CN114287521B - Compound enzymolysis preparation method of laminarin for replacing fish meal for cottonseed protein compound feed and application thereof - Google Patents
Compound enzymolysis preparation method of laminarin for replacing fish meal for cottonseed protein compound feed and application thereof Download PDFInfo
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Abstract
The invention discloses a compound enzymolysis preparation method of laminarin for replacing fish meal for compound feeding with cottonseed protein and application thereof. According to the invention, raw materials are pretreated by a solid fermentation method, and the algin lyase and the mannanase are subjected to compound enzymolysis, so that the viscosity of kelp substrates is reduced, the concentration of reaction substrates is increased, the enzymolysis rate is greatly improved, and the kelp polysaccharide with low molecular weight and good solubility and growth promoting effect is produced by an environment-friendly and efficient process method. The compound feeding of laminarin and cottonseed protein for prawns has the advantages that laminarin can improve the digestion and absorption capacity of intestinal tracts to nutrient substances, reduce the adverse effect of plant protein feed, improve the survival rate of prawns, have good growth promoting effect, and finally realize the effect of replacing fish meal by compound use with plant proteins such as cottonseed protein and the like, thereby reducing the feeding cost.
Description
Technical Field
The invention relates to an extraction method for preparing laminarin by composite enzymolysis, in particular to a method for preparing laminarin with functions of promoting nutrition absorption and aquatic product growth by composite enzymolysis of laminarin by alginate lyase and mannanase.
Background
Fish meal has a vital role as a major protein source in aquaculture feed formulations. However, as marine environmental pollution is serious and marine fishery resources are excessively caught, fish meal resources are drastically reduced, the price of the fish meal resources is continuously increased, and the cost of the feed is greatly increased. The resource limitation of fish meal severely restricts the growth of the aquatic product word stock industry, thereby influencing the sustainable development of the marine fish farming industry.
However, after the fish meal dosage is reduced in the compound feed, the problems of slow growth, low disease resistance and the like of the cultured aquatic products can occur. Therefore, the search for improved growth and survival rates of water under low fish meal conditions has become a hot spot in recent years for nutritional research in water production. It is now common in aquaculture to use plant-derived proteins to replace part of the fish meal. However, plant protein feed has more anti-nutritional factors or nondigestible and absorbable components, and the increase of the content of the plant protein feed in the feed can lead to the increase of the feed coefficient of aquatic products, and the production of the aquatic products is influenced to a certain extent. Therefore, there is a need to find an effective strategy that allows plant-derived proteins to be better used in aquaculture.
Laminarin is a biological macromolecular substance naturally existing in kelp, has special effects of promoting lead discharge, regulating blood lipid, enhancing immunity, promoting growth, and improving nutrition absorption, and has no toxic side effects. Namely, laminarin can play roles in protecting organisms, promoting nutrient absorption and the like in aquaculture. Therefore, there is a need to investigate the use of laminarin and vegetable proteins in aquaculture.
Disclosure of Invention
The invention provides a method for producing laminarin by composite enzymolysis and an application method of composite cottonseed protein in prawn culture. The laminarin prepared by the method for producing laminarin by composite enzymolysis provided by the invention is used in combination with cottonseed protein, can replace fish meal in conventional aquaculture, and improves the survival rate and growth performance of prawns.
