Method for separating protein, grease and chitin from hermetia illucens larvae and application

Abstract

The invention belongs to the technical field of extraction and separation, and relates to a method for separating protein, grease and chitin of hermetia illucens larvae and application. The invention provides a method for separating protein, grease and chitin from hermetia illucens larvae, which comprises the following steps: mixing monobasic organic acid, binary organic acid, ternary organic acid and an alcohol compound, and heating in a water bath to obtain an ionic solution; the hermetia illucens larvae are smashed, dried and then placed in the ionic solution, the mixture is divided into three layers after being heated in a water bath, the upper-layer substance is grease, the middle-layer substance is the ionic solution containing protein, and the lower-layer substance is chitin and inorganic salt; the upper-layer substance, the middle-layer substance and the lower-layer substance are separated, the upper-layer substance is grease, the middle-layer substance is taken and separated to obtain protein, and the lower-layer substance is taken and inorganic salt is removed to obtain chitin. According to the separation method provided by the invention, the use of toxic chemical solvents is avoided, so that the hermetia illucens larvae are fully recycled.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and separation, and relates to a method for separating protein, oil and chitin from black soldier fly larvae and application thereof. Background Art

[0002] The black soldier fly (Hermetia illucensL) is an edible resource insect. Its larvae are rich in active proteins, insect fat, probiotics, antimicrobial peptides and chitin. They can be used as staple food or feed supplements for animals to help digestion, promote growth, enhance immunity and make the meat delicious. The black soldier fly larvae are saprophagous, have a varied diet, a large appetite, strong resistance to adversity, overlapping life histories and great flexibility, high nutritional value of prepupae, and have migration characteristics before pupation. The black soldier fly is the industry development direction of organic waste treatment. It has unique advantages in treating urban food waste, so a large number of black soldier fly larvae are produced, which need to be further recycled.

[0003] The existing industry separation and extraction scheme for the components of black soldier fly larvae is mainly to extract the dried insect powder with n-hexane, dissolve the oil in n-hexane, and obtain crude insect oil after distillation and recovery of n-hexane. The protein and insect shell, inorganic salts and other substances insoluble in n-hexane in the insect become a crude protein mixture. CN118027247A discloses a method for extracting chitin from black soldier fly larvae, which uses dichloromethane and petroleum ether as organic solvents to extract the oil from the insect powder to reduce the oil content in the insect powder, remove the oil in the insect powder, and then extract chitin. CN117486967A discloses a method for ultrasonic-assisted alkaline extraction and separation of black soldier fly larvae protein, which uses an ultrasonic cell disruptor combined with an alkaline method to extract protein. Although the above methods have well achieved the extraction of protein, oil and chitin from black soldier fly larvae, the above methods cannot achieve the simultaneous separation of protein, oil and chitin in black soldier fly larvae. Some other extraction methods involve the use of toxic organic solvents during extraction, which is not conducive to the subsequent conversion of resource recovery products; some extraction methods use solid-solid separation, which has poor separation efficiency, or involve large and expensive instruments. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the above-mentioned technical problems. By using it, the protein, oil and chitin in the black soldier fly larvae can be separated synchronously in a simple and low-cost manner using a non-toxic organic solvent. To this end, the present invention provides a method for separating protein, oil and chitin in black soldier fly larvae and its application to solve this need in the art.

[0005] On the one hand, the present invention relates to a method for separating protein, oil and chitin of black soldier fly larvae, which comprises: mixing a monobasic organic acid, a dibasic organic acid, a tribasic organic acid and an alcohol compound, and heating in a water bath to obtain an ion solution;

[0006] The black soldier fly larvae are crushed and dried, and then placed in the ion solution, and then heated in a water bath to separate into three layers, wherein the upper layer is oil, the middle layer is an ion solution containing protein, and the lower layer is chitin and inorganic salts;

[0007] The upper layer material, the middle layer material and the lower layer material are separated, the upper layer material is oil, the middle layer material is separated to obtain protein, and the lower layer material is removed of inorganic salts to obtain chitosan.

[0008] Furthermore, in the method for separating black soldier fly larvae protein, oil and chitin provided by the present invention, the monobasic organic acid is selected from acetic acid, propionic acid or benzoic acid, the dibasic organic acid is selected from oxalic acid, malic acid or tartaric acid, the tribasic organic acid is selected from citric acid, isophthalic acid or cyclohexanetrilic acid, and the alcohol compound is selected from ethanol, ethylene glycol, diethylene glycol or glycerol.

