阅读说明：本技术 一种动物多肽粉及其制备方法与应用 (Animal polypeptide powder and preparation method and application thereof ) 是由 聂静洁 于 2021-08-17 设计创作，主要内容包括：本发明属于动物蛋白提取技术领域,具体涉及一种动物多肽粉及其制备方法与应用。本发明对鲣鱼内脏进行脱脂,得到鱼油；然后将脱脂后的鲣鱼内脏进行酶解,将收集的酶解液进行分子量分段,收集分子量介于5000～10000Da的鲣鱼多肽液,真空干燥,得到鲣鱼多肽粉,该方法操作简单,成本低,制备得到的鲣鱼多肽粉可进一步作为壁材制备微胶囊。(The invention belongs to the technical field of animal protein extraction, and particularly relates to animal polypeptide powder and a preparation method and application thereof. The method comprises the steps of degreasing bonito viscera to obtain fish oil; and then carrying out enzymolysis on degreased bonito viscera, carrying out molecular weight segmentation on the collected enzymolysis liquid, collecting a bonito polypeptide liquid with the molecular weight of 5000-10000 Da, and carrying out vacuum drying to obtain the bonito polypeptide powder.)

1. A preparation method of animal polypeptide powder is characterized by comprising the following steps:

(1) dividing skipjack viscera into small pieces, homogenizing at low temperature, mixing with petroleum ether, defatting, centrifuging at low temperature to obtain organic phase at the uppermost layer and defatted skipjack viscera as the rest; evaporating the organic phase to remove petroleum ether in the organic phase to obtain fish oil;

(2) putting the degreased bonito viscera into a sodium chloride solution, and adjusting the pH of the system to 7.0-7.5; then adding compound protease for enzymolysis; centrifuging after enzymolysis, and collecting enzymolysis liquid;

(3) ultrafiltering the collected enzymolysis liquid with filter membrane with cut-off molecular weight of 5000Da, and collecting protein liquid with molecular weight greater than 5000 Da; and then, performing ultrafiltration by using a filter membrane with the molecular weight cutoff of 10000Da, collecting bonito polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain the bonito polypeptide powder.

2. The method for preparing animal polypeptide powder according to claim 1, wherein:

the mass ratio of the bonito viscera to the petroleum ether in the step (1) is 1: (0.5 to 1).

3. The method for preparing animal polypeptide powder according to claim 1, wherein:

the degreasing treatment in the step (1) is carried out for 4-6 h at room temperature.

4. The method for preparing animal polypeptide powder according to claim 1, wherein:

the mass ratio of the degreased bonito viscera to the sodium chloride solution in the step (2) is 1: (1-3).

5. The method for preparing animal polypeptide powder according to claim 1, wherein:

the mass fraction of the sodium chloride solution in the step (2) is 8-10%.

6. The method for preparing animal polypeptide powder according to claim 1, wherein:

the compound protease in the step (2) is a mixture of trypsin and papain;

the dosage of the trypsin is 2000-2500U/g;

the dosage of the papain is 1000-1500U/g.

7. The method for preparing animal polypeptide powder according to claim 1, wherein:

and (3) carrying out enzymolysis for 5-6 h at 45-55 ℃ under the enzymolysis condition in the step (2).

8. An animal polypeptide powder characterized by being prepared by the preparation method of any one of claims 1 to 7.

9. A microcapsule containing animal polypeptide powder, which is characterized by being prepared by the following method:

(1) mixing a soluble wall material and the bonito polypeptide powder of claim 8 in a mass ratio of (4-5): 1, and dissolving the mixture in water to obtain a composite wall material solution with the total mass fraction of 3-5%;

(2) mixing the fish oil as the core material in the claim 1 with an emulsifier, adding the mixture into the composite wall material solution prepared in the step (1), fully emulsifying, homogenizing, and spray-drying to obtain the microcapsule containing the animal polypeptide powder.

10. Microcapsules containing animal polypeptide powder according to claim 9, characterized in that:

the soluble wall material in the step (1) is at least one of modified starch, Arabic gum, chitosan, soybean protein and cyclodextrin.

Technical Field

The invention belongs to the technical field of animal protein extraction, and particularly relates to animal polypeptide powder and a preparation method and application thereof.