The technical scheme adopted by the invention for solving the technical problems is as follows: an extraction method for preparing laminarin by composite enzymolysis comprises the following steps:
step 1: pulverizing kelp powder: pulverizing herba Zosterae Marinae powder in pulverizer, and sieving with 100 mesh sieve;
step 2: solid fermentation of kelp powder: putting the sieved kelp powder into a homogenizer, adding corn starch accounting for 10% of the mass of the kelp powder, then adding water according to a feed-liquid ratio of 1:1, uniformly mixing and stirring, inoculating fermentation bacteria into the material, fermenting for 48 hours, and stirring once every 12 hours;
wherein the fermentation strain is Aspergillus niger;
step 3: homogenizing enzymolysis liquid: putting the fermented kelp powder into a dissolving tank, adding water according to a solid content of 1:10, opening a stirring paddle, homogenizing uniformly, and heating to 50 ℃;
step 4: algin lyase enzymolysis: the algin lyase is used for enzymolysis, the enzyme activity is 25000-30000U/g, the enzyme addition amount is 250-300U/g kelp substrate, the enzymolysis temperature is 45-60 ℃, the pH is regulated to 7.0 by sodium hydroxide, and the action time is 3 hours;
step 5: mannanase enzymolysis: adjusting the pH of the enzymolysis liquid to 4.5 after the enzymolysis in the step 4 by hydrochloric acid, and then performing enzymolysis by using mannase with the enzyme activity of 40000-50000U/g and the enzyme addition amount of 200-250U/g kelp substrate, wherein the enzymolysis temperature is 45-60 ℃ and the action time is 2 hours;
step 6: enzyme deactivation: heating the enzymolysis liquid to 95 ℃ and keeping for 15min for enzyme deactivation;
step 7: and (3) centrifuging: adding absolute ethyl alcohol into the enzymolysis liquid prepared in the step 3 according to 10-12% of the volume of the enzymolysis liquid to enable macromolecular saccharide substances to settle, and centrifuging to remove enzymolysis residues;
step 8: filtering the enzymolysis liquid by using a filter membrane to remove impurities and bacteria, wherein the aperture specification of the filter membrane is 0.45-0.95 mu m;
step 9: spray drying: and (3) concentrating the supernatant after centrifugal impurity removal by suspension distillation until the solid content is more than or equal to 25%, adding maltodextrin according to 20-25% of the solid content, and performing spray drying to complete the preparation. Preferably, the spray drying inlet temperature of the step 9 is 170-190 ℃, the air outlet temperature is 90-110 ℃, and the flow rate is 8-12mL/min, so as to obtain laminarin dry powder.
The application method of laminarin prepared by composite enzymolysis is characterized by comprising the following steps: the application method of laminarin for replacing fish meal for the compound feed with the cottonseed protein concentrate comprises the following steps:
method 1: adding laminarin into feed according to the proportion of 1% -2%, uniformly mixing, processing and preparing according to the required feed form, and directly feeding;
method 2: the laminarin is dissolved into daily drinking water of animals according to the proportion of 1% -2% for use.
Compared with the prior art, the invention has the beneficial effects that:
(1) The kelp raw material is pretreated by a solid state fermentation method, and then the algin lyase and the mannanase are used for compound enzymolysis, so that the viscosity of a kelp substrate can be obviously reduced, the enzymolysis rate is greatly improved, and the kelp polysaccharide with low molecular weight and good solubility and growth promoting effect is produced by an environment-friendly and efficient process method. Has the advantages of mild reaction condition, simple operation and low cost.
(2) The laminarin obtained by the invention is compounded with cottonseed protein for feeding prawns, can play a role in replacing fish meal in conventional feed, improves the survival rate of the prawns, and has excellent growth promoting effect.
Drawings
Fig. 1: effect of combined laminarin and cottonseed protein on weight gain rate of litopenaeus vannamei fed by substituting fish meal
Fig. 2: effect of combination of laminarin and cottonseed protein on specific growth rate of litopenaeus vannamei fed by substituting fish meal
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Step 1: pulverizing kelp powder: the kelp powder is put into a pulverizer for pulverization, and the pulverized kelp powder is sieved by a 100-mesh sieve.
Step 2: solid fermentation of kelp powder: putting the sieved kelp powder into a homogenizer, adding corn starch accounting for 10% of the kelp powder by mass, then adding water according to a feed-liquid ratio of 1:1, mixing and stirring uniformly, inoculating fermentation bacteria into the material, fermenting for 48 hours, and stirring once every 12 hours.
Wherein the fermentation strain is Aspergillus niger.
Step 3: homogenizing enzymolysis liquid: and (3) putting the fermented kelp powder into a dissolving tank, adding water according to a solid content of 1:10, opening a stirring paddle, homogenizing uniformly, and heating to 50 ℃.
