CN118240905A - Preparation method and application of hermetia illucens water-soluble protein powder - Google Patents
Preparation method and application of hermetia illucens water-soluble protein powder Download PDFInfo
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- CN118240905A CN118240905A CN202410320845.2A CN202410320845A CN118240905A CN 118240905 A CN118240905 A CN 118240905A CN 202410320845 A CN202410320845 A CN 202410320845A CN 118240905 A CN118240905 A CN 118240905A
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- Prior art keywords
- hermetia illucens
- water
- soluble protein
- protein powder
- preparation
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 62
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
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- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
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- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Birds (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a preparation method of hermetia illucens water-soluble protein powder and application of the hermetia illucens water-soluble protein powder in laying hen production, and relates to the technical field of insect protein processing. The method provided by the invention is to directly pulp and defat the live black soldier fly, reduce the drying cost, reasonably mix the components and weight ratio of the compound enzyme preparation and the compound fermentation bacteria liquid, and obtain the water-soluble protein powder of the black soldier fly by adopting the technology of enzymolysis before fermentation. The water-soluble protein powder of the hermetia illucens provided by the invention can obviously improve the production performance and the egg quality of laying hens in the laying hen breeding process.
Description
Technical Field
The invention relates to the technical field of insect protein processing, in particular to a preparation method of water-soluble protein powder of hermetia illucens and application of the water-soluble protein powder in laying hen production.
Background
The hermetia illucens is a diptera and hermetia illucens insect, and is one of hot spots for research in the field of insect resources in recent years. Along with the wide popularization of the recycling treatment of kitchen waste and livestock manure of the hermetia illucens larvae, the application technology of the feed conversion of the hermetia illucens larvae is also focused and studied.
At present, the processing mode of microwave drying is mainly adopted for the hermetia illucens larvae, and a great amount of documents report that the microwave drying of the hermetia illucens powder is used for replacing protein raw materials in the livestock industry, but the cost of the microwave drying mode is too high (2000-3000 yuan/ton), so that the application of the hermetia illucens larvae in livestock production is severely limited. At present, the yield of the hermetia illucens powder is very small, and the application of the hermetia illucens powder as a protein raw material cannot meet the market demand. The modern bioengineering technology is utilized to excavate new substances from the insect bodies, and the creation of new products is an effective way for efficiently, safely and sustainably utilizing insect animal resources, and is an important guarantee for promoting environmental protection, healthy development of animal husbandry and food safety. Currently, peptide powder is produced in an enzymolysis mode in actual production, for example, publication No. CN107012193A discloses a method for preparing tryptone by using hermetia illucens, and trypsin is adopted to carry out enzymolysis on hermetia illucens larva powder; publication number CN117070274a discloses a method for preparing grease by using hermetia illucens larvae, which also adopts an enzyme preparation mixed by neutral protease, lipase and bromelain to carry out enzymolysis on the hermetia illucens larva pulp; however, the obtained enzymolysis product often has obvious bitter taste, and has the problems of incomplete enzymolysis, low protein content, low nutritive value and the like.
The active peptide segments which produce the synergistic effect of a plurality of enzymes are prepared by utilizing the action of beneficial bacteria and other microorganisms on the black soldier fly larva raw materials, so that the enzymolysis efficiency is improved, and the bitter taste influence of the simple enzymolysis products is effectively reduced. At present, a method for treating hermetia illucens larvae by utilizing a method of combining enzymolysis and microbial fermentation to obtain protein powder with high protein and high polypeptide content does not exist.