[0009] Furthermore, in the method for separating black soldier fly larvae protein, oil and chitin provided by the present invention, the molar ratio of the monobasic organic acid, dibasic organic acid, tribasic organic acid and alcohol compound is 1:1-10:1-2:6-10.

[0010] Furthermore, in the method for separating protein, oil and chitin from black soldier fly larvae provided by the present invention, the mass ratio of the crushed and dried black soldier fly larvae to the ion solution is 1:3-10.

[0011] Furthermore, in the method for separating protein, oil and chitin from black soldier fly larvae provided by the present invention, the prepared ion solution is heated in a water bath to reach the eutectic temperature.

[0012] Furthermore, in the method for separating protein, oil and chitin from black soldier fly larvae provided by the present invention, the black soldier fly larvae are crushed and dried and then mixed with the ion solution, which is then heated in a water bath to fully dissolve the protein and then centrifuged to separate into three layers.

[0013] Furthermore, in the method for separating black soldier fly larvae protein, oil and chitin provided by the present invention, the centrifugal conditions are 3000-20000 rpm, 10-30 min.

[0014] Furthermore, in the method for separating black soldier fly larvae protein, oil and chitin provided by the present invention, after the middle layer material is mixed with ethanol, the ionic solution and ethanol form a homogeneous phase, the protein is precipitated in the ethanol system, the protein is cooled and precipitated, and then centrifuged to obtain the protein, and the remaining liquid is evaporated to separate the ethanol and the ionic solution for recycling.

[0015] Furthermore, in the method for separating protein, oil and chitosan from black soldier fly larvae provided by the present invention, the lower layer material is separated by filtration, and the lower layer material is washed with ethanol to remove inorganic salts and separate chitosan.

[0016] On the other hand, the present invention relates to the application of the method for separating protein, oil and chitin of black soldier fly larvae in resource recovery of black soldier fly larvae.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0018] (1) The present invention provides a method for separating protein, oil and chitosan from black soldier fly larvae. The method adopts a method of preparing an ion solution by mixing a monobasic organic acid, a dibasic organic acid, a tribasic organic acid and an alcohol compound, thereby efficiently separating protein, oil and chitosan from the black soldier fly larvae, thereby achieving full resource recovery and utilization of the black soldier fly larvae.

[0019] (2) In the process of material separation and extraction, it is crucial to choose a suitable solvent. The melting point of the solvent is an important physical property that directly affects the temperature range of the solvent. For ionic liquids, their melting points are usually much lower than traditional organic solvents or water, which allows ionic liquids to remain liquid at low temperatures, thus having a wider temperature range of use. Specifically in material separation, the role of the low melting point of ionic liquids is mainly reflected in the following aspects: reducing the operating temperature: Due to the low melting point of ionic liquids, material separation operations can be carried out at lower temperatures, which helps to reduce the impact of high temperature on the structure and activity of the material to be separated and protect the integrity of the material to be separated; improving solubility: ionic liquids generally have good solubility for many organic and inorganic compounds, including proteins in black soldier fly larvae. At a lower melting point, ionic liquids may exhibit better solubility, thereby more effectively dissolving and separating proteins; reducing energy consumption: operating at a lower temperature can significantly reduce energy consumption. This is particularly important for large-scale industrial production, because the reduction in energy consumption can be directly converted into a reduction in production costs; broadening the scope of application: the low melting point of ionic liquids allows them to be used in a wider temperature range, thereby broadening the scope of application of ionic liquids in protein separation and other fields. The present invention selects monobasic organic acids, dibasic organic acids, ternary organic acids and alcohol compounds to mix and prepare ionic solutions, wherein the monobasic organic acids, dibasic organic acids, ternary organic acids and alcohol compounds can not only serve as ion donors, but also as ion receptors, and through appropriate component selection, they have a lower melting point, suitable viscosity and higher solubility in proteins.

[0020] (3) The method provided by the present invention uses an ionic solution formed by a natural eutectic compound, all of which are natural products, low in price, and readily available raw materials, avoiding the use of n-hexane solvent, and is environmentally friendly. The scheme has high separation efficiency, simple operation steps, and can separate three products in one step, which has broad industrial application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is the chitosan product obtained by separation.

[0023] Figure 2 It is the oil and fat product obtained by separation.

[0024] Figure 3It is the protein powder product obtained by separation. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention is described below in conjunction with the examples, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials are available on the market unless otherwise specified, and the % in the following examples are all mass percentages unless otherwise specified.

[0026] Example 1

[0027] This embodiment provides a method for separating protein, oil and chitin from black soldier fly larvae.

