CN1415757A - Method for extracting protein and chitin from fly maggot by using enzyme hydrolysis as well as preparing chitosan from chitin - Google Patents
Method for extracting protein and chitin from fly maggot by using enzyme hydrolysis as well as preparing chitosan from chitin Download PDFInfo
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Abstract
A process for extracting protein and chitin from fly maggot and preparing chitosan from the chitin incldues such steps as extracting hydrolytic protein by enzyme hydrolysis, removing stink and enhancing fragrance by Maillard reaction, enzymolyzing, removing yellow substances and black spot, etc. The quality of produced protein and chitin conforms with food grade standard.
Description
Technical Field
The invention relates to a method for extracting protein and chitin from fly maggots, in particular to a method for extracting protein and chitin from fly maggots by an enzymatic hydrolysis method and preparing chitosan by using chitin, belonging to the technical field of biological enzymatic hydrolysis of fly maggot proteins.
Background
The fly maggot is an insect with high reproduction speed and high nutritive value, and contains high-quality protein and chitin resources. The level of large-scale fly maggot culture can be formed at present. However, the fly maggots are mainly used as substitute feed at present, the technical content of the product is low, the economic benefit is poor, and the development of the fly maggot breeding industry is seriously hindered. In recent years, with the deep understanding of the nutritional value and functional activity of fly maggots, the utilization of fly maggots has been developed deeply from alternative feeds to high value-added applications such as food, health products, medicines and cosmetics.
At present, the fly maggot protein powder is mainly produced by a mechanical method or a chemical method, such as wet crushing and direct drying of the fly maggots; or drying fly maggot and pulverizing. The fly maggot protein powder product has poor water solubility, poor color and heavy fishy smell, is only used for an animal feed formula or used as an animal feed additive, and has great limitation on the application field. The extraction of fly maggot chitin mainly utilizes mechanical crushing and cleaning to obtain fly maggot skin, then uses dilute acid dilute alkali treatment to remove protein, decolour and decalcify so as to obtain the invented product. However, the maggot skin prepared by a mechanical method has high protein content, complicated subsequent processing, long processing time, great waste of protein resources and serious pollution in the production process. More importantly, no method for effectively removing black oral cavity and yellow internal organs exists in the processing process, which affects the polymerization degree, color and impurity degree of chitin and chitosan products and reduces the product quality.
As seen from the related documents, although published documents of using the fly maggots in animal protein powder formulations and as animal protein feed additives and published documents of chemically extracting fly maggot chitin from the fly maggots are reported, such as Chinese patents 971164908, 981034462, 991193911, 001002651 and the like, the reports of extracting protein from the fly maggots by an enzyme hydrolysis method and recovering chitin are not seen at home and abroad.
Disclosure of Invention
(1) Technical problem to be solved
The invention aims to solve the technical problems of extracting protein from fly maggots and recycling chitin by using an enzymatic hydrolysis method; deodorizing and flavoring the fly maggot hydrolyzed protein; removing yellow substances and black spots (fly larvae oral cavity) in fly larvae chitin by using enzymatic hydrolysis and combination physical screening; the chitosan is prepared from the fly maggot chitin, so that the deacetylation degree of the chitosan is more than or equal to 85 percent and reaches the food sanitation standard.
(2) Technical scheme
A method for extracting protein from fly maggot is provided. It is characterized by that it utilizes the enzyme hydrolysis method to extract fly maggot protein, then utilizes Maillard reaction to remove fishy smell and raise fragrance. The process flow diagram is shown in the attached drawing. The invention utilizes a biotechnology enzyme hydrolysis method to extract hydrolyzed protein from fly maggots, uses enzyme hydrolysis to change macromolecular protein into water-soluble polypeptide and amino acid, and simultaneously prevents bitter taste generated by enzymolysis by selecting specific enzyme species and optimizing conditions. The flavor of the fly maggot protein obtained by enzymolysis is modified by utilizing Maillard reaction, and the fishy smell is eliminated, so that the solubility and the flavor of the protein can be greatly improved. Fresh fly maggot is used as raw material, water is added to carry out colloid milling and crushing, heating and heat preservation pretreatment are carried out, enzyme is added to carry out enzymatic hydrolysis reaction, enzyme is killed, filtration is carried out, the filtrate is carried out Maillard reaction to remove fishy smell and increase fragrance, concentration or no concentration is carried out, and spray drying is carried out to prepare the product of the fly maggot protein hydrolysate powder.