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides a preparation method of water-soluble protein powder of hermetia illucens and application of the water-soluble protein powder in laying hen production. According to the invention, the live black soldier fly is directly pulped and defatted, so that the drying cost is reduced, and then the enzymolysis-before-fermentation technology is adopted to degrade the somatic proteins into various functional polypeptides and unknown growth factors, so that the economic value of the black soldier fly larvae is maximized, and the application efficacy of the water-soluble protein powder of the black soldier fly in the laying hen breeding is evaluated, so that the production performance and the egg quality of the laying hen are improved.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme:
A preparation method of hermetia illucens water-soluble protein powder, which comprises the following steps:
(1) Flushing the living hermetia illucens larvae with flowing water to remove impurities on the surfaces of the larvae;
(2) Steaming cleaned hermetia illucens larva in 80-90deg.C water for 30min, pulping with a refiner, and dehydrating with a centrifuge for 15-20min to obtain solid concentrate of hermetia illucens larva;
(3) Adding 3-5L of degreasing solvent into dehydrated hermetia illucens solid concentrate for degreasing for 3-5 times according to each kilogram to remove crude fat in the concentrate;
(4) Removing degreasing solvent by a centrifuge and recovering grease to obtain defatted hermetia illucens larva solids and grease;
(5) Mixing defatted hermetia illucens larva solid with 4-5L of water per kg, adding a compound enzyme preparation according to 3-5% of the solid weight, and performing enzymolysis reaction at 40-50deg.C for 4-5h; obtaining an enzymolysis product;
(6) Adding 3-4% glucose and 20-30% composite fermentation bacteria liquid according to the liquid amount of the fermentation product after the enzymolysis reaction, performing fermentation reaction at 35-37 ℃ for 20-24 hours, and sterilizing at 85-90 ℃ for 10-15 minutes after the fermentation; obtaining an enzymolysis fermentation product;
(7) And centrifuging the enzymolysis and fermentation product, collecting supernatant, and performing spray drying to obtain the hermetia illucens water-soluble protein powder.
Specifically, in the step (3), the degreasing solvent is one of n-hexane, petroleum ether and ethanol.
Specifically, in the step (5), the complex enzyme preparation is one or more of neutral protease, trypsin, alkaline protease, flavourzyme, papain and acid protease.
Specifically, in the step (5), the complex enzyme preparation is alkaline protease and flavourzyme with the mass ratio of 1-3:1-2.
Specifically, in the step (5), the complex enzyme preparation is alkaline protease and flavourzyme with a mass ratio of 1:1.
Specifically, in the step (5), the addition amount of the complex enzyme preparation is 3-5% of the weight of the solid matters.
Specifically, in the step (6), the composite fermentation bacteria liquid is lactobacillus plantarum seed liquid with the volume ratio of 4-8:2-4:1-2: bacillus subtilis seed liquid: the saccharomyces cerevisiae seed liquid consists of saccharomyces cerevisiae seed liquid.
Specifically, in the step (6), the composite fermentation bacteria liquid is lactobacillus plantarum seed liquid with the volume ratio of 4:2:1: bacillus subtilis seed liquid: the saccharomyces cerevisiae seed liquid consists of saccharomyces cerevisiae seed liquid.
Specifically, in the step (6), the addition amount of the composite fermentation bacteria liquid is 15-20% of the liquid amount of the fermentation product.
The water-soluble protein powder of the hermetia illucens prepared by the method is used for preparing functional feed raw materials of laying hens.
(III) beneficial effects
The invention provides a preparation method of hermetia illucens water-soluble protein powder and application thereof in laying hen production, and the preparation method has the following advantages:
1. The invention directly pulps and degreases the live black soldier fly, reduces the drying cost, reasonably matches the components and weight ratio of the compound enzyme preparation and the compound fermentation bacteria liquid, adopts the enzymolysis-before-fermentation technology to obtain the water-soluble protein powder of the black soldier fly, and compared with the conventional enzymolysis treatment, the method obviously improves the content of functional polypeptide and unknown growth factors after the degradation of the insect protein, reduces the pH of the product, and maximizes the economic value of the black soldier fly larvae.
2. The water-soluble protein powder of the black soldier flies can obviously improve the productivity and the egg quality of laying hens in the laying hen cultivation, and the addition of the water-soluble protein powder of the black soldier flies in the later daily ration of the laying hens can improve the eggshell thickness and the eggshell strength, so that the broken egg proportion is reduced, the laying rate is improved, and the problems of the reduction of the eggshell quality and the egg quality of the laying hens in the later period of a large-scale chicken farm can be effectively solved.