[0028] By selecting different compounds and their proportions, ionic solutions with different characteristics such as melting point, polarity, viscosity, etc., experiments have shown that they have different solubility capabilities for black soldier fly insect protein.

[0029] In the experimental batch of black soldier fly larvae, every 50g of larvae contained an average of 4.6-5.3g of protein. When dissolved in 100mL of ionic solution, those that were completely dissolved were considered to have excellent solubility, those with less than 1g of undissolved protein at the bottom were considered to have good solubility, those with a bottom undissolved protein amount between 1g and 2.5g were considered to be medium, and those with a bottom undissolved protein amount greater than 2.5g were considered to have poor solubility.

[0030] Table 1. Properties of ionic solutions composed of different compounds

[0031]

[0032] As shown in Table 1, different compounds have different melting points, viscosities and solubilities of ionic solutions. Among them, alcohol compounds have the greatest contribution to the formation of ionic solutions. Lack of alcohol compounds will result in the inability to form ionic solutions; monobasic organic acids, dibasic organic acids, and tribasic organic acids are indispensable. The lack of any organic acid will lead to a decrease in the melting point and solubility of ionic solutions. Through experiments adjusting different ratios, it can be seen that the increase of monobasic organic acids, dibasic organic acids, and tribasic organic acids will increase the melting point and viscosity of ionic solutions; alcohol compounds can effectively reduce the viscosity and melting point of the solution, but are not conducive to dissolving proteins. The addition of a small amount of tribasic organic acid can significantly improve the solubility of ionic solutions for black soldier fly insect protein, but the addition of a slightly excessive amount of tribasic organic acid will cause the viscosity to rise sharply, which is not conducive to the solubility of black soldier fly insect protein.

[0033] In addition, this example also tests the performance of the ionic solution obtained when the alcohol compound is replaced with other low melting point organic solvents. The test results are shown in Table 2.

[0034] Table 2. Properties of ionic solutions composed of other low melting point organic solvents

[0035] Monobasic organic acid Dibasic organic acid Tribasic organic acid Organic solvents Molar Ratio Melting point(℃) Solubility Acetic acid / Citric Acid Choline chloride 1:1:2 90 medium Acetic acid Malic Acid Citric Acid Choline chloride 1:1:1:2 97 medium

[0036] As shown in Table 2, after the alcohol compound is replaced by choline chloride, the melting point of its ionic solution is higher and the solubility of the black soldier fly insect protein is poorer.

[0037] Example 2

[0038] This embodiment provides a method for the simultaneous separation of protein, oil and chitin from black soldier fly larvae.

[0039] Step 1: Weigh acetic acid, malic acid, citric acid, and ethylene glycol in a molar ratio of 1:6:1:8, with a total mass of 500 g, add them into a round-bottom flask in sequence, place in a water bath at 80°C and stir continuously for 0.5 h to form a transparent liquid, which is the ionic solution.

[0040] Step 2: Dry the black soldier fly insects in an oven at 100°C to constant weight, grind the dried black soldier fly insects into powder using a grinder, and pass through a 40-mesh medical sieve to obtain dry insect powder.

[0041] Step 3: Weigh 100g of dry insect powder and add it to the ionic solution prepared above, react in a water bath at 100℃ for 2h, centrifuge to dissolve the protein in the ionic solution, centrifuge at 10000rpm for 3min to separate the fat and ionic solution, chitin and inorganic salts are insoluble in the ionic solution and precipitate at the bottom. Use a separatory funnel to separate the upper layer of fat and weigh it, filter the lower layer with a 60-mesh medicine sieve, wash the retained chitin with water: ethanol = 1:1 (volume ratio), and wash away some inorganic salts.

[0042] Step 4: Collect the ion solution and add 1000 mL of ethanol. The ion solution and ethanol form a homogeneous phase. The protein precipitates in a large amount of ethanol system. Place it in a refrigerated place at 4°C for precipitation for 2 hours. Centrifuge it at 10,000 rpm for 5 minutes, collect the precipitated protein, and separate the ethanol and ion solution of the mixed solvent by rotary evaporation, and recycle it for reuse.

[0043] The collected chitosan and protein were dried, and the yields of insect protein, oil and chitosan were 90.2%, 95.5% and 92.4% respectively. The ash content in chitosan was 1.2%, and the nitrogen content was 4.4%, meeting the requirements of industrial-grade chitosan. Figures 1 to 3 The protein content and nitrogen content in chitosan were measured by Kjeldahl method, the ash content of chitosan was measured by weight of the residue after burning in a muffle furnace, and the chitosan yield and oil yield were measured by weight after drying.