Step 4: algin lyase enzymolysis: the algin lyase is used for enzymolysis, the enzyme activity is 25000-30000U/g, the enzyme addition amount is 250-300U/g kelp substrate, the enzymolysis temperature is 45-60 ℃, the pH is regulated to 7.0 by sodium hydroxide, and the action time is 3 hours;
step 5: mannanase enzymolysis: adjusting the pH of the enzymolysis liquid to 4.5 after the enzymolysis in the step 4 by hydrochloric acid, and then performing enzymolysis by using mannase with the enzyme activity of 40000-50000U/g and the enzyme addition amount of 200-250U/g kelp substrate, wherein the enzymolysis temperature is 45-60 ℃ and the action time is 2 hours;
step 6: enzyme deactivation: heating the enzymolysis liquid to 95 ℃ and keeping for 15min for enzyme deactivation
Step 7: and (3) centrifuging: and (3) adding absolute ethyl alcohol into the enzymolysis liquid prepared in the step (3) according to 10-12% of the volume of the enzymolysis liquid, settling macromolecular saccharide substances, and centrifuging to remove enzymolysis residues.
Step 8: filtering the enzymolysis liquid by using a filter membrane, removing impurities and sterilizing, wherein the pore size specification of the filter membrane is 0.45-0.95 mu m.
Step 9: spray drying: concentrating supernatant after centrifugal impurity removal by suspension distillation until the solid content is more than or equal to 25%, adding maltodextrin according to 20-25% of the solid content, and spray drying to obtain the final product
Wherein the spray drying inlet temperature is 170-190 ℃, the air outlet temperature is 90-110 ℃, and the flow rate is 8-12mL/min, thus obtaining laminarin dry powder.
The application method of laminarin prepared by composite enzymolysis is characterized by comprising the following steps: the application method of laminarin for replacing fish meal for the compound feed with the cottonseed protein concentrate comprises the following steps:
method 1: adding laminarin into feed according to the proportion of 1% -2%, uniformly mixing, processing and preparing according to the required feed form, and directly feeding;
method 2: the laminarin is dissolved into daily drinking water of animals according to the proportion of 1% -2% for use.
The invention is illustrated in detail below with reference to examples:
example 1:
1) And (3) putting the dried kelp into a pulverizer to pulverize, and sieving the kelp powder with a 100-mesh sieve after the pulverization is finished.
2) Weighing 150g of sieved kelp powder, putting the sieved kelp powder into a homogenizer, adding 15g of corn starch, then adding 165mL of water, inoculating zymophyte into the materials, uniformly mixing, transferring into a fermentation bag, fermenting at 28 ℃ for 48 hours, and stirring every 12 hours.
Wherein the zymophyte is Aspergillus niger.
3) Transferring the fermented kelp powder into a tissue homogenizer, adding 1650mL of water, homogenizing, and heating to 50deg.C
4) And regulating the pH value of the reaction solution to 7.0 by using sodium hydroxide, adding algin lyase according to the amount of 300U/g kelp powder substrate, performing enzymolysis, stirring the reaction kettle at the rotating speed of 50r/min, and performing enzymolysis for 3 hours at the temperature of 50 ℃.
5) Adjusting the pH value of the reaction solution after the enzymolysis of the 4) to 4.5 by using hydrochloric acid, adding mannase according to the amount of 200U/g kelp powder substrate, carrying out enzymolysis, stirring the reaction kettle at the rotating speed of 50r/min, and carrying out enzymolysis for 2h at the temperature of 50 ℃.
6) And heating the enzymolysis liquid to 95 ℃ and keeping for 15min to inactivate enzyme.
7) After the enzymolysis liquid is cooled, 200mL of absolute ethyl alcohol is added, the mixture is kept stand for 15min, and then the insoluble residues are removed by centrifugation at 6000rpm/min of a centrifuge for 20 min.
8) And filtering the centrifuged enzymolysis liquid by using a glass core funnel to remove the bacterial residues, wherein the aperture of the glass core funnel is 0.65 mu m.
9) Concentrating the supernatant by rotary evaporation to about 750mL, measuring the solid content in the concentrated solution, adding maltodextrin according to 20% of the solid content, dissolving uniformly, and performing spray drying to obtain laminarin. Wherein the air inlet temperature of spray drying is 170-180 ℃, the air outlet temperature is 80-90 ℃, the flow rate is 8-10mL/min, and the yield of laminarin is about 83%.