The enzymatic hydrolysis process comprises the following steps:
grinding by a rubber mill: 1 part of fly maggot is added with 1-3 parts of water (w/w, weight ratio, the same below) to be ground by a rubber mill to obtain 2-4 parts of fly maggot slurry, and water can be added according to the processing requirement to adjust the concentration of the fly maggot slurry.
Heating and heat preservation pretreatment: and heating the fly maggot slurry at 60-90 ℃ for 10-60 minutes, and cooling to below 60 ℃.
And (3) performing enzymatic hydrolysis reaction: selecting proper enzyme species for hydrolyzing the fly maggot protein from a plurality of proteases by taking the size of the protein hydrolysis degree and the bitter taste as indexes. The enzyme is alkaline protease, AS.1398 protease, papain, trypsin, flavor enzyme, neutral protease, or complex enzyme of two enzymes. The process conditions are analyzed by orthogonal experiments, and the main and secondary relations of all factors influencing the hydrolysis degree are that the temperature is more than the time and the enzyme-substrate ratio is more than the pH value. The enzymolysis condition is that the temperature is 30-55 ℃, the time is 1-4 hours, the enzyme-substrate ratio (u/g) is 50-700, and the pH value is 6.5-8.5. And (5) inactivating enzyme after the reaction is finished.
And (3) filtering: the filtrate is fly maggot protein liquid, and the filter residue is fly maggot skin. And (5) performing Maillard reaction on the filtrate to remove fishy smell and increase flavor.
Deodorizing and flavoring the enzymolysis protein liquid: the post-modification is carried out by adopting the Maillard reaction, thereby achieving the purposes of increasing aroma and removing fishy smell. The reaction was carried out using amino acids produced by enzymatic hydrolysis and adding a small amount of sugar according to the following ratio.
Raw materials and proportion thereof
| Raw materials | Use amount (w) |
| Fly maggot hydrolysate Amino acids Glucose | 100 0.05～1.5 0.2～0.5 |
Reaction conditions are as follows: the temperature is 100-125 ℃, and the time is 30-100 minutes.
Preparing protein powder: concentrating the reaction liquid, and spray drying to obtain the product, namely the maggot protein hydrolysate powder. The protein content in the product is more than or equal to 60 percent.
Or directly carrying out Maillard reaction on the enzymolysis liquid to remove fishy smell and increase fragrance under the same process conditions without filtering after enzyme deactivation of the enzymolysis liquid, concentrating or not, and carrying out spray drying to prepare the maggot hydrolyzed protein powder, wherein the protein content is more than or equal to 54 percent.
A method for extracting Chitin (Chitin) from fly maggot is provided. After the fly maggots are subjected to enzymatic hydrolysis, fly maggot skins obtained by filtering and separating fly maggot protein liquid and subjecting filter residues to enzymatic hydrolysis are taken as raw materials, and the fly maggot chitin finished product is prepared by deproteinization and decalcification. The process flow diagram is shown in the attached drawing. After the fly maggot protein is extracted by enzymatic hydrolysis, the main component of the fly maggot skin is chitin, and a small amount of protein, fat, ash (calcium carbonate salt) and the like are also contained. Therefore, the adoption of reasonable deproteinization and decalcification process conditions is extremely important for improving the quality of the chitin. The protein in the fly maggot skin is removed by applying a biological technology enzymatic hydrolysis method, the black mouthparts and the yellow internal organs in the fly maggot skin can be hydrolyzed simultaneously, and the impurities in the chitin product can be effectively removed by a physical method. The process for extracting the chitin comprises the following steps: and (3) screening and washing the fly maggot skin, boiling in alkali to remove protein, washing to be neutral, leaching in acid to remove calcium, washing to be neutral, and drying to obtain the fly maggot chitin finished product.
Deproteinization: deproteinizing with NaOH solution, wherein the concentration of alkali liquor is 2-6% (w/w), the temperature is 60-100 ℃, the liquid-solid ratio (ml/g) is 3-10, and the reaction time is 1-5 hours.