Background

Bonito (Katsuwonus pelamis) is a fish belonging to Osteichthyes, Perciformes, Scombridae, and Katsuwonus, and has spindle shape and nearly circular cross section; the kiss is short and the front end is sharp; 2 dorsal fins, wherein the second dorsal fin and the hip fin are smaller and lower, and 7-8 small separating fins are arranged behind the second dorsal fin and the hip fin respectively; a tail fin crescent shape; the back side of the body is blue black, the abdomen is silvery white, and 4-6 obvious black longitudinal bands are arranged on the abdomen side; the maximum body length is 100 cm, and is usually 50-60 cm. Bonito is one of the important economic fishes in tuna fishery in the world, and fish has the advantages of high protein, low fat, rich nutrition and the like, and can be made into firewood fish, cans and raw fish slices.

A large amount of leftover byproducts such as head and tail sections, internal organs and the like can be generated in the skipjack deep processing process, the skipjack leftover byproducts are comprehensively utilized, resources can be fully utilized, the economic value of a fish body is improved, the discharge of waste water and waste can be reduced, and the method has positive significance for protecting the environment. The bonito viscera contains abundant fish oil and active protein, and can be used for development and production of nutritional formula, active peptide and functional protein. At present, the research on the skipjack leftover byproduct at home and abroad is mostly to search different enzymolysis conditions of head and internal organs, research on the functional characteristics of enzymolysis liquid, and research on the extraction and refining of tuna head fish oil and the characteristics of fish skin collagen and gel. CN103540638A discloses a method for preparing antioxidant peptide from skipjack processing by-products, which comprises mixing and pulverizing skipjack head and tail sections and other by-products as processing objects with cooking liquor, then performing synchronous three-phase separation to obtain a protein source capable of being directly subjected to enzymolysis, adding flavourzyme, performing enzymolysis for 4 hours at a pH of 6.5 and a temperature of 50 ℃, purifying the enzymolysis liquid by ultrafiltration, activated carbon adsorption and desalination, and spray-drying to prepare peptide powder. CN108004221A discloses a method for extracting protease from bonito viscera, which comprises the following steps: crushing skipjack viscera into meat paste, mixing the meat paste into a buffer solution containing copper sulfate, adding EDTA (ethylene diamine tetraacetic acid), and then carrying out fractional salting-out protease by adopting ammonium sulfate with the saturation of 35-45%; and purifying the protease by ion exchange chromatography after salting out, and finally purifying by a molecular sieve by a Sephadex G-100 gel layer. The method has the advantages of complex operation and high cost, and the polypeptide or protein prepared by the protein has low stability.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a preparation method of animal polypeptide powder.

The invention also aims to provide the animal polypeptide powder prepared by the preparation method.

Still another object of the present invention is to provide the use of the above animal polypeptide powder.

It is still another object of the present invention to provide a microcapsule comprising the above animal polypeptide powder.

The purpose of the invention is realized by the following technical scheme:

a preparation method of animal polypeptide powder comprises the following steps:

(1) dividing skipjack viscera into small pieces, homogenizing at low temperature, mixing with petroleum ether, defatting, centrifuging at low temperature to obtain organic phase at the uppermost layer and defatted skipjack viscera as the rest; evaporating the organic phase to remove petroleum ether in the organic phase to obtain fish oil;

(2) putting the degreased bonito viscera into a sodium chloride solution, and adjusting the pH of the system to 7.0-7.5; then adding compound protease for enzymolysis; centrifuging after enzymolysis, and collecting enzymolysis liquid;

(3) ultrafiltering the collected enzymolysis liquid with filter membrane with cut-off molecular weight of 5000Da, and collecting protein liquid with molecular weight greater than 5000 Da; then, performing ultrafiltration by using a filter membrane with the molecular weight cutoff of 10000Da, collecting skipjack polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain skipjack polypeptide powder;

the mass ratio of the bonito viscera to the petroleum ether in the step (1) is preferably 1: (0.5 to 1);

the low-temperature homogenizing condition in the step (1) is preferably 0-4 ℃;

the condition of the degreasing treatment in the step (1) is preferably degreasing treatment at room temperature for 4-6 h;

the mass ratio of the degreased bonito viscera to the sodium chloride solution in the step (2) is 1: (1-3);

the mass fraction of the sodium chloride solution in the step (2) is preferably 8-10%;

the compound protease in the step (2) is preferably a mixture of trypsin and papain;

the dosage of the trypsin is preferably 2000-2500U/g;

the dosage of the papain is preferably 1000-1500U/g;

the enzymolysis condition in the step (2) is preferably 45-55 ℃ for 5-6 h;

an animal polypeptide powder is prepared by the above preparation method;

the application of the animal polypeptide powder in the food field;

a microcapsule containing the animal polypeptide powder is prepared by the following method:

(1) mixing a soluble wall material and the skipjack polypeptide powder according to a mass ratio of (4-5): 1, and dissolving the mixture in water to obtain a composite wall material solution with the total mass fraction of 3-5%;

(2) mixing the fish oil serving as a core material with an emulsifier, adding the mixture into the composite wall material solution prepared in the step (1), fully emulsifying, homogenizing, and spray-drying to obtain microcapsules containing animal polypeptide powder;

the soluble wall material in the step (1) is preferably at least one of modified starch, Arabic gum, chitosan, soybean protein and cyclodextrin;

the emulsifier in the step (2) is preferably tween 80, octyl, tricaprin or enzymolysis soybean phospholipid;

the core-wall ratio of the core material to the composite wall material in the step (2) is preferably (0.6-1): 2;

a microcapsule containing animal polypeptide powder is prepared by the above preparation method;

compared with the prior art, the invention has the following advantages and effects:

(1) the traditional fish oil and protein extraction method usually adopts an enzymolysis method, the method utilizes protease to hydrolyze protein in raw materials, so that fat combined with the protein is dissociated, and then the fish oil at the upper layer is obtained through centrifugation, so that the method has the characteristics of mild reaction and the like, but the method is long in time consumption, and in the subsequent centrifugal separation process, a separation system is sequentially divided into four layers from top to bottom: the fish oil purification device comprises crude fish oil, an emulsified protein layer, an enzymolysis protein layer and an impurity sediment layer, wherein the second emulsified protein layer is a uniform and stable dispersion system formed by the fish oil and protein, and the existence of the emulsified protein layer affects the effective separation of the first crude fish oil layer on the one hand and also causes the reduction of the extraction rate of the fish oil on the other hand, and the existence of the layer of fish oil can further affect the separation and purification of subsequent enzymolysis protein.

(2) The invention adopts salt solution and compound protease to extract protein in degreased bonito viscera, and utilizes a plurality of enzyme digestion stable point technologies of the compound protease to obtain more kinds of too short free amino groups, so that the obtained polypeptide has better free radical scavenging capacity.

(3) According to the invention, the enzymolysis liquid after enzymolysis is subjected to molecular weight segmentation in an ultrafiltration mode to obtain bonito polypeptide with a specific molecular weight segment, on one hand, protease does not need to be inactivated, on the other hand, the polypeptide not only has good free radical scavenging capacity, but also can be used as a wall material to stabilize emulsion.

(4) The invention adopts the soluble wall material and the skipjack polypeptide powder as the composite wall material, and the prepared fish oil is used as the core material, on one hand, the byproduct of skipjack leftovers is comprehensively utilized, so that the added value of the byproduct is greatly increased, on the other hand, the composite wall material coats the fish oil, so that the fish oil is not required to be deodorized and deodorized, the oxidation of the fish oil is avoided, the application range is expanded, and the consumer acceptance degree is improved.

(5) The invention opens up a new direction for industrial development of the skipjack byproduct reprocessing, and provides a new research idea.

Drawings

FIG. 1 is a diagram showing a product of bonito polypeptide powder obtained in example 1.

FIG. 2 is a diagram showing the product of bonito polypeptide powder obtained in example 2.

FIG. 3 is a graph showing the results of the oxidative stability of microcapsules.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

In the examples, papain (100 ten thousand U/g), product lot number: 201201016, trypsin (5 ten thousand U/g), product batch number: 210104024, available from Biotech, Inc., Henghua, Dongning.

Example 1

(1) Adopting skipjack viscera imported from Japan as raw materials, unfreezing, removing impurities, cutting the skipjack viscera into small blocks, homogenizing at a low temperature of 4 ℃, mixing 1000g of homogenized skipjack viscera with 1000ml of petroleum ether, performing extraction and degreasing treatment at room temperature for 4h, centrifuging at a low temperature, wherein the uppermost layer is an organic phase, and the rest is degreased skipjack viscera; evaporating the organic phase to remove petroleum ether in the organic phase to obtain fish oil;

(2) taking 700g of degreased bonito internal organs prepared in the step (1), placing the degreased bonito internal organs in 700ml of sodium chloride solution with the mass fraction of 10%, and adjusting the pH of the system to be 7.5; adding trypsin and papain for enzymolysis at 45 deg.C for 6h, wherein the amount of trypsin is 2500U/g, and the amount of papain is 1500U/g; centrifuging after enzymolysis, and collecting enzymolysis liquid;