Drawings
FIG. 1 shows the DPPH radical scavenging activity of the water-soluble protein powder of hermetia illucens.
FIG. 2 effects of different proteases on the degree of hydrolysis of the hermetia illucens larva solids and the yield of polypeptides.
FIG. 3 shows the effect of different kinds of complex enzyme preparation on the hydrolysis degree of the solid matter of the hermetia illucens larva and the yield of the polypeptide.
FIG. 4 shows the effect of different weight ratios of alkaline protease and flavourzyme on the degree of hydrolysis of hermetia illucens larva solids and the yield of polypeptides.
FIG. 5 shows the effect of pH after treatment of the solid matter of the black soldier fly larvae with different compound fermentation broths.
FIG. 6 shows the effect of polypeptide yield after solid treatment of black soldier fly larvae with different compound fermentation broths.
FIG. 7 effects of two different preparation methods, example 2 and comparative example 1, on pH of enzymatic or enzymatic fermentation broth and yield of polypeptide.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are 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.
Example 1
1. Preparation of lactobacillus plantarum seed solution:
Selecting a small amount of frozen lactobacillus plantarum strain to inoculate on a sterilized MRS agar culture medium, culturing at a constant temperature of 37 ℃ for 24 hours for strain activation, then inoculating the activated strain in the sterilized MRS broth culture medium, placing in a constant temperature shaking table, culturing at a temperature of 37 ℃ at a rotating speed of 160r/min for 4-6 hours, and preparing seed liquid.
2. Preparing bacillus subtilis seed liquid:
Selecting a small amount of frozen bacillus subtilis strain, inoculating the bacillus subtilis strain to a sterilized nutrient agar culture medium, culturing at a constant temperature of 30 ℃ for 24 hours for strain activation, inoculating the activated strain to the sterilized nutrient broth culture medium, placing the culture medium in a constant temperature shaking table, and culturing at a rotating speed of 160r/min at a temperature of 30 ℃ for 12 hours to prepare seed liquid.
3. Preparing saccharomyces cerevisiae seed liquid:
Inoculating a small amount of frozen Saccharomyces cerevisiae strain to sterilized YPD medium, culturing at 28deg.C for 48 hr for strain activation, inoculating the activated strain to sterilized YPD medium (without agar), placing in a constant temperature shaking table, and culturing at 28deg.C at 160r/min for 18 hr to obtain seed solution.
The specific composition of the culture medium involved is as follows:
MRS medium: 10.0g of peptone, 10.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 0.2g of magnesium sulfate, 5.0g of sodium acetate, 2.0g of triammonium citrate, 2.0g of dipotassium hydrogen phosphate, 0.04g of manganese sulfate, 1.0g of tween 80 and 20.0g of agar (liquid culture medium is free), and 1.0L of distilled water; sterilizing at 121deg.C for 20min at pH 5.7+ -0.2.
Nutrient agar medium: beef extract 3.0g, peptone 10.0g, naCl 5.0g, agar 20.0g (liquid medium does not contain), distilled water 1.0L, pH 7.0. Sterilizing at 121deg.C for 20 min.
YPD medium: yeast extract 10g, glucose: 20.0g, peptone: 20.0g, agar 20.0g (liquid medium free), distilled water 1.0L, pH: 6.2+/-0.2, 121 ℃ and sterilizing for 20 min.