Decalcification: deproteinizing fly maggot skins, removing impurities by using a sieve, cleaning the left residues to be neutral, centrifuging to remove water, decalcifying by using a dilute hydrochloric acid solution, wherein the concentration of hydrochloric acid is 0.5-1.5 mol/L, the liquid-solid ratio is 2.0-9.0 ml/g, the reaction time is 20-60 minutes, the temperature is room temperature, cleaning to be neutral by using pure water, and drying at low temperature to obtain the fly maggot chitin.
A method for preparing Chitosan (Chitosan) from fly maggot chitin is provided. The chitosan product is prepared by taking fly maggot chitin extracted by the enzymatic hydrolysis method as a raw material and performing deacetylation reaction in concentrated alkali liquor.
Chitosan is a cationic straight-chain amino polysaccharide, and is a product formed by removing acetyl groups in molecules of chitin. The preparation process of chitosan is actually the alkaline hydrolysis process of amide polysaccharide chitin, namely deacetylation reaction. The reaction formula of the deacetylation reaction is as follows:
At present, the chitosan is mainly prepared from shrimp and crab chitin as raw materials, and is generally prepared by treating chitin by strong alkali at high temperature to remove most acetyl groups. Compared with shrimp and crab chitin, the fly maggot chitin has less ash content, low heavy metal content and stable quality. The degree of deacetylation is one of the main performance indicators of chitosan. Food grade chitosan requires a higher degree of deacetylation, i.e. the degree of deacetylation is more than or equal to 85%.
The chitosan prepared by the invention is a chitosan product prepared by taking the fly maggot chitin extracted by the method as a raw material and performing alkaline hydrolysis, namely deacetylation reaction. The raw materials adopt non-crushed fly maggot chitin, the alkali liquor adopts NaOH solution, the alkali concentration is 30-55% (w/w), the reaction temperature is 30-140 ℃, the reaction time is 2-30 hours, and the liquid-solid ratio is 5-20 ml/g. Under the optimized process conditions, the deacetylation degree of the product can reach more than 88 percent.
(3) Advantageous effects
The invention provides a new way for the deep processing and comprehensive utilization of fly maggot resources by researching the enzymatic hydrolysis process conditions of fly maggot proteins and the preparation process conditions of chitin and chitosan in detail.
The comparison of the extraction rate and the dissolution rate of the protein shows that the extraction rate and the dissolution rate of the protein after the enzymatic treatment reach more than 92 percent and 80 percent, while the mechanical separation is only 64 percent and 24 percent. The enzymatic method obviously improves the solubility of the protein, improves the dissolution rate of the protein and increases the extraction rate of the protein.
By adopting Maillard reaction, the protein liquid achieves the purposes of increasing aroma and removing fishy smell, and the product has aromatic flavor and the fishy smell disappears under the optimized process condition.
The content of protein in the fly maggot skin after enzyme hydrolysis is lower than that of the fly maggot skin obtained by mechanical crushing. Greatly saves the consumption of alkali and the reaction time in the deproteinization process. The black mouthparts and the yellow viscera in the fly maggot skin after the enzymatic hydrolysis are also subjected to enzymolysis and then are subjected to screening and washing, wherein the most part of the black mouthparts and the yellow viscera can be easily removed, a small amount of impurities remained in the fly maggot skin are further removed by screening and washing after being subjected to alkali treatment, and the removal rate of microscopic black spots reaches more than 98%.
The fly larvae chitosan is prepared from fly larvae chitin. Under the optimized process conditions, the deacetylation degree and the yield of the chitosan are high. The product obtained is confirmed to be chitosan by infrared spectrum and x-ray diffraction analysis.
The fly maggot protein has high nutritive value. According to the analysis of nutritional ingredients of the fly maggots at home and abroad, the fly maggots (dry basis) contain 54-65% of crude protein, 10-14% of fat, 8-10% of chitin, abundant vitamins, trace elements and the like, and the nutritional ingredients are relatively comprehensive. The amino acids in the fly maggot protein are relatively complete, and the provided amino acids can meet the recommended amino acid requirements of children and adults. A large number of animal experiments show that: the maggot protein powder prepared from maggot has obvious immunoregulation effect, antifatigue effect, radioprotective effect, and antiaging effect. The maggot protein hydrolysate powder prepared by enzyme hydrolysis changes macromolecular protein into water-soluble polypeptide and amino acid, which can be absorbed by human body more easily after eating.