(3) ultrafiltering the collected enzymolysis liquid with filter membrane with cut-off molecular weight of 5000Da, and collecting protein liquid with molecular weight greater than 5000 Da; then, performing ultrafiltration by using a filter membrane with the molecular weight cutoff of 10000Da, collecting skipjack polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain skipjack polypeptide powder;

(4) respectively weighing 16g of soluble wall material beta-cyclodextrin and 4g of bonito polypeptide powder prepared in the step (3), mixing and dissolving in 500ml of water to obtain a composite wall material solution with the total mass fraction of 4%;

(5) and (2) mixing 10g of the fish oil prepared in the step (1) as a core material with 5g of Tween 80 as an emulsifier, adding the mixture into the composite wall material solution prepared in the step (4), fully emulsifying, homogenizing and spray-drying to obtain the bonito oil/polypeptide composite microcapsule, wherein the core-wall ratio of the core material to the composite wall material is 1: 2.

example 2

(1) Adopting skipjack viscera imported from Japan as raw materials, unfreezing, removing impurities, cutting the skipjack viscera into small blocks, homogenizing at a low temperature of 4 ℃, mixing 500g of homogenized skipjack viscera with 760ml of petroleum ether, performing extraction and degreasing treatment at room temperature for 5h, centrifuging at a low temperature, wherein the uppermost layer is an organic phase, and the rest is degreased skipjack viscera; evaporating the organic phase to remove petroleum ether in the organic phase to obtain fish oil;

(2) taking 320g of degreased bonito internal organs prepared in the step (1), placing the degreased bonito internal organs into 700ml of sodium chloride solution with the mass fraction of 10%, and adjusting the pH value of the system to be 7.5; adding trypsin and papain for enzymolysis at 50 deg.C for 6h, wherein the amount of trypsin is 2500U/g and the amount of papain is 1200U/g; centrifuging after enzymolysis, and collecting enzymolysis liquid;

(3) ultrafiltering the collected enzymolysis liquid with filter membrane with cut-off molecular weight of 5000Da, and collecting protein liquid with molecular weight greater than 5000 Da; then, performing ultrafiltration by using a filter membrane with the molecular weight cutoff of 10000Da, collecting skipjack polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain skipjack polypeptide powder;

(4) respectively weighing 8.33g of soluble wall material beta-cyclodextrin and 1.67g of bonito polypeptide powder prepared in the step (3), mixing and dissolving in 200ml of water to obtain a composite wall material solution with the total mass fraction of 5%;

(5) and (2) mixing 5g of the fish oil prepared in the step (1) as a core material with 2g of Tween 80 as an emulsifier, adding the mixture into the composite wall material solution prepared in the step (4), fully emulsifying, homogenizing and spray-drying to obtain the bonito oil/polypeptide composite microcapsule, wherein the core-wall ratio of the core material to the composite wall material is 1: 2.

example 3

(1) Taking skipjack viscera imported from Japan as a raw material, unfreezing, removing impurities, cutting the skipjack viscera into small blocks, homogenizing at a low temperature of 4 ℃, mixing 1200g of homogenized skipjack viscera with 1500ml of petroleum ether, performing extraction and degreasing treatment at room temperature for 6h, centrifuging at a low temperature, wherein the uppermost layer is an organic phase, and the rest is degreased skipjack viscera; evaporating the organic phase to remove petroleum ether in the organic phase to obtain fish oil;

(2) placing 800g of degreased bonito viscera prepared in the step (1) in 700ml of sodium chloride solution with the mass fraction of 10%, and adjusting the pH of the system to 7.0; adding trypsin and papain for enzymolysis at 55 ℃ for 5h, wherein the dosage of the trypsin is 2000U/g, and the dosage of the papain is 1000U/g; centrifuging after enzymolysis, and collecting enzymolysis liquid;

(3) ultrafiltering the collected enzymolysis liquid with filter membrane with cut-off molecular weight of 5000Da, and collecting protein liquid with molecular weight greater than 5000 Da; then, performing ultrafiltration by using a filter membrane with the molecular weight cutoff of 10000Da, collecting skipjack polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain skipjack polypeptide powder;

(4) respectively weighing 12g of soluble wall material beta-cyclodextrin and 3g of bonito polypeptide powder prepared in the step (3), mixing and dissolving in 500ml of water to obtain a composite wall material solution with the total mass fraction of 3%;