Example 2
The water-soluble protein powder of hermetia illucens is prepared according to the following steps:
(1) Flushing the living hermetia illucens larvae with flowing water to remove impurities on the surfaces of the larvae;
(2) Steaming cleaned hermetia illucens larva in 80-90deg.C water for 30min, pulping with a refiner, and dehydrating with a centrifuge for 15-20min to obtain solid concentrate of hermetia illucens larva;
(3) Adding n-hexane into the dehydrated hermetia illucens solid concentrate according to a ratio of 1:3, heating to 50 ℃, heating for 2 hours, filtering the filtrate, performing the above operation on the solid concentrate again, degreasing for 3-5 times, and removing crude fat in the concentrate;
(4) Removing degreasing solvent by a centrifuge and recovering grease to obtain defatted hermetia illucens larva solids and grease;
(5) Mixing the defatted hermetia illucens larva solid with water according to a ratio of 1:4-5 (Kg/L), adding a compound enzyme preparation according to 3% of the solid weight, and performing enzymolysis reaction at 50 ℃ for 4-5h; obtaining an enzymolysis product; the compound enzyme preparation is alkaline protease and flavourzyme with the mass ratio of 1:1;
(6) Adding 3-4% glucose and 20% composite fermentation bacteria liquid according to the liquid amount of the fermentation product after the enzymolysis reaction is finished, performing fermentation reaction at 35-37 ℃ for 20-24 hours, and sterilizing at 85 ℃ for 10 minutes after the fermentation is finished; obtaining an enzymolysis fermentation product; the composite fermentation bacteria liquid is lactobacillus plantarum seed liquid with the volume ratio of 4:2:1: bacillus subtilis seed liquid: the saccharomyces cerevisiae seed liquid consists of;
(7) And centrifuging the enzymolysis and fermentation product, collecting supernatant, and performing spray drying to obtain the hermetia illucens water-soluble protein powder.
The amino acid composition of the water-soluble protein powder of hermetia illucens prepared by the method is shown in table 1.
TABLE 1 amino acid composition in Water-soluble protein powder of hermetia illucens
Through detection, the hermetia illucens water-soluble protein powder contains more aspartic acid, glutamic acid and histidine, and the amino acids have great influence on the biological activity of the polypeptide.
The in-vitro antioxidant activity of the water-soluble protein powder of the hermetia illucens prepared by the method is measured, and the test result is shown in figure 1, which shows that the clearance rate of DPPH free radicals is gradually increased along with the increase of the concentration of the water-soluble protein powder of the hermetia illucens, and the clearance rate is 86.7% when the concentration reaches 8mg/mL, so that the water-soluble protein of the hermetia illucens has better antioxidant effect.
Comparative example 1
The water-soluble protein powder of hermetia illucens is prepared according to the following steps:
(1) Flushing the living hermetia illucens larvae with flowing water to remove impurities on the surfaces of the larvae;
(2) Steaming cleaned hermetia illucens larva in 80-90deg.C water for 30min, pulping with a refiner, and dehydrating with a centrifuge for 15-20min to obtain solid concentrate of hermetia illucens larva;
(3) Adding n-hexane into the dehydrated hermetia illucens solid concentrate according to a ratio of 1:3, heating to 50 ℃, heating for 2 hours, filtering the filtrate, performing the above operation on the solid concentrate again, degreasing for 3-5 times, and removing crude fat in the concentrate;
(4) Removing degreasing solvent by a centrifuge and recovering grease to obtain defatted hermetia illucens larva solids and grease;
(5) Mixing the defatted hermetia illucens larva solid with water according to a ratio of 1:4-5 (Kg/L), adding a compound enzyme preparation according to 3% of the solid weight, and performing enzymolysis reaction at 50 ℃ for 4-5h; obtaining an enzymolysis product; the compound enzyme preparation is alkaline protease and flavourzyme with the mass ratio of 1:1;
(6) And centrifuging the enzymolysis product, collecting supernatant, and performing spray drying to obtain the water-soluble protein powder of the hermetia illucens.
