阅读说明：本技术 一种牡蛎肽的提取方法 (Method for extracting oyster peptide ) 是由 林润暄 陈钰淇 林灼华 于 2021-08-09 设计创作，主要内容包括：本申请公开了一种牡蛎肽的提取方法,其包括以下步骤：(1)粉碎：取洗净干燥后的牡蛎壳和牡蛎肉,粉碎成牡蛎细粉；(2)分散：将牡蛎细粉、碳酸二氢钙、碳酸氢钠、微晶纤维素和水按质量比1:0.02～0.04:0.04～0.06:0.01～0.03:23～28混合,搅拌,分散均匀得到分散液；(3)酶解：向分散液中加入分解酶,使分散液酶解；(4)去重金属：向酶解后的分散液中加入EDTA二钠,搅拌反应25～35min；(5)过滤：将去金属后的分散液过滤,取滤液；(6)浓缩、干燥、包装；具有去重金属、提高蛋白回收率的优点。(The application discloses an extraction method of oyster peptide, which comprises the following steps: (1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder; (2) dispersing: mixing the oyster fine powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water according to the mass ratio of 1: 0.02-0.04: 0.04-0.06: 0.01-0.03: 23-28, stirring, and uniformly dispersing to obtain a dispersion liquid; (3) enzymolysis: adding a lytic enzyme to the dispersion to allow the dispersion to undergo enzymatic hydrolysis; (4) removing heavy metals: adding EDTA disodium into the dispersion liquid after enzymolysis, and stirring for reaction for 25-35 min; (5) and (3) filtering: filtering the dispersion liquid after removing the metal, and taking filtrate; (6) concentrating, drying and packaging; has the advantages of removing heavy metals and improving the recovery rate of protein.)

1. The method for extracting the oyster peptide is characterized by comprising the following steps of:

(1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder;

(2) dispersing: mixing the fine oyster powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the oyster fine powder to the monocalcium carbonate to the sodium bicarbonate to the microcrystalline cellulose to the water is 1: 0.02-0.04: 0.04-0.06: 0.01-0.03: 23-28;

(3) enzymolysis: adding a lytic enzyme to the dispersion to allow the dispersion to undergo enzymatic hydrolysis;

(4) removing heavy metals: adding EDTA disodium into the dispersion liquid after enzymolysis, and stirring for reaction for 25-35 min;

(5) and (3) filtering: filtering the dispersion liquid after removing the metal, and taking filtrate;

(6) concentrating, drying, and packaging.

2. The method for extracting oyster peptides according to claim 1, wherein in the step (3), the lytic enzymes comprise neutral protease and lipase, and the enzymolysis step comprises the following two steps:

adjusting the pH value of the dispersion liquid to 6.8-7.2, heating to 50-55 ℃, adding neutral protease, stirring for enzymolysis for 5-7 hr, heating to 75-85 ℃ after enzymolysis, and keeping the temperature for 8-12 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.2-0.4;

adjusting the pH value of the dispersion liquid to 6.8-7.2, heating to 38-42 ℃, adding lipase, stirring for enzymolysis for 2-4 hr, heating to 85-95 ℃ after enzymolysis, and keeping the temperature for 4-5 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the lipase is 100: 0.1-0.3.

3. The method for extracting oyster peptide according to claim 2, wherein in the step (4), besides the disodium EDTA is added to the dispersion, microcrystalline cellulose and ethyl acetate are added in the step (4); in the step (4), the mass ratio of the dispersion liquid after enzymolysis, EDTA disodium, microcrystalline cellulose and ethyl acetate is 100: 0.02-0.04: 0.08-0.11: 7-9.

4. The method for extracting oyster peptides according to claim 3, comprising the following steps:

(1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder;

(2) dispersing: mixing the fine oyster powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the oyster fine powder to the calcium dihydrogen carbonate to the sodium bicarbonate to the microcrystalline cellulose to the water is 1:0.03:0.05:0.02: 25;

(3) enzymolysis:

regulating pH of the dispersion to 7.0, heating to 52 deg.C, adding neutral protease, stirring for enzymolysis for 6hr, heating to 80 deg.C after enzymolysis, and keeping the temperature for 10min to inactivate enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.3;

adjusting pH of the dispersion to 7.0, heating to 40 deg.C, adding lipase, stirring for enzymolysis for 3hr, heating to 90 deg.C after enzymolysis, and keeping the temperature for 5min to inactivate enzyme; the mass ratio of the dispersion liquid to the lipase is 100: 0.2;