(5) and (3) mixing 7.5g of the fish oil prepared in the step (1) as a core material with 5g of Tween 80 as an emulsifier, adding the mixture into the composite wall material solution prepared in the step (4), fully emulsifying, homogenizing and spray-drying to obtain the bonito fish oil/polypeptide composite microcapsule, wherein the core-wall ratio of the core material to the composite wall material is 1: 2.

comparative example 1

(1) Same as example 1, step (1);

(2) same as example 1, step (2);

(3) directly vacuum drying the collected enzymolysis liquid without ultrafiltration to obtain skipjack polypeptide powder;

(4) same as example 1, step (4);

(5) same as example 1, step (5).

Comparative example 2

(1) Taking skipjack viscera imported from Japan as a raw material, unfreezing, removing impurities, cutting the skipjack viscera into small blocks, homogenizing at a low temperature of 4 ℃, putting 1000g of the homogenized skipjack viscera into 1000ml of sodium chloride solution with a mass fraction of 10%, and adjusting the pH value of the system to be 7.5; adding trypsin and papain for enzymolysis at 45 deg.C for 6h, wherein the amount of trypsin is 2500U/g, and the amount of papain is 1500U/g; centrifuging after enzymolysis, collecting the fish oil at the uppermost layer, discarding the residue at the lowermost layer, and taking the rest as enzymolysis liquid;

(2) ultrafiltering the collected enzymolysis liquid with 5000Da filter membrane, and collecting protein liquid with molecular weight greater than 5000 Da; then, performing ultrafiltration by a 10000Da filter membrane, collecting a skipjack polypeptide liquid with the molecular weight of 5000-10000 Da, and performing vacuum drying to obtain skipjack polypeptide powder;

(3) respectively weighing 16g of soluble wall material beta-cyclodextrin and 4g of bonito polypeptide powder prepared in the step (3), mixing and dissolving in 500ml of water to obtain a composite wall material solution with the total mass fraction of 4%;

(4) and (2) mixing 10g of the fish oil prepared in the step (1) as a core material with 5g of Tween 80 as an emulsifier, adding the mixture into the composite wall material solution prepared in the step (4), fully emulsifying, homogenizing and spray-drying to obtain the bonito oil/polypeptide composite microcapsule, wherein the core-wall ratio of the core material to the composite wall material is 1: 2.

example 4

(1) Detection of encapsulation efficiency of microcapsules

The microcapsule embedding rate is the percentage of the fish oil encapsulated in the microcapsule to the total amount of the fish oil, and the calculation formula is as follows:

and (3) the microcapsule embedding rate (%) (total oil content of the microcapsule product-surface oil content of the microcapsule product)/total oil content of the microcapsule product multiplied by 100, wherein the total oil content of the microcapsule product and the surface oil content of the microcapsule product are detected according to a conventional method.

(2) Detection of peroxide number

The oxidation stability of the fish oil and the bonito oil/polypeptide composite microcapsules prepared in example 1 and the comparative example were measured, and the specific method was SC/T3505.2006.

(3) Analysis of results

The difference between the comparative example 1 and the example 1 is that the enzymolysis solution is directly dried in vacuum without ultrafiltration, the prepared bonito polypeptide powder has a large molecular weight distribution range, and the microcapsule prepared by using the bonito polypeptide powder as a wall material influences the embedding rate of fish oil on one hand, and on the other hand, the capability of removing free radicals of the bonito polypeptide powder is poor, as can be seen from table 1, the embedding rate of the microcapsule prepared by the comparative example 1 is obviously lower than that of the examples 1-3, and further, the embedding rate of the microcapsule can be improved by carrying out molecular weight segmentation on the enzymolysis solution in an ultrafiltration mode.

TABLE 1 embedding rates of skipjack fish oil/polypeptide composite microcapsules prepared in examples 1 to 3 and comparative examples 1 to 2

Examples	Embedding rate
		Example 1	92.5％
Example 2	94.3％
		Example 3	91.7％
Comparative example 1	83.4％
		Comparative example 2	87.1％

The difference between the comparative example 2 and the example 1 is that the fish oil and the polypeptide are directly extracted by enzymolysis, and although the fish oil and the polypeptide can be separated to a certain extent by enzymolysis, the extraction rate and purity of the fish oil and the polypeptide are reduced due to the unobvious system layering, and the oxidation stability of the capsule is influenced to a certain extent (figure 3).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.