Test example 1
Influence of different proteases and complex enzyme preparations on hydrolysis degree and polypeptide yield of hermetia illucens larva after solid treatment
The test selects six proteases such as neutral protease, trypsin, alkaline protease, flavourzyme, papain and acid protease to carry out enzymolysis reaction on the hermetia illucens larva solids respectively, and the enzymolysis conditions are as follows: the ratio of the feed to the water is 1:4-5, the temperature is 50 ℃, the time is 5 hours, the natural pH value is high, and the addition amount of protease is 3%. And after the enzymolysis is finished, inactivating enzyme for 10 minutes at 80-90 ℃, and measuring the hydrolysis degree and the polypeptide yield of an enzymolysis product, wherein the result is shown in figure 2. From the results of FIG. 2, trypsin, alkaline protease and flavourzyme have a higher degree of hydrolysis than neutral protease, papain and acid protease, wherein the degree of hydrolysis of alkaline protease is highest, 33.52%, followed by flavourzyme, and the degree of hydrolysis is 33.22%; the yield of the polypeptide of the hydrolyzed alkaline protease group is 27.10 percent which is higher than that of other proteases, and the alkaline protease is the optimal protease preparation of the method in combination with two indexes of hydrolysis degree and polypeptide content.
Test example 2
Based on the test result of test example 1, the influence of the complex enzyme preparation on the hydrolysis degree and the polypeptide yield of the treated hermetia illucens larva solids is further studied.
And (3) selecting composite enzyme preparations with different compositions and proportions to perform enzymolysis reaction on the hermetia illucens larva solids respectively, and determining the hydrolysis degree and the polypeptide yield of the enzymolysis products. The compositions of the different complex enzyme preparations are shown in Table 1. The results of the influence of different complex enzyme preparations on the degree of hydrolysis and the yield of the polypeptide are shown in fig. 3 and 4.
Firstly, researching the influence of different kinds of compound enzyme preparations on the hydrolysis degree of the black soldier fly larva solids and the polypeptide yield, in the test, respectively mixing alkaline protease with neutral protease, trypsin, flavourzyme, papain and acid protease according to the mass ratio of 1:1 to be used as an enzymolysis compound enzyme, respectively adding enzyme preparations 1-5 according to 3% of the weight of the black soldier fly larva solids, carrying out enzymolysis for 5 hours at 50 ℃, inactivating enzyme for 10 minutes at 80-90 ℃ after the enzymolysis is finished, and measuring the hydrolysis degree of an enzymolysis product and the polypeptide yield. The effect of different complex enzyme preparations 1-5 on the hydrolysis degree of the solid matter of the hermetia illucens larva and the yield of the polypeptide are shown in figure 3. From the results of fig. 3, the hydrolysis degree and the polypeptide yield of the enzyme preparation 3 (alkaline protease and flavourzyme) are far higher than those of other combinations, and under the enzymolysis condition, the hydrolysis degree of the soldier fly larvae is 38.29%, and the polypeptide yield is 35.32%, so that the alkaline protease and flavourzyme are selected as the optimal double-enzyme combination.
Based on the above experiments, we have further explored the weight ratio of alkaline protease to flavourzyme, and the weight ratio of alkaline protease to flavourzyme is shown in Table 1. Respectively adding an enzyme preparation into the black soldier fly larvae according to 3% of the solid weight of the black soldier fly larvae, carrying out enzymolysis for 5 hours at 50 ℃, inactivating enzyme for 10 minutes at 80-90 ℃ after the enzymolysis is finished, and measuring the hydrolysis degree and the polypeptide yield of the enzymolysis products. The results of the effect of different complex enzyme preparations 3, 6-10 on the degree of hydrolysis and the yield of polypeptide are shown in FIG. 4. As can be seen from the results of FIG. 4, the hydrolysis degree and polypeptide yield of the hermetia illucens protein by the enzyme preparation 3 and the enzyme preparations 6-8 are above 30%, and the enzyme preparations 9 and 10 are below 30, and the results show that the weight ratio of alkaline protease to flavourzyme provided by the invention is 1-3:1-2, can realize better hydrolysis effect and polypeptide yield of hermetia illucens protein; the hydrolysis degree of the enzyme preparation 3 on the hermetia illucens larva solids is 38.29%, the polypeptide yield is 35.32%, and the polypeptide yield is far higher than that of other enzyme preparations, which shows that the enzymolysis effect on the hermetia illucens larva is best when the weight ratio of alkaline protease to flavourzyme is 1:1.