The fly maggot protein prepared by the enzymatic hydrolysis method has high nutritive value, and can be widely applied to the industries of food, medicine, cosmetics and the like due to good solubility, water retention, emulsibility, oxidation resistance and the like.
In the food industry, the hydrolyzed protein is a seasoning base material which can well enhance the natural flavor of food, can be applied to various flavor type seasonings and nutrient supplements of the food, can also improve the nutritional value and the storage safety of the food, and can be widely applied to various foods as a natural unlimited food additive, such as meat product processing, protein beverage, baked products and the like. In the cosmetic industry, hydrolyzed protein has good emulsifying property, lubricity and surface activity, and is one of the high-quality base materials of skin care products and hair care products. In the pharmaceutical industry, oral administration of hydrolyzed protein can be applied to special people who need to eat high protein, and for patients who lack protein and can not take orally due to gastrointestinal dysfunction, severe burns and excessive blood loss, the injection of hydrolyzed protein is an extremely effective method.
Chitosan is one of the most important derivatives of chitin, because its molecule contains free-NH2The active group, when dissolved in an acidic solution, can be protonated to take a positive charge, is the only basic polysaccharide in natural polysaccharides, thus having many special physicochemical properties and physiological and pharmacological functions, and being praised as the sixth vital element necessaryfor human body by many researchers.
A large number of researches prove that the chitin and the chitosan have multiple functions, such as reducing cholesterol, protecting liver, improving immunity of organisms, reducing uric acid in vivo, inhibiting gastric acid secretion, regulating and balancing trace element levels of human bodies, reducing food calories, inhibiting bacteria and preventing corrosion and the like. Health foods containing chitin and chitosan as main ingredients are being widely developed, and have health functions of regulating immunity, regulating blood lipid, lowering blood sugar, etc.
In conclusion, the invention has important economic benefit and social benefit.
Drawings
The attached figure shows the process flow schematic diagram of extracting fly maggot protein and chitin and preparing chitosan by using chitin.
The specific implementation mode is as follows:
example 1
The enzyme hydrolysis method extracts hydrolyzed protein and chitin from fly maggot. Fresh fly maggots are ground by a rubber mill, slurry is heated and treated, and then hydrolyzed for 3 hours by adopting AS.1398 protease under the conditions of enzyme base ratio of 500u/g, 45 ℃ and pH7.0. At this time, the degree of hydrolysis was 18%, the protein elution rate was 81%, and the hydrolysate was not bitter.
Enzyme deactivation of the enzyme hydrolysate, filtration, Maillard reaction of the filtrate at 120 deg.c for 60 min to obtain aromatic product with no fishy smell. Spray drying to obtain maggot protein powder.
The residue fly maggot skin was used to extract chitin by the same method as in example 4 below.
Example 2
The enzyme hydrolysis method extracts hydrolyzed protein and chitin from fly maggot. Adopts AS.1398 protease and flavor enzyme, the two are enzymolyzed by complex enzyme composed of 1: 1(w/w), and the rest conditions are the same as the example 1. The degree of hydrolysis and the protein leaching rate after hydrolysis can also achieve satisfactory results.
Example 3
The enzyme hydrolysis method is used for extracting hydrolyzed protein from fly maggots. After the fly larvae are subjected to enzymolysis, the enzymatic hydrolysate is directly subjected to Maillard reaction for enhancing the flavor, and spray drying is carried out to obtain fly larva protein powder, wherein the protein content is more than or equal to 60%.
Example 4
The chitin is extracted from fly maggots by an enzyme hydrolysis method. After the maggot skin obtained after enzyme hydrolysis is sieved and cleaned to remove impurities, deproteinization is carried out by using a 5% NaOH solution, boiling is carried out for 3 hours at 100 ℃, the liquid-solid ratio is 9ml/g, and the maggot skin is cleaned to be neutral; decalcification is carried out by reacting for 50 minutes with 1mol/L hydrochloric acid solution with liquid-solid ratio of 6ml/g, and washing to neutrality.
Amino acid analysis of the deproteinized chitin proves that the protein removal rate is 97 percent (based on the total amount of amino acid). The ash content of the decalcified chitin is 0.2%.
Example 5
Chitosan is prepared by deacetylation of fly maggot chitin. Performing deacetylation reaction on fly maggot chitin in concentrated alkali. The temperature is 120 ℃, the time is 3 hours, the alkali concentration is 45 percent, and the liquid-solid ratio is 10 ml/g. The degree of deacetylation of the product at this point was 88% with a yield of 75%.
