Enzymolysis and fermentation combined method for optimizing nutritional composition of hermetia illucens powder and application

Abstract

The invention belongs to the technical field of biology, particularly relates to an enzymolysis and fermentation combined method for optimizing nutritional composition of hermetia illucens powder and application, and aims to solve the problems that existing hermetia illucens powder is high in fat content, saturated fatty acid is mainly used in fatty acid composition, in addition, the content of chitin in the hermetia illucens powder is high, and the nutritional composition of the hermetia illucens powder is poor. In order to solve the problem of low digestion and absorption efficiency of nutrient substances such as protein in the prior art, the invention provides the following scheme: the method comprises the following steps: S1, degreasing hermetia illucens powder by using an organic solvent extraction method, the mass volume ratio of the hermetia illucens powder to an organic solvent is (10-35): 90, the pressure is 15-30MPa, each time lasts for 3-5 hours, and the degreasing is performed for 2-3 times to obtain degreased hermetia illucens powder; when the degreased hermetia illucens powder treated by the method is used in largemouth bass feed, the growth performance, the feed efficiency and the immune performance of fishes are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder and its application. Background Art

[0002] The black soldier fly (Hermetia illucens), belonging to Diptera insects, has the advantages of large biomass, rich nutrient content, and low price. In recent years, black soldier fly powder has been regarded as a new protein source with great potential and plays an important role in replacing fish meal in aquatic feed. However, compared with high-quality protein sources such as fish meal, the black soldier fly powder has a high oil content and an unbalanced fatty acid composition, especially mainly saturated fatty acids, and also has a high chitin content. These nutritional characteristics result in poor palatability, unbalanced nutritional composition, and low digestion, absorption, and utilization efficiency of the black soldier fly powder, severely restricting its wide application in the industry. Our previous research found that replacing more than 15% of fish meal with black soldier fly powder in carnivorous fish will significantly reduce the body growth performance and feed efficiency. Analyzing the nutritional composition of black soldier fly powder, it is found that the black soldier fly powder has a high fat content and its fatty acid composition is mainly saturated fatty acids. In addition, the black soldier fly powder has a high chitin content, resulting in low digestion and absorption efficiency of nutrients such as protein. Therefore, there is an urgent need to develop a new technology method that can optimize the nutritional composition of black soldier fly powder, improve the palatability and nutritional value of black soldier fly powder, and improve the utilization efficiency of nutrients in black soldier fly powder to make it highly utilized in aquatic feed. Summary of the Invention

[0003] The purpose of the present invention is to develop an enzymatic hydrolysis and fermentation combined technology for optimizing the nutritional composition of black soldier fly powder, which can effectively improve the palatability and nutritional composition of black soldier fly powder, achieve the purpose of improving the utilization efficiency of nutrients in black soldier fly powder and enabling it to highly replace fish meal or soybean meal in aquatic feed, thereby making up for the deficiencies of the existing technology.

[0004] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0005] An enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder, comprising the following steps:

[0006] S1: Degrease the black soldier fly powder by using the method of organic solvent extraction, wherein the mass-volume ratio of the insect powder to the organic solvent is 10 - 35:90, the pressure is 15 - 30 MPa, each time for 3 - 5 h, and perform 2 - 3 times to obtain degreased black soldier fly powder;

[0007] S2: Add 2.5 - 6.0 enzyme units / g of the material of complex enzyme to the degreased black soldier fly powder material, enzymatically hydrolyze at 40 - 42 °C and pH 7.0 for 3 - 6 h. During this period, the material should be turned every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by reducing the temperature;

[0008] S3: Add the material with enzymatic hydrolysis terminated with 1.0 - 3.0% by mass of the mixed strains in the logarithmic growth phase, ferment at 32 - 35 °C and pH 7.0 for 5 - 7 d. During this period, the material should be turned every 6 h to ensure the ventilation volume.

[0009] Preferably, in S2, the complex enzyme includes bromelain, papain and chitinase.

[0010] Preferably, the ratio of bromelain, papain and chitinase is 4:2:7.

[0011] Preferably, in S3, the mixed strains include: Bacillus, Saccharomyces cerevisiae and Mortierella alpina.