[0044] Example 3

[0045] This embodiment provides a method for the simultaneous separation of protein, oil and chitin from black soldier fly larvae.

[0046] Step 1: Weigh propionic acid, oxalic acid, tribenzoic acid, and ethanol in a molar ratio of 1:1:1:6 to a total mass of 500 g, add them into a round-bottom flask in sequence, place it in a water bath at 75°C and reflux and stir for 0.5 h to form a transparent liquid, which is the ionic solution.

[0047] Step 2: Dry the black soldier fly insects in an oven at 100°C to constant weight, grind the dried black soldier fly insects into powder using a grinder, and pass through a 40-mesh medical sieve to obtain dry insect powder.

[0048] Step 3: Weigh 100g of dry insect powder and add it to the ionic solution prepared above. React in a water bath at 100℃ for 2h. After centrifugation, part of the protein is dissolved in the ionic solution. After centrifugation at 10000rpm for 3min, the fat and the ionic solution are separated. Chitosan and inorganic salts are insoluble in the ionic solution and precipitate at the bottom. Use a separatory funnel to separate the upper fat and weigh it. Use a 60-mesh medicine sieve to filter the reactant in the lower layer. The retained chitosan is washed with water: ethanol = 1:1 (volume ratio), and part of the inorganic salt is washed away.

[0049] Step 4: Collect the ion solution and add 1000 mL of ethanol. The ion solution and ethanol form a homogeneous phase. The protein precipitates in a large amount of ethanol system. Place it in a refrigerated place at 4°C for precipitation for 2 hours. Centrifuge it at 10,000 rpm for 5 minutes, collect the precipitated protein, and separate the ethanol and ion solution of the mixed solvent by rotary evaporation, and recycle it for reuse.

[0050] The collected chitosan and protein were dried, and the yields of insect protein, oil and chitosan were 3.6%, 92.0% and 88.1% respectively, the ash content in chitosan was 3.3%, and the nitrogen content was 6.9%. The protein content and the nitrogen content in chitosan were measured by Kjeldahl nitrogen determination method, the ash content of chitosan was measured by weight of the residue after burning in a muffle furnace, and the chitosan yield and oil yield were measured by weight after drying.

[0051] Example 4

[0052] This embodiment provides a method for the simultaneous separation of protein, oil and chitin from black soldier fly larvae.

[0053] Step 1: Weigh benzoic acid, tartaric acid, cyclohexanetricarboxylic acid, and diethylene glycol in a molar ratio of 1:10:2:10, with a total mass of 500 g, add them into a round-bottom flask in sequence, place it in a water bath at 90°C and reflux and stir for 0.5 h to form a transparent liquid, which is the ionic solution.

[0054] Step 2: Dry the black soldier fly insects in an oven at 100°C to constant weight, grind the dried black soldier fly insects into powder using a grinder, and pass through a 40-mesh medical sieve to obtain dry insect powder.

[0055] Step 3: Weigh 100g of dry insect powder and add it to the ionic solution prepared above. React in a water bath at 100℃ for 2h. After centrifugation, part of the protein is dissolved in the ionic solution. After centrifugation at 10000rpm for 3min, the fat and the ionic solution are separated. Chitosan and inorganic salts are insoluble in the ionic solution and precipitate at the bottom. Use a separatory funnel to separate the upper fat and weigh it. Use a 60-mesh medicine sieve to filter the reactant in the lower layer. The retained chitosan is washed with water: ethanol = 1:1 (volume ratio), and part of the inorganic salt is washed away.

[0056] Step 4: Collect the ion solution and add 1000 mL of ethanol. The ion solution and ethanol form a homogeneous phase. The protein precipitates in a large amount of ethanol system. Place it in a refrigerated place at 4°C for precipitation for 2 hours. Centrifuge it at 10,000 rpm for 5 minutes, collect the precipitated protein, and separate the ethanol and ion solution of the mixed solvent by rotary evaporation, and recycle it for reuse.

[0057] The collected chitosan and protein were dried, and the yields of insect protein, oil and chitosan were 19.7%, 76.2% and 85.5% respectively. The ash content in chitosan was 2.9% and the nitrogen content was 6.2%. The protein content and the nitrogen content in chitosan were measured by Kjeldahl nitrogen determination method, the ash content of chitosan was measured by weight of the residue after burning in a muffle furnace, and the chitosan yield and oil yield were measured by weight after drying.