Example 2
1) And (3) putting the dried kelp into a pulverizer to pulverize, and sieving the kelp powder with a 100-mesh sieve by using a vibration sieving machine after the pulverization is finished.
2) Weighing 7.5kg of sieved kelp powder, putting the sieved kelp powder into a homogenizer, adding 0.75kg of corn starch, then adding 8.5L of water, inoculating fermentation bacteria into the material, uniformly mixing and homogenizing, transferring into a large fermentation bag, fermenting at 28 ℃ for 48 hours, and stirring once every 12 hours.
Wherein the zymophyte is Aspergillus niger.
3) Transferring the fermented kelp powder into an enzymolysis tank, adding about 80L of water, homogenizing, introducing steam into a jacket of the enzymolysis tank, and heating the enzymolysis liquid to 50 ℃.
4) And regulating the pH value of the enzymolysis liquid to 7.0 by using sodium hydroxide, adding algin lyase according to the amount of 300U/g kelp powder substrate, performing enzymolysis, stirring the reaction kettle at the rotating speed of 50r/min, and performing enzymolysis for 3 hours at the temperature of 50 ℃.
5) Adjusting the pH value of the reaction solution after the enzymolysis of the 4) to 4.5 by using hydrochloric acid, adding mannase according to the amount of 200U/g kelp powder substrate, carrying out enzymolysis, stirring the reaction kettle at the rotating speed of 50r/min, and carrying out enzymolysis for 2h at the temperature of 50 ℃.
5) And (3) introducing steam into the jacket layer of the enzymolysis tank to heat the enzymolysis liquid to 95 ℃ and keeping the temperature for 15min for enzyme deactivation.
6) And (3) adding 10L of absolute ethyl alcohol after cooling the enzymolysis liquid, standing for 15min, and centrifuging at 6000rpm/min by a disc centrifuge to remove insoluble residues.
7) The enzymatic hydrolysate after centrifugation was filtered with a 0.45 μm filter membrane.
8) Concentrating the supernatant by rotary evaporation to 40L, measuring the solid content in the concentrated solution, adding maltodextrin according to 20% of the solid content, dissolving uniformly, and spray drying to obtain laminarin. Wherein the air inlet temperature of spray drying is 170-180deg.C, the air outlet temperature is 80-90deg.C, the flow rate is 8-10mL/min, and the yield of laminarin is about 85%.
Example 3
The compound use of laminarin and cottonseed protein prepared by the technology replaces the influence of partial fish meal on the growth condition of prawns. Laminarin according to the present technology was prepared according to example 2. Weighing 30kg of sieved kelp powder, putting the sieved kelp powder into a homogenizer, adding 3kg of corn starch, then adding 33L of water, inoculating Aspergillus niger into the material, uniformly mixing and homogenizing, and putting the material into a vibrating solid fermentation tank for fermentation for 48 hours. After fermentation, transferring the materials into an enzymolysis tank, adding 330L of water, homogenizing, introducing steam into a jacket in the dissolution tank, heating the materials to 50 ℃, adjusting the pH of the reaction solution to 7.0 by using sodium hydroxide, adding algin lyase according to the amount of 300U/g kelp powder substrate, performing enzymolysis, and stirring at the rotating speed of 50r/min and the temperature of 50 ℃ for 3 hours. After the enzymolysis of the algin lyase is finished, hydrochloric acid is used for adjusting the pH value of the reaction solution to 4.5, mannanase is added according to the amount of 200U/g kelp powder substrate, the enzymolysis is carried out, the rotating speed of a stirring paddle is 50r/min, and the enzymolysis is carried out for 2 hours at 50 ℃. After the enzymolysis is finished, the enzymolysis liquid is heated to 95 ℃ and kept for 15min for enzyme deactivation. And (3) adding 40L of absolute ethyl alcohol after cooling the enzymolysis liquid, standing for 15min, and centrifuging at 6000rpm/min by using a disc centrifuge to remove insoluble residues. The enzymatic hydrolysate after centrifugation was sterilized by filtration using a 0.45 μm pore size filter. Concentrating the supernatant by rotary evaporation to about 150L, measuring the solid content in the concentrated solution, adding maltodextrin according to 20% of the solid content, dissolving uniformly, and spray drying to obtain laminarin. Wherein the air inlet temperature of spray drying is 170-180 ℃, the air outlet temperature is 80-90 ℃, the flow rate is 8-10mL/min, and the product yield is above 85%. The analysis of laminarin product composition is shown in Table 1.