[0012] Preferably, the ratio of Bacillus: Saccharomyces cerevisiae: Mortierella alpina = 1.2:1.0:2.5.

[0013] Preferably, in S1, the defatting of black soldier fly powder is carried out by using the method of organic solvent extraction, where the mass - volume ratio of the powder to the organic solvent is 15 - 30:90, the pressure is 15 - 25 MPa, each time for 4 - 5 h, and it is carried out 2 - 3 times to obtain defatted black soldier fly powder.

[0014] Preferably, in S2, 3 - 6.0 enzyme units / g of the material of the complex enzyme is added to the defatted black soldier fly powder material, and enzymatic hydrolysis is carried out at 40 - 41 °C and pH 7.0 for 3 - 5 h. During this period, the material should be turned every half hour. After the enzymatic hydrolysis time is up, the enzyme is inactivated by reducing the temperature.

[0015] Preferably, in S3, the material with enzymatic hydrolysis terminated is added with 2.0 - 3.0% by mass of the mixed strains in the logarithmic growth phase, ferment at 33 - 35 °C and pH 7.0 for 6 - 7 d. During this period, the material should be turned every 6 h to ensure the ventilation volume.

[0016] Preferably, in S3, the material with enzymatic hydrolysis terminated is added with 3.0% by mass of the mixed strains in the logarithmic growth phase, ferment at 35 °C and pH 7.0 for 7 d. During this period, the material should be turned every 6 h to ensure the ventilation volume.

[0017] Application of the combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder: After analyzing the nutritional composition of the defatted black soldier fly powder, it is experimentally evaluated in aquatic animal feed.

[0018] Mortierella alpina:

[0019] Mortierella alpina is a strain capable of fermenting and producing polyunsaturated fatty acids (PUFAs). By optimizing the fermentation conditions for PUFAs production by the strain through single-factor experiments and orthogonal experiments, the oil yield and biomass can be increased, providing a theoretical basis for the industrial production of PUFAs.

[0020] The main components of fatty acids in black soldier fly oil are saturated fatty acids, mainly lauric acid (C12:0) and palmitic acid (C16:0), and their total sum accounts for about 80% of the saturated fatty acids. The polyunsaturated fatty acids in black soldier fly oil are mainly linoleic acid (C18:2n-6). In addition, the content of n-3 series polyunsaturated fatty acids in black soldier fly oil is relatively low, basically within 4% of the total fatty acid content.

[0021] In solid-state fermentation (SSF), Mortierella alpina can bioconvert hydrophobic substrates such as animal fat by-products into high-value fermentation bioproducts rich in arachidonic acid (ARA).

[0022] Compared with untreated cottonseed protein, the cottonseed protein treated by the present invention has the following advantages:

[0023] 1. The defatted black soldier fly powder treated by the present invention increases the contents of amino acids such as lysine and methionine;

[0024] 2. The defatted black soldier fly powder treated by the present invention significantly increases the content of unsaturated fatty acids;

[0025] 3. The defatted black soldier fly powder treated by the present invention significantly increases the apparent digestibility;

[0026] 4. The defatted black soldier fly powder treated by the present invention significantly improves the growth performance, feed efficiency and immune performance of fish when used in the feed of largemouth bass. Specific Embodiments

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0028] Example 1

[0029] An enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder includes the following steps:

[0030] S1: The black soldier fly powder is defatted by using an organic solvent extraction method, where the mass-volume ratio of the powder to the organic solvent is 10:90, the pressure is 15 MPa, for 3 h each time, and carried out 2 times to obtain defatted black soldier fly powder;

[0031] S2: Add a compound enzyme with 2.5 enzyme units / g of material to the defatted black soldier fly powder material, and carry out enzymatic hydrolysis at 40 °C and pH 7.0 for 3 h. During this period, the material should be stirred every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by reducing the temperature; the compound enzyme includes bromelain, papain, and chitinase; the ratio of bromelain, papain, and chitinase is 4:2:7;

[0032] S3: Add 1.0% by mass fraction of a mixed bacterial strain in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, and ferment at 32 °C and pH 7.0 for 5 d. During this period, the material should be stirred every 6 h to ensure the ventilation volume; the mixed bacterial strain includes: Bacillus, yeast, and Mortierella alpina, and the ratio of Bacillus: yeast: Mortierella alpina = 1.2:1.0:2.5.