[0058] Example 5

[0059] This embodiment provides a method for the simultaneous separation of protein, oil and chitin from black soldier fly larvae.

[0060] Step 1: Weigh acetic acid, malic acid, citric acid, and glycerol in a molar ratio of 1:4:1:8 to a total mass of 500 g, add them into a round-bottom flask in sequence, place it in a water bath at 100°C and reflux and stir for 0.5 h to form a transparent liquid, which is the ionic solution.

[0061] Step 2: Dry the black soldier fly insects in an oven at 100°C to constant weight, grind the dried black soldier fly insects into powder using a grinder, and pass through a 40-mesh medical sieve to obtain dry insect powder.

[0062] Step 3: Weigh 100g of dry insect powder and add it to the ionic solution prepared above. React in a water bath at 100℃ for 2h. After centrifugation, part of the protein is dissolved in the ionic solution. After centrifugation at 10000rpm for 3min, the fat and the ionic solution are separated. Chitosan and inorganic salts are insoluble in the ionic solution and precipitate at the bottom. Use a separatory funnel to separate the upper fat and weigh it. Use a 60-mesh medicine sieve to filter the reactant in the lower layer. The retained chitosan is washed with water: ethanol = 1:1 (volume ratio), and part of the inorganic salt is washed away.

[0063] Step 4: Collect the ion solution and add 1000 mL of ethanol. The ion solution and ethanol form a homogeneous phase. The protein precipitates in a large amount of ethanol system. Place it in a refrigerated place at 4°C for precipitation for 2 hours. Centrifuge it at 10,000 rpm for 5 minutes, collect the precipitated protein, and separate the ethanol and ion solution of the mixed solvent by rotary evaporation, and recycle it for reuse.

[0064] The collected chitosan and protein were dried, and the yields of insect protein, oil and chitosan were 70.5%, 89.2% and 90.0% respectively. The ash content in chitosan was 1.5%, and the nitrogen content was 4.6%. The protein content and the nitrogen content in chitosan were measured by Kjeldahl nitrogen determination method, the ash content of chitosan was measured by weight of the residue after burning in a muffle furnace, and the chitosan yield and oil yield were measured by weight after drying.

[0065] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred implementation modes of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A method for separating protein, oil and chitin from black soldier fly larvae, characterized in that: include: The monobasic organic acid, the dibasic organic acid, the tribasic organic acid and the alcohol compound are mixed and heated in a water bath to prepare an ion solution; The black soldier fly larvae are crushed and dried, and then placed in the ion solution, and then heated in a water bath to separate into three layers, wherein the upper layer is oil, the middle layer is an ion solution containing protein, and the lower layer is chitin and inorganic salts; The upper layer material, the middle layer material and the lower layer material are separated, the upper layer material is oil, the middle layer material is separated to obtain protein, and the lower layer material is removed of inorganic salts to obtain chitosan.

2. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: The monobasic organic acid is selected from acetic acid, propionic acid or benzoic acid, the dibasic organic acid is selected from oxalic acid, malic acid or tartaric acid, the ternary organic acid is selected from citric acid, isophthalic acid or cyclohexanetrilic acid, and the alcohol compound is selected from ethanol, ethylene glycol, diethylene glycol or glycerol.

3. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: In terms of molar ratio, the ratio of the monobasic organic acid, the dibasic organic acid, the tribasic organic acid and the alcohol compound is 1:1-10:1-2:6-10.

4. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: The mass ratio of the crushed and dried black soldier fly larvae to the ion solution is 1:3-10.

5. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: The prepared ion solution is heated in a water bath to reach the eutectic temperature.

6. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: The black soldier fly larvae are crushed and dried, mixed with the ion solution, heated in a water bath to fully dissolve the protein, and then centrifuged to separate into three layers.

7. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 6, characterized in that: The centrifugal conditions are 3000-20000 rpm, 10-30 min.

8. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: After the middle layer material and ethanol are mixed, the ionic solution and ethanol form a homogeneous phase, the protein is precipitated in the ethanol system, the protein is cooled and precipitated, and then centrifuged to obtain the protein, and the remaining liquid is evaporated to separate the ethanol and the ionic solution for recycling.

9. The method for separating protein, oil and chitin from black soldier fly larvae according to claim 1, characterized in that: The lower layer material is separated by filtration, and the lower layer material is washed with ethanol to remove inorganic salts and obtain chitosan.

10. Use of the method for separating protein, oil and chitin from black soldier fly larvae according to any one of claims 1 to 9 in resource recovery of black soldier fly larvae.

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