(4) removing heavy metals: adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the dispersion liquid after enzymolysis, and stirring to react for 30 min; in the step (4), the mass ratio of the dispersion liquid after enzymolysis, EDTA disodium, microcrystalline cellulose and ethyl acetate is 100:0.03:0.10: 8;

(5) and (3) filtering: vacuum filtering the dispersion solution after removing metals with 0.5 μm filter membrane, and collecting filtrate;

(6) vacuum concentrating to solid content of 50 wt%, spray drying, and packaging.

Technical Field

The invention relates to an extraction method of oyster peptide.

Background

The marine food oyster has great edible value and medicinal value, is the first large cultured shellfish in the world, and is cultured in coastal areas such as Zhejiang province in China in a large scale. Zhejiang Ninghai is known as the 'village of oysters', and 700 years of oyster cultivation has been known. The oyster has high nutritive value, the protein content in the oyster is up to 45-57%, and the amino acid composition is complete. According to the evaluation of the world food and agricultural organization, the completeness and the mass ratio of the essential amino acid in the oyster meat are superior to those of human milk and cow milk. Ancient people called oyster as the "most expensive of aquatic products", ancient Roman praised it as "delicious fish in the sea", Western people called "magic stone" and "milk in the sea", Japanese did the reputation of "root source".

Modern marine organisms such as oysters contain considerable heavy metals due to marine pollution, and thus this technical problem needs to be considered when handling oysters.

Disclosure of Invention

The invention aims to provide an extraction method of oyster peptide, which has the advantage of removing heavy metals.

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

a method for extracting oyster peptide comprises the following steps:

(1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder;

(2) dispersing: mixing the fine oyster powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the oyster fine powder to the monocalcium carbonate to the sodium bicarbonate to the microcrystalline cellulose to the water is 1: 0.02-0.04: 0.04-0.06: 0.01-0.03: 23-28;

(3) enzymolysis: adding a lytic enzyme to the dispersion to allow the dispersion to undergo enzymatic hydrolysis;

(4) removing heavy metals: adding EDTA disodium into the dispersion liquid after enzymolysis, and stirring for reaction for 25-35 min;

(5) and (3) filtering: filtering the dispersion liquid after removing the metal, and taking filtrate;

(6) concentrating, drying, and packaging.

Further, in the step (3), the lytic enzyme comprises neutral protease and lipase, and the enzymolysis step comprises the following two parts:

adjusting the pH value of the dispersion liquid to 6.8-7.2, heating to 50-55 ℃, adding neutral protease, stirring for enzymolysis for 5-7 hr, heating to 75-85 ℃ after enzymolysis, and keeping the temperature for 8-12 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.2-0.4;

adjusting the pH value of the dispersion liquid to 6.8-7.2, heating to 38-42 ℃, adding lipase, stirring for enzymolysis for 2-4 hr, heating to 85-95 ℃ after enzymolysis, and keeping the temperature for 4-5 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the lipase is 100: 0.1-0.3.

Further, in the step (4), besides adding disodium EDTA to the dispersion, microcrystalline cellulose and ethyl acetate are added in the step (4); in the step (4), the mass ratio of the dispersion liquid after enzymolysis, EDTA disodium, microcrystalline cellulose and ethyl acetate is 100: 0.02-0.04: 0.08-0.11: 7-9.

Further, the method comprises the following steps:

(1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder;

(2) dispersing: mixing the fine oyster powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the oyster fine powder to the calcium dihydrogen carbonate to the sodium bicarbonate to the microcrystalline cellulose to the water is 1:0.03:0.05:0.02: 25;

(3) enzymolysis:

regulating pH of the dispersion to 7.0, heating to 52 deg.C, adding neutral protease, stirring for enzymolysis for 6hr, heating to 80 deg.C after enzymolysis, and keeping the temperature for 10min to inactivate enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.3;

adjusting pH of the dispersion to 7.0, heating to 40 deg.C, adding lipase, stirring for enzymolysis for 3hr, heating to 90 deg.C after enzymolysis, and keeping the temperature for 5min to inactivate enzyme; the mass ratio of the dispersion liquid to the lipase is 100: 0.2;

(4) removing heavy metals: adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the dispersion liquid after enzymolysis, and stirring to react for 30 min; in the step (4), the mass ratio of the dispersion liquid after enzymolysis, EDTA disodium, microcrystalline cellulose and ethyl acetate is 100:0.03:0.10: 8;

(5) and (3) filtering: vacuum filtering the dispersion solution after removing metals with 0.5 μm filter membrane, and collecting filtrate;

(6) vacuum concentrating to solid content of 50 wt%, spray drying, and packaging.