[0033] Example 2

[0034] A combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder, comprising the following steps:

[0035] S1: Use the method of organic solvent extraction to defat the black soldier fly powder, where the mass-to-volume ratio of the insect powder to the organic solvent is 35:90, the pressure is 30 MPa, for 5 h each time, and carry out 3 times to obtain defatted black soldier fly powder;

[0036] S2: Add a compound enzyme with 6.0 enzyme units / g of material to the defatted black soldier fly powder material, and carry out enzymatic hydrolysis at 42 °C and pH 7.0 for 6 h. During this period, the material should be stirred every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by reducing the temperature; the compound enzyme includes bromelain, papain, and chitinase; the ratio of bromelain, papain, and chitinase is 4:2:7;

[0037] S3: Add 3.0% by mass fraction of a mixed bacterial strain in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, and ferment at 35 °C and pH 7.0 for 7 d. During this period, the material should be stirred every 6 h to ensure the ventilation volume; the mixed bacterial strain includes: Bacillus, yeast, and Mortierella alpina, and the ratio of Bacillus: yeast: Mortierella alpina = 1.2:1.0:2.5.

[0038] Example 3

[0039] A combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder, comprising the following steps:

[0040] S1: Use the method of organic solvent extraction to defat the black soldier fly powder, where the mass-to-volume ratio of the insect powder to the organic solvent is 15:90, the pressure is 15 MPa, for 4 h each time, and carry out 2 times to obtain defatted black soldier fly powder;

[0041] S2: Add a complex enzyme of 3 enzyme units / g of material to the defatted black soldier fly powder material, enzymatically hydrolyze it at 40 °C and pH 7.0 for 3 h. During this period, the material should be stirred every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by lowering the temperature. The complex enzyme includes bromelain, papain, and chitinase; the ratio of bromelain, papain, and chitinase is 4:2:7;

[0042] S3: Add 2.0% by mass fraction of a mixed strain in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, ferment at 33 °C and pH 7.0 for 6 d. During this period, the material should be stirred every 6 h to ensure the ventilation volume. The mixed strain includes: Bacillus, yeast, and Mortierella alpina, and the ratio of Bacillus: yeast: Mortierella alpina = 1.2:1.0:2.5.

[0043] Example 4

[0044] An enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder, comprising the following steps:

[0045] S1: Use the method of organic solvent extraction to defat the black soldier fly powder, where the mass-volume ratio of the powder to the organic solvent is 30:90, the pressure is 25 MPa, each time for 5 h, and perform it 3 times to obtain defatted black soldier fly powder;

[0046] S2: Add a complex enzyme of 6.0 enzyme units / g of material to the defatted black soldier fly powder material, enzymatically hydrolyze it at 41 °C and pH 7.0 for 5 h. During this period, the material should be stirred every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by lowering the temperature. The complex enzyme includes bromelain, papain, and chitinase; the ratio of bromelain, papain, and chitinase is 4:2:7;

[0047] S3: Add 3.0% by mass fraction of a mixed strain in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, ferment at 35 °C and pH 7.0 for 7 d. During this period, the material should be stirred every 6 h to ensure the ventilation volume. The mixed strain includes: Bacillus, yeast, and Mortierella alpina, and the ratio of Bacillus: yeast: Mortierella alpina = 1.2:1.0:2.5.