TABLE 1 composition and weight ratio relationship of different Complex enzyme preparations
Test example 3
Based on the results of test example 2, we have also made further studies on the effects of pH and polypeptide yield after treatment of the solid matter of the hermetia illucens larvae in the composite fermentation broth. Adding an enzyme preparation 3 according to 3% of the solid content of the black soldier fly larvae, carrying out enzymolysis for 5 hours at 50 ℃, inactivating enzyme for 10 minutes at 80-90 ℃ after the enzymolysis is finished, adding 3-4% of glucose and 15% of compound fermentation bacteria liquid according to the liquid content of an enzymolysis product after the enzymolysis is finished, fermenting at 35-37 ℃ for 20 hours, sterilizing for 10 minutes at 80-90 ℃ after the fermentation is finished, and measuring the pH value and the polypeptide yield of the fermentation liquid. Wherein the composite fermentation bacteria liquid is lactobacillus plantarum seed liquid: bacillus subtilis seed liquid: the saccharomyces cerevisiae seed liquid is respectively configured according to the ratio of 1:1:1, 2:2:1, 3:2:1, 4:2:1 and 5:2:1. The results are shown in fig. 5 and 6. When lactobacillus plantarum seed solution: bacillus subtilis seed liquid: saccharomyces cerevisiae seed solutions are = 4:2:1, the pH and the polypeptide yield tend to be stable, the pH value is 5.66, and the polypeptide yield is 38.52%.
Test example 4
The pH and polypeptide yield of the hermetia illucens enzymatic hydrolysis or enzymatic hydrolysis fermentation broth prepared by the two methods of example 2 and comparative example 1 were measured, and the results are shown in FIG. 7. Compared with the simple enzymolysis, the method adopting enzymolysis and composite probiotics fermentation can obviously reduce the pH value of fermentation liquor, improve the polypeptide yield and improve the palatability, and provide conditions for the application of the water-soluble protein powder of hermetia illucens in animal husbandry.
Test example 5
Application of water-soluble protein powder of hermetia illucens prepared by the invention in laying hen production
1 Materials and methods
1.1 Design of experiments
The test selects 405 chicken eggs with good body condition and similar weight and with the age of 58 weeks and randomly divided into 3 groups, wherein 3 repeats are arranged in each group, and 45 chickens are in each repeat. The pre-trial period was 1 week and the positive trial period was 4 weeks. The control group is fed with basic diet, and the test group is respectively added with 0.5% and 1.0% of water-soluble protein powder of hermetia illucens in the basic diet. The diets of each test group were prepared by referring to NY/T33-2004 chicken raising Standard, and the diet compositions are shown in Table 2.
The feeding management conditions of the groups of laying hens are the same, and a closed henhouse and three-layer ladder type cage cultivation are adopted; the nipple type drinking bowl can drink water freely, and is fed twice daily (7:30, 17:30) for free feeding; artificial illumination is adopted, the constant illumination time is 16h/d, and the illumination intensity is 10-20 lx; ventilating longitudinally under negative pressure, wherein the temperature is 20-25 ℃ and the relative humidity is 40-60%; picking up eggs at 2:30 pm every day, cleaning the henhouse once every day, sterilizing the henhouse once every week, cleaning manure once every two days, and performing epidemic prevention according to a conventional epidemic prevention program.
Table 2 test diet composition (air drying basis)
Note that: the premix is provided for each kilogram of diet with vitamin A9800 IU, vitamin D 3 IU, vitamin E28 IU, vitamin K 3 2.4.4 mg, vitamin B 1 1.6.6 mg, vitamin B 2 7.0.0 mg, vitamin B 6 4.5.5 mg, vitamin B 12 0.03.03 mg, biotin 0.18mg, folic acid 1.2mg, nicotinic acid 40.0mg, pantothenic acid 10.0mg, iron 90mg, copper 15mg, manganese 100mg, zinc 75mg, selenium 0.3mg, iodine 0.8mg and phytase 1000IU.