The technical effects of the invention are mainly reflected in the following aspects:

in the oyster processing process, EDTA disodium is used for carrying out heavy metal treatment, so that the heavy metal content in the product is reduced, and the food safety is improved; in the treatment process, because of the existence of heavy metals, polypeptide and heavy metals in the process have the possibility of chelation, so that chelated heavy metals exist in the product, calcium bicarbonate and the action of the heavy metals under the alkaline condition are utilized to promote and combine the obstruction of microcrystalline cellulose on dispersion and chelation of the calcium bicarbonate and the heavy metals, so that the chelation of the heavy metals and the polypeptide is reduced, the heavy metals are in a free state, the action of the heavy metals and EDTA disodium is facilitated, the heavy metal removal effect is improved, and the loss of the polypeptide is also reduced;

the method has the advantages that the emulsification stability is good in the enzymolysis liquid obtained after enzymolysis of the oysters, the separation is not ideal, the separation time and the product quality are affected, the fat is subjected to enzymolysis through lipase, the interaction of protein and fat is destroyed, and the stability of the emulsion is reduced, so that oil and water are separated, the separation effect is improved through the action of ethyl acetate and microcrystalline cellulose, the process is accelerated, and the protein recovery rate is improved.

Detailed Description

Example 1: a method for extracting oyster peptide comprises the following steps:

(1) crushing: pulverizing cleaned and dried Concha Ostreae and Carnis Ostreae into fine powder;

(2) dispersing: mixing the fine oyster powder, calcium dihydrogen carbonate, sodium bicarbonate, microcrystalline cellulose and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the oyster fine powder to the calcium dihydrogen carbonate to the sodium bicarbonate to the microcrystalline cellulose to the water is 1:0.03:0.05:0.02: 25;

(3) enzymolysis:

regulating pH of the dispersion to 7.0, heating to 52 deg.C, adding neutral protease, stirring for enzymolysis for 6hr, heating to 80 deg.C after enzymolysis, and keeping the temperature for 10min to inactivate enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.3;

adjusting pH of the dispersion to 7.0, heating to 40 deg.C, adding lipase, stirring for enzymolysis for 3hr, heating to 90 deg.C after enzymolysis, and keeping the temperature for 5min to inactivate enzyme; the mass ratio of the dispersion liquid to the lipase is 100: 0.2;

(4) removing heavy metals: adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the dispersion liquid after enzymolysis, and stirring to react for 30 min; in the step (4), the mass ratio of the dispersion liquid after enzymolysis, EDTA disodium, microcrystalline cellulose and ethyl acetate is 100:0.03:0.10: 8;

(5) and (3) filtering: vacuum filtering the dispersion solution after removing metals with 0.5 μm filter membrane, and collecting filtrate;

(6) vacuum concentrating to solid content of 50 wt%, spray drying, and packaging.

Example 1 molecular weight distribution of oyster peptide is shown in table 1.

TABLE 1

Molecular weight Range (Dalton)	Percentage peak area (%, lambda 220nm)
		M＞3000	0.15
2000＜M≤3000	0.44
		1000＜M≤2000	2.03
180＜M≤1000	17.25
		M≤180	80.13

Example 2: influence of step (2) on oyster peptide extraction method

Test objects: example 1, control 1-5.

Control group 1: referring to example 1, the difference from example 1 is that the input mass ratio of the oyster fine powder, monocalcium carbonate, sodium bicarbonate, microcrystalline cellulose and water in step (2) is 1:0.02:0.04:0.01: 25.

Control group 2: referring to example 1, the difference from example 1 is that the input mass ratio of the oyster fine powder, monocalcium carbonate, sodium bicarbonate, microcrystalline cellulose and water in step (2) is 1:0.04:0.06:0.03: 25.

