilized acidic protease prepared in the step (b 3) and the fish leftover fermentation liquor prepared in the step (a 2) into a sea cucumber culture water area according to the feed-liquid ratio of 1: 100.
The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme provided by the embodiment has the advantages of reasonable process and reliable operation. The immobilized enzyme has high stability and long half-life cycle, slowly carries out enzymolysis on fish leftover fermentation liquor, releases sea cucumber bait, simultaneously plays a role in degrading residual bait deposited on the sea bottom or the pool bottom, reduces the discharge of nitrogen and phosphorus in the aquaculture industry, and adopts chitosan which also plays a role in purifying water quality and protects the aquaculture ecological environment. It reduces the times of throwing baits and the labor cost. The invention makes full use of marine wastes, improves the utilization rate of marine resources and reduces environmental pollution.
Claims (1)
1. The method for preparing the slow-release sea cucumber bait by using the fish leftovers and the immobilized enzyme is characterized by comprising the following steps of: which comprises the following steps:
(a) fermentation treatment of fish leftovers
(a1) Culture of zymocyte liquid
Inoculating a W2 strain into a DAE culture medium, controlling the temperature to be 20-25 ℃, and culturing for 2-3 days under the condition of 150-180 r/min to obtain a seed bacterial liquid, wherein the OD600 of the seed bacterial liquid is 1.0-1.2; wherein, the formula of the DAE culture medium is as follows: 0.4-0.8 g of peptone, 0.05-0.2 g of yeast extract, 0.01-0.02 g of iron phosphate and 90-100 ml of seawater; the strain W2 is a protease-producing strain separated and screened from a fish intestinal tract, and the inoculation ratio of the strain W2 to the DAE culture medium is 1: 300-500;
(a2) preparation of fish leftover fermentation liquor
Selecting fish leftovers as raw materials, and grinding the fish leftovers into a meat paste; weighing minced fish leftovers, putting the minced fish leftovers into the seed bacterial liquid prepared in the step (a1), controlling the temperature to be 20-25 ℃, culturing for 2-3 d under the condition of 150-180 r/min, and taking fermentation liquid to obtain fish leftovers fermentation liquid; wherein the feed-liquid ratio of the minced fish leftovers to the seed bacterial liquid is 1: 20-25;
(b) preparation of immobilized enzymes
(b1) Preparation of the support
Weighing chitosan, and dissolving the chitosan in a hydrochloric acid solution with the concentration of 20-30% to prepare a chitosan solution; then injecting the prepared chitosan solution into a mixed solution containing 15% of sodium hydroxide and 30% of methanol, and condensing into hollow spherical chitosan; wherein the material-liquid ratio of the chitosan to the hydrochloric acid solution is 1: 3-5, and the deacetylation degree of the chitosan is 80-85%;
(b2) activation of the support
Putting the hollow spherical chitosan prepared in the step (b 1) into a glutaraldehyde aqueous solution with the concentration of 4-8%, stirring and reacting for 4-6 h at room temperature, standing overnight at 4 ℃, removing supernatant, washing for 2-3 times with water, and removing residual glutaraldehyde to obtain glutaraldehyde-activated hollow spherical chitosan; wherein the material-liquid ratio of the hollow spherical chitosan to the glutaraldehyde aqueous solution is 1: 2-3;
(b3) encapsulation of enzymes
Adding the glutaraldehyde-activated hollow spherical chitosan prepared in the step (b 2) into neutral protease, alkaline protease and acid protease preparations prepared from buffers with different pH values respectively, stirring and reacting for 2-3 h at room temperature, standing overnight at 4 ℃, and washing with water to remove free enzymes to obtain immobilized neutral protease, alkaline protease and acid protease; wherein the pH value of the buffer solution of the neutral protease is 6.8-7.5; the pH value of the buffer solution of the alkaline protease is 8.5-10.5; the pH value of the buffer solution of the acid protease is 4.0-4.5;
(c) throw in
And (c) putting the immobilized neutral protease, the immobilized alkaline protease and the immobilized acidic protease prepared in the step (b 3) and the fish leftover fermentation liquor prepared in the step (a 2) into the sea cucumber culture water area according to the feed-liquid ratio of 1: 50-100.
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