1.2 Measurement of production Properties
During the positive test period, the number of eggs laid, the weight of eggs, the number of soft broken eggs and the number of abnormal eggs in each group (in repeated units) were recorded daily. And (5) carrying out material caking once per week, and calculating the laying rate, daily feed intake, average egg weight and material-egg ratio in the statistical test period.
1.3 Determination of egg quality
Measuring the egg weight, the egg white height, the egg yolk color and the Hash unit by adopting an egg quality automatic analyzer; measuring eggshell strength by using an eggshell strength tester; and (3) after the eggshell membrane is stripped, measuring eggshell thicknesses at three positions of an eggshell tip, an equator and a blunt end by using a vernier caliper, and taking an average value of 3 measurement results as an eggshell thickness final value.
1.4 Evaluation of egg flavor
The evaluation is carried out from the color and luster of the egg white (1-5 min), the flavor (1-5 min), the taste of the egg white (1-5 min) and the taste of the egg yolk (1-5 min), and the comprehensive score (0-9 min) is evaluated.
1.5 Statistics and analysis
Test data were collated with Excel 2016, single-factor analysis of variance (one-way ANOVA) with SPSS 26.0 software, multiple comparison with Duncan's test, P <0.05 indicated significant differences, P <0.01 indicated very significant differences, and results were expressed as (mean ± standard deviation).
2 Results
2.1 Influence of Water-soluble protein powder of hermetia illucens on production performance of laying hens
TABLE 3 Effect of hermetia illucens water-soluble protein powder on laying hen production performance
| Group of | Laying rate/% | Average egg weight/g | Broken egg rate/% | Ratio of feed to egg | Average daily feed intake/g |
| Control group | 89.63±0.36b | 60.02±0.45 | 1.18±0.06A | 2.08±0.04 | 124.47±3.01 |
| 0.5% Group | 90.98±0.43a | 60.16±0.33 | 0.82±0.03B | 2.02±0.02 | 121.37±0.81 |
| 1.0% Group | 91.10±0.68a | 60.27±0.29 | 0.81±0.04B | 2.02±0.04 | 121.50±1.71 |
Note that: the same column of shoulder marks different lowercase letters represent that the difference is significant (P < 0.05), the different uppercase letters represent that the difference is extremely significant (P < 0.01), and the unlabeled letters represent that the difference is not significant (P > 0.05). The table below is the same.
As shown in Table 3, compared with the control group, the egg laying rate of the water-soluble protein powder added with 0.5% and 1.0% of the hermetia illucens in the later daily ration of the laying hen is improved by 1.51% (P < 0.05) and 1.64% (P < 0.05), and the egg breaking rate is reduced by 30.51% (P < 0.01) and 31.36% (P < 0.01), respectively. The test result shows that the water-soluble protein powder of the hermetia illucens can improve the laying rate of the laying hens, greatly reduce the broken egg rate and improve the economic benefit of the laying hen breeding enterprises.
2.2 Influence of the Water-soluble protein powder of hermetia illucens on the quality of eggs of laying hens
TABLE 4 influence of Water-soluble protein powder of hermetia illucens on egg quality of laying hen
As shown in Table 4, the addition of 0.5% and 1.0% of the water-soluble black soldier fly protein powder to the later daily ration of the laying hen increased the eggshell strength and eggshell thickness (P < 0.05) compared with the control group. The eggshell strength and eggshell thickness are increased, thereby reducing the proportion of broken eggs.