According to experimental requirements, basic ration of prawns is used as a main feed component, and red fish powder and cottonseed protein are used as main protein sources. Wherein, the positive control group uses the feed: the content of fish meal in the feed is 25% based on the ration of the prawn and the red fish meal. The negative control group used feed: the shrimp basic ration, red fish powder and cottonseed protein are used for replacing 30% of fish meal protein in the feed. The feed used in the test group is based on the negative control group, and laminarin with different contents is used for replacing the prawn basic ration. Test group 1: the prawn basic ration, red fish powder and cottonseed protein, and laminarin replaces 1% of the prawn basic ration; test group 2: the prawn basic ration, red fish meal and cottonseed protein, and laminarin replaces 2% of the prawn basic ration; test group 3: the prawn basic ration, red fish meal and cottonseed protein, and laminarin replaces 3% of the prawn basic ration; test group 4: the prawn basic ration, red fish powder and cottonseed protein, and laminarin replaces 4% of the prawn basic ration. All the raw materials are fully and evenly mixed, extruded to prepare hard pellet feed with the diameter of 1.0mm, and the feed is air-dried at room temperature until the water content is about 20 percent and stored at the temperature of minus 20 ℃ for standby. The raw materials and the nutritional composition of the experimental feed are shown in Table 2.
The 720-tail litopenaeus vannamei larvae with uniform size, healthy physical constitution and initial weight of 0.74 g are randomly divided into 6 groups, 3 repeats are arranged in each group, 40-tail litopenaeus vannamei larvae are fed into a glass fiber barrel with the volume of 0.3 cubic meter by taking the repeats as a unit, and the culture test lasts for 56 days. Feeding the feed according to 8% -20% of the weight, wherein the feeding is carried out for 1 time in each of 07:00, 11:00, 17:00 and 21:00; feeding for 1h, observing feeding condition, and properly adjusting feeding amount according to weather, water quality and the like. Water is changed every day at the initial stage of the test, water is changed every day 2 weeks before the culture test is finished, and the water change amount is 1/3-1/2 of the total water amount. Fasted for 24 hours after the end of the cultivation test, counted in duplicate units and weighed. The growth indexes of the prawns, namely Survival Rate (SR), weight gain rate (WG), specific Growth Rate (SGR) and Feed Coefficient (FCR) are calculated as follows:
survival rate (survivinval rate, SR,%) =100 x final mantissa/initial mantissa;
weight gain rate (WG,%) =100 x (final average weight-initial average weight)/initial average weight;
specific growth rate (SGR,% 1 d) =100 x (1 n terminal average weight-ln initial average weight)/days of feeding:
feed coefficient (feed conversion rate, FCR) =feeding dry weight/(final body weight-initial body weight).
As can be seen from Table 3, the survival rate of the prawns was reduced by 2.8% in the negative control group compared with the positive control group, and none of the prawns in test group 1, test group 2, test group 3 and test group 4 died. In addition, compared with the positive control group, the feed coefficient of the negative control group is increased by 9.8%, the feed coefficient of the test group added with laminarin has no obvious difference from the positive control group, and the feed coefficient of the test group 4 is reduced by 2%. The weight gain rate of the prawn is shown in figure 1, the specific growth rate of the prawn is shown in figure 2, the weight gain rate WG and the specific growth rate SGR reach the significance maximum value (P < 0.05) in the test group 2, and the values of other treatment groups are slightly improved as compared with the positive control group although the values of the other treatment groups are not significantly different. This shows that the use of cottonseed protein to replace part of fish meal in prawn feed can reduce the survival rate of prawn and raise the feed coefficient of prawn culture. The addition of laminarin while using cottonseed protein to replace fish meal can maintain and improve the survival rate of prawns, does not affect the utilization rate of feed, and can improve the production performance of prawns. In particular, 2% of laminarin is compounded with cottonseed protein, so that the growth performance of the prawns can be obviously improved. The invention can eliminate the adverse effect of cottonseed protein feeding on prawns, maintain and improve the survival rate of the prawns, and has good growth promoting effect by improving the digestion and absorption capacity of intestinal tracts on nutrient substances, thereby achieving the effect of replacing fish meal by compounding laminarin with plant proteins such as cottonseed protein and the like and reducing the feeding cost.