[0048] Example 5

[0049] A new technical method provided by the present invention can optimize the nutritional composition of black soldier fly powder, improve the palatability and nutritional value of black soldier fly powder. Add the composite enzyme of the present invention (bromelain: papain: chitinase = 4:2:7) with 4.50 enzyme units / g of material to the obtained defatted black soldier fly powder material, enzymatically hydrolyze at 40 - 42 °C and pH 7.0 for 4.5 h. During this period, the material should be stirred every half hour. After the enzymolysis time, inactivate the enzyme by reducing the temperature; then add 2.2% mass fraction of the mixed bacteria in the logarithmic growth phase (Bacillus: Saccharomyces cerevisiae: Mortierella alpina = 1.2:1.0:2.5) to the material after terminating the enzymolysis, ferment at 32 - 35 °C and pH 7.0 for 5 d. During this period, the material should be stirred every 6 h to ensure the ventilation volume. Dry the raw material after enzymatic hydrolysis and fermentation at 45 °C, and respectively detect the nutritional composition and apparent digestibility of the untreated defatted black soldier fly powder (negative control), the defatted black soldier fly powder only treated with enzymatic hydrolysis (enzymolysis group), the defatted black soldier fly powder only treated with fermentation (fermentation group), and the defatted black soldier fly powder treated by the method of the present invention (the present invention).

[0050] On this basis, a fish feeding experiment was carried out to verify the utilization efficiency of the defatted black soldier fly powder treated by the method of the present invention in feed. In the basal diet (positive control group), use the defatted black soldier fly powder treated by the method of the present invention (the present invention group), the defatted black soldier fly powder only treated with enzymatic hydrolysis (enzymolysis group), and the defatted black soldier fly powder only treated with fermentation (fermentation group) to replace 40% of fish meal respectively. Select juvenile fish with good vitality, bright color and regular specifications (average specification: 20.0 ± 0.12 g), and feed twice a day (7:00 and 18:00). The experiment has 4 treatment groups, with 4 parallels in each group, and 60 fry are placed in each net cage, and a comparative experiment is carried out for 8 weeks. After the breeding experiment is over, collect tissues such as whole fish and plasma for subsequent analysis. After 8 weeks of the experiment, a challenge experiment is carried out, acutely infect Vibrio harveyi, and calculate the survival rate based on the number of deaths 7 days after the challenge.

[0051] The test results are as follows:

[0052] (1) Differences in the conventional nutritional composition of the defatted black soldier fly powder (un)treated by the method of the present invention

[0053] Table 1: Conventional nutritional composition (% dry matter)

[0054]

[0055] The results show that the present invention can increase the protein content of the defatted black soldier fly powder, indicating that the present invention can effectively optimize the nutritional composition of the defatted black soldier fly powder.

[0056] Table 2: Amino acid composition (% dry matter)

[0057]

[0058]

[0059] The results show that the present invention can increase the contents of lysine and methionine in defatted black soldier fly powder, indicating that the present invention can effectively optimize the amino acid nutritional composition of defatted black soldier fly powder.

[0060] Table 3: Fatty acid composition (% dry matter)

[0061]

[0062] The results show that the present invention can increase the content of unsaturated fatty acids in defatted black soldier fly powder, indicating that the present invention can effectively optimize the fatty acid nutritional composition of defatted black soldier fly powder.

[0063] (2) Table 4: Apparent digestibility of defatted black soldier fly powder (not) treated by the method of the present invention

[0064]

[0065]

[0066] The results show that the present invention can increase the apparent digestibility of dry matter, crude protein and crude fat in defatted black soldier fly powder, indicating that the present invention can effectively improve the utilization efficiency of the nutrition of defatted black soldier fly powder.

[0067] (3) Differences in growth performance and feed utilization of largemouth bass when defatted black soldier fly powder (not) treated by the method of the present invention replaces fish meal

[0068] Table 5: Growth performance and feed efficiency of largemouth bass after cultivation

[0069]

[0070] The results show that compared with the enzymatic hydrolysis group and the fermentation group, the present invention can significantly increase the indexes for evaluating growth performance of farmed fish, including final weight, weight gain rate and specific growth rate, and can also significantly increase the feed efficiency and carcass ratio, indicating that the present invention has greater potential to replace fish meal in the feed of largemouth bass.

[0071] Table 6: Whole fish nutritional composition of largemouth bass after cultivation (% dry matter)

[0072]

[0073] The results showed that, compared with the enzymatic hydrolysis group and the fermentation group, the present invention could significantly increase the deposition of nutrients such as protein and fat in cultured fish, and there was no significant difference in the protein deposition amount compared with the positive control, indicating that the present invention could improve the deposition efficiency of nutrients in largemouth bass.