2.3 Influence of Water-soluble protein powder of hermetia illucens on egg flavor
TABLE 5 Effect of hermetia illucens water-soluble protein powder on egg flavor
| Group of | Color and luster of protein | Fragrance of Chinese medicine | Taste of protein | Yolk mouthfeel | Comprehensive score |
| Control group | 3.40±0.54 | 3.96±0.32 | 4.20±0.28 | 4.10±0.14 | 7.90±0.26 |
| 0.5% Group | 3.54±0.23 | 4.24±0.18 | 4.30±0.21 | 4.06±0.09 | 8.26±0.23 |
| 1.0% Group | 3.56±0.25 | 4.24±0.29 | 4.30±0.22 | 4.10±0.38 | 8.28±0.15 |
As shown in table 4, the addition of 0.5% and 1.0% of the black soldier fly water-soluble protein powder to the later daily ration of the laying hen can improve the flavor and total score of the eggs, but the differences are not significant.
Conclusion 3
The water-soluble protein powder of the hermetia illucens can obviously improve the production performance and the egg quality of laying hens in the laying hen breeding process, and the thickness and the strength of eggshells can be improved by adding the water-soluble protein powder of the hermetia illucens into the later daily ration of the laying hens, so that the broken egg proportion is reduced, the laying rate is improved, and the problems of the quality of eggshells and the reduction of the egg quality of the laying hens in the later period of a large-scale chicken farm can be effectively solved.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. The preparation method of the hermetia illucens water-soluble protein powder is characterized by comprising the following steps of:
(1) Flushing the living hermetia illucens larvae with flowing water to remove impurities on the surfaces of the larvae;
(2) Steaming cleaned hermetia illucens larva in 80-90deg.C water for 30min, pulping with a refiner, and dehydrating with a centrifuge for 15-20min to obtain solid concentrate of hermetia illucens larva;
(3) Adding 3-5L of degreasing solvent into dehydrated hermetia illucens solid concentrate for degreasing for 3-5 times according to each kilogram to remove crude fat in the concentrate;
(4) Removing degreasing solvent by a centrifuge and recovering grease to obtain defatted hermetia illucens larva solids and grease;
(5) Mixing defatted hermetia illucens larva solid with 4-5L of water per kg, adding a compound enzyme preparation according to 3-5% of the solid weight, and performing enzymolysis reaction at 40-50deg.C for 4-5h; obtaining an enzymolysis product;
(6) Adding 3-4% glucose and 20-30% composite fermentation bacteria liquid according to the liquid amount of the fermentation product after the enzymolysis reaction, performing fermentation reaction at 35-37 ℃ for 20-24 hours, and sterilizing at 85-90 ℃ for 10-15 minutes after the fermentation; obtaining an enzymolysis fermentation product;
(7) And centrifuging the enzymolysis and fermentation product, collecting supernatant, and performing spray drying to obtain the hermetia illucens water-soluble protein powder.
2. The method for preparing the water-soluble protein powder of hermetia illucens as claimed in claim 1, wherein in the step (3), the degreasing solvent is one of n-hexane, petroleum ether and ethanol.
3. The method for preparing the water-soluble protein powder of hermetia illucens according to claim 1, wherein in the step (5), the complex enzyme preparation is one or more of neutral protease, trypsin, alkaline protease, flavourzyme, papain and acid protease.
4. The preparation method of the hermetia illucens water-soluble protein powder according to claim 1, wherein in the step (5), the compound enzyme preparation is alkaline protease and flavourzyme with a mass ratio of 1-3:1-2.
5. The preparation method of the hermetia illucens water-soluble protein powder according to claim 1, wherein in the step (5), the addition amount of the compound enzyme preparation is 3-5% of the weight of solids.
6. The preparation method of the hermetia illucens water-soluble protein powder according to claim 1, wherein in the step (6), the composite fermentation bacteria liquid is lactobacillus plantarum seed liquid with a volume ratio of 4-8:2-4:1-2: bacillus subtilis seed liquid: the saccharomyces cerevisiae seed liquid consists of saccharomyces cerevisiae seed liquid.
7. The preparation method of the hermetia illucens water-soluble protein powder according to claim 1, wherein in the step (6), the addition amount of the composite fermentation broth is 15-20% of the liquid amount of a fermentation product.
8. The water-soluble protein powder of hermetia illucens prepared by the method of claim 1 is used for preparing functional feed raw materials for laying hens.
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