TABLE 1 analysis of laminarin product composition (%)
TABLE 2 basic feed composition and nutrient level (%)
TABLE 3 Effect of kelp polysaccharide and cottonseed protein combination to replace fish meal on Litopenaeus vannamei survival and feed utilization
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. The foregoing is merely illustrative of the preferred embodiments of this invention, and it is noted that there is objectively no limit to the specific structure disclosed herein, since numerous modifications, adaptations and variations can be made by those skilled in the art without departing from the principles of the invention, and the above-described features can be combined in any suitable manner; such modifications, variations and combinations, or the direct application of the inventive concepts and aspects to other applications without modification, are contemplated as falling within the scope of the present invention.
Claims (2)
1. A compound enzymolysis preparation method of laminarin for replacing fish meal for compound feeding with cotton seed concentrated protein is characterized by comprising the following steps:
step 1: pulverizing kelp powder: pulverizing herba Zosterae Marinae powder in pulverizer, and sieving with 100 mesh sieve;
step 2: solid fermentation of kelp powder: putting the sieved kelp powder into a homogenizer, adding corn starch accounting for 10% of the mass of the kelp powder, then adding water according to a feed-liquid ratio of 1:1, mixing and stirring uniformly, inoculating fermentation bacteria into the materials, and fermenting for 48 hours;
wherein the zymophyte is aspergillus niger;
step 3: homogenizing enzymolysis liquid: putting the fermented kelp powder into a dissolving tank, adding water according to a solid content of 1:10, opening a stirring paddle, homogenizing uniformly, and heating to 50 ℃;
step 4: algin lyase enzymolysis: the algin lyase is used for enzymolysis, the enzyme activity is 25000-30000U/g, the enzyme addition amount is 250-300U/g kelp substrate, the enzymolysis temperature is 45-60 ℃, the pH is regulated to 7.0 by sodium hydroxide, and the action time is 3 hours;
step 5: mannanase enzymolysis: adjusting the pH of the enzymolysis liquid to 4.5 after the enzymolysis in the step 4 by hydrochloric acid, and then performing enzymolysis by using mannase with the enzyme activity of 40000-50000U/g and the enzyme addition amount of 200-250U/g kelp substrate, wherein the enzymolysis temperature is 45-60 ℃ and the action time is 2 hours;
step 6: enzyme deactivation: heating the enzymolysis liquid to 95 ℃ and keeping for 15min for enzyme deactivation;
step 7: and (3) centrifuging: adding absolute ethyl alcohol into the enzymolysis liquid prepared in the step 6 according to 10-12% of the volume of the enzymolysis liquid to enable macromolecular saccharide substances to settle, and centrifuging to remove enzymolysis residues;
step 8: filtering the enzymolysis liquid by using a filter membrane to remove impurities and bacteria, wherein the aperture specification of the filter membrane is 0.45-0.95 mu m;
step 9: spray drying: concentrating the supernatant after centrifugal impurity removal by rotary evaporation until the solid content is more than or equal to 25%, adding maltodextrin according to 20-25% of the solid content, and performing spray drying to finish the preparation; wherein the spray drying inlet temperature is 170-190 ℃, the air outlet temperature is 90-110 ℃, and the flow rate is 8-12mL/min, thus obtaining laminarin dry powder.
2. An application method of laminarin prepared by the composite enzymolysis preparation method of claim 1, which is characterized in that: the kelp polysaccharide is used for replacing fish meal in the compound feed of the cottonseed concentrated protein;
the application method comprises the following steps:
adding laminarin into feed according to the proportion of 1% -2%, uniformly mixing, processing and preparing according to the required feed form, and directly feeding.
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