[0074] (4) Differences in the immune performance of largemouth bass with defatted black soldier fly larvae powder (un)treated by the method of the present invention replacing fish meal:

[0075] Table VII: Survival rate after Vibrio harveyi infection

[0076]

[0077] The results showed that, compared with the enzymatic hydrolysis group and the fermentation group, the present invention could significantly increase the survival rate of fish after Vibrio harveyi infection, indicating that the present invention could significantly improve the disease resistance of largemouth bass.

[0078] Table VIII: Changes in immune-related indexes in plasma after Vibrio harveyi infection

[0079]

[0080] The results showed that, compared with the enzymatic hydrolysis group and the fermentation group, the present invention could significantly reduce the contents of globulin, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase in plasma, indicating that the present invention could effectively improve the body immunity of largemouth bass, form a defense barrier to protect the host from pathogen invasion, improve the immune system, and enhance the disease resistance.

[0081] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. An enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder, characterized in that, It includes the following steps: S1: Defat the black soldier fly larvae powder by using the method of organic solvent extraction, where the mass-volume ratio of the larvae powder to the organic solvent is 10 - 35:90, the pressure is 15 - 30 MPa, for 3 - 5 hours each time, and perform 2 - 3 times to obtain defatted black soldier fly larvae powder; S2: Add a complex enzyme of 2.5 - 6.0 enzyme units / g of the material to the defatted black soldier fly larvae powder material, and carry out enzymatic hydrolysis at 40 - 42 °C and pH 7.0 for 3 - 6 hours. During this period, the material should be turned every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by reducing the temperature; S3: Add a mixed strain with a mass fraction of 1.0 - 3.0% in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, and ferment at 32 - 35 °C and pH 7.0 for 5 - 7 days. During this period, the material should be turned every 6 hours to ensure the ventilation volume.

2. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 1, wherein In the above S2, the complex enzyme includes bromelain, papain, and chitinase.

3. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 2, wherein The ratio of bromelain, papain, and chitinase is 4:2:

7.

4. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 1, characterized in that, In the above S3, the mixed strain includes: Bacillus, Saccharomyces cerevisiae, and Mortierella alpina.

5. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 4, characterized in that The ratio of Bacillus:Saccharomyces cerevisiae:Mortierella alpina = 1.2:1.0:2.

5.

6. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 1, wherein In the above S1, defat the black soldier fly larvae powder by using the method of organic solvent extraction, where the mass-volume ratio of the larvae powder to the organic solvent is 15 - 30:90, the pressure is 15 - 25 MPa, for 4 - 5 hours each time, and perform 2 - 3 times to obtain defatted black soldier fly larvae powder.

7. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 1, characterized in that, In the above S2, add a complex enzyme of 3 - 6.0 enzyme units / g of the material to the defatted black soldier fly larvae powder material, and carry out enzymatic hydrolysis at 40 - 41 °C and pH 7.0 for 3 - 5 hours. During this period, the material should be turned every half hour. After the enzymatic hydrolysis time is up, inactivate the enzyme by reducing the temperature.

8. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 1, characterized in that, In the above S3, add a mixed strain with a mass fraction of 2.0 - 3.0% in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, and ferment at 33 - 35 °C and pH 7.0 for 6 - 7 days. During this period, the material should be turned every 6 hours to ensure the ventilation volume.

9. The enzymatic hydrolysis and fermentation combined method for optimizing the nutritional composition of black soldier fly powder according to claim 8, characterized in that, In the above S3, add a mixed strain with a mass fraction of 3.0% in the logarithmic growth phase to the material after terminating the enzymatic hydrolysis, and ferment at 35 °C and pH 7.0 for 7 days. During this period, the material should be turned every 6 hours to ensure the ventilation volume.

10. Application of the combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder, wherein the combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder is the combined enzymatic hydrolysis and fermentation method for optimizing the nutritional composition of black soldier fly powder according to any one of claims 1-9, characterized in that, Conduct a nutritional composition analysis on the defatted black soldier fly larvae powder and then conduct an experimental evaluation in aquatic animal feed.

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