Claims (4)
1. A method for extracting protein from fly maggot, its characteristic is to apply the enzyme hydrolysis method to extract the fly maggot protein, reuse Maillard reaction to remove the fishy smell and fragrance, its process is to regard fresh fly maggot as raw materials, add water and carry on the rubber mill to break, heat and preserve heat the preconditioning, add enzyme to carry on the enzymatic hydrolysis reaction, enzyme deactivation, filter, filtrate carry on Maillard reaction to remove fishy smell and fragrance, spray drying, make the fly maggot protein hydrolysate powder of product;
(1) the enzymatic hydrolysis process comprises the following steps:
grinding by a rubber mill: 1 part of fly maggot is added with 1-3 parts of water (w/w, weight ratio, the same below) to be ground by a rubber mill to obtain 2-4 parts of fly maggot slurry, and water is added to adjust the concentration of the fly maggot slurry;
heating and heat preservation pretreatment: heating the fly maggot slurry at 60-90 ℃ for 10-60 minutes, and cooling to below 60 ℃;
and (3) performing enzymatic hydrolysis reaction: the enzyme seeds adopt single enzyme of alkaline protease, AS.1398 protease, papain, trypsin, flavor enzyme and neutral protease or compound enzyme of two enzymes, the enzymolysis condition is that the temperature is 30-55 ℃, the time is 1-4 hours, the enzyme substrate ratio is 50-700 u/g, the pH value is 6.5-8.5, and the enzyme deactivation is finished after the reaction;
filtering, wherein the filtrate is fly maggot protein liquid, and the filter residue is fly maggot skin;
(2) deodorizing and flavoring the enzymolysis protein liquid:
the filtrate is subjected to Maillard reaction to remove fishy smell and enhance flavor, and the raw materials and the proportion are as follows:
raw materials and proportion thereof Raw materials
Use amount (w)
Fly maggot hydrolysate
Amino acids
Glucose
100
0.05～1.5
0.2～0.5
Reaction conditions are as follows: the temperature is 100-125 ℃, and the time is 30-100 minutes;
(3) preparing protein powder:
concentrating or not concentrating the reaction solution, and spray drying to obtain the product, namely the maggot hydrolyzed protein powder.
2. The method as claimed in claim 1, wherein the protein is extracted from fly maggots by adding enzyme for enzyme hydrolysis, deactivating enzyme, performing Maillard reaction on the enzymatic hydrolysate to remove fishy smell and increase flavor, and spray drying to obtain fly maggot hydrolyzed protein powder.
3. A method for extracting chitin from fly maggot, its characteristic is after the enzyme hydrolysis of fly maggot, regard fly maggot skin of enzyme hydrolysis of filter residue after filtering and separating fly maggot protein liquid as raw materials, its process is that fly maggot skin sieve washes, alkali boils to deproteinize, wash to neutrality, acid leaching decalcification, wash to neutrality, dry, make fly maggot chitin finished product;
(1) deproteinization: deproteinizing with NaOH solution, wherein the concentration of alkali liquor is 2-6% (w/w), the temperature is 60-100 ℃, the liquid-solid ratio is 3-10 ml/g, and the reaction time is 1-5 hours;
(2) decalcification: deproteinizing fly maggot skins, removing impurities by using a sieve, cleaning the left residues to be neutral, centrifuging to remove water, decalcifying by using a dilute hydrochloric acid solution, wherein the concentration of hydrochloric acid is 0.5-1.5 mol/L, the liquid-solid ratio is 2.0-9.0 ml/g, the reaction time is 20-60 minutes, the temperature is room temperature, cleaning to be neutral by using pure water, and drying to obtain the fly maggot chitin.
4. A method for preparing chitosan from fly maggot chitin is characterized in that the fly maggot chitin extracted by an enzymatic hydrolysis method is used as a raw material, deacetylation reaction is carried out in concentrated alkali liquor to prepare a chitosan product, the raw material is the fly maggot chitin, the alkali liquor is NaOH solution, the alkali concentration is 30-55% (w/w), the reaction temperature is 30-140 ℃, the reaction time is 2-30 hours, and the liquid-solid ratio is 5-20 ml/g.
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