Control group 3: referring to example 1, the difference from example 1 is that calcium bicarbonate, sodium bicarbonate or microcrystalline cellulose is not added to the oyster fine powder in step (2), only water is added, and the input mass ratio of the oyster fine powder to the water is 1: 25.

Control group 4: referring to example 1, the difference from example 1 is that in step (2), microcrystalline cellulose is not added to the oyster fine powder, and the input mass ratio of the oyster fine powder, monocalcium carbonate, sodium bicarbonate and water is 1:0.03:0.05: 25.

Control group 5: referring to example 1, the difference from example 1 is that the input mass ratio of the oyster fine powder, monocalcium carbonate, sodium bicarbonate, microcrystalline cellulose and water in step (2) is 1:0.06:0.08:0.05: 25.

The test contents are as follows: the crude protein and the total amount of the three heavy metals of lead, cadmium and chromium are respectively measured on the test object.

Determination of crude protein: measured by a Kjeldahl method, and the standard is GB 5009.5-2010. The protein recovery (%) is the product protein content x 100%/raw material protein content, i.e., the protein content in the product obtained after spray drying in step (6), and the raw material protein content is the protein content of the oyster fine powder obtained in step (1).

And (3) heavy metal determination: referring to GB15618-1995, the test object is the product obtained after spray drying in step (6).

The test results are shown in table 2. Table 2 shows:

(1) referring to the data of the protein recovery rate, along with the increase of the contents of the oyster fine powder, the monocalcium carbonate, the sodium bicarbonate and the microcrystalline cellulose in the step (2), the protein recovery rate shows a trend of increasing first and then reducing smoothly; when the oyster fine powder, the monocalcium carbonate, the sodium bicarbonate and the microcrystalline cellulose are 1:0.03:0.05:0.02 and 1:0.04:0.06:0.03, the protein recovery rate is over 85 percent;

(2) referring to the data of the total amount of lead, cadmium and chromium, the total amount of lead, cadmium and chromium tends to decrease first and then increase along with the increase of the contents of the oyster fine powder, the monocalcium carbonate, the sodium bicarbonate and the microcrystalline cellulose in the step (2); when the ratio of oyster fine powder to calcium bicarbonate, sodium bicarbonate and microcrystalline cellulose is 1:0.03:0.05:0.02, the total amount of lead, cadmium and chromium is 0.

TABLE 2

Example 3: influence of step (3) and step (4) on oyster peptide extraction method

Test objects: example 1, control 6-9.

Control group 6: referring to example 1, the difference from example 1 is that the enzymatic step of step (3) includes only one of the following: adjusting pH of the dispersion to 7.0, heating to 52 deg.C, adding neutral protease, stirring for enzymolysis for 6hr, heating to 80 deg.C after enzymolysis, and keeping the temperature for 10min to inactivate enzyme; the mass ratio of the dispersion liquid to the neutral protease is 100: 0.3.

Control group 7: referring to example 1, the difference from example 1 is that only disodium EDTA is added in step (4), and microcrystalline cellulose or ethyl acetate is not added.

Control group 8: referring to example 1, the difference from example 1 is that only disodium EDTA and microcrystalline cellulose are added in step (4), and ethyl acetate is not added.

Control group 9: referring to example 1, the difference from example 1 is that only disodium EDTA and ethyl acetate were added in step (4), and microcrystalline cellulose was not added.

The test contents are as follows: and (3) respectively measuring the time consumed by vacuum filtration of the test object, namely taking 1kg of the dispersion liquid treated in the step (4), carrying out vacuum filtration by using a 0.5-micron filter membrane, wherein the suction filtration pressure is 0.1MPa, the suction filtration temperature is 25 ℃, counting from the beginning of the suction filtration until the filtrate is completely filtered, and recording the total time consumed.

Determination of crude protein: measured by a Kjeldahl method, and the standard is GB 5009.5-2010. The protein recovery (%) is the product protein content x 100%/raw material protein content, i.e., the protein content in the product obtained after spray drying in step (6), and the raw material protein content is the protein content of the oyster fine powder obtained in step (1).

The test results are shown in table 3. Table 3 shows: compared with the control group 6 which is not subjected to the two-step treatment in the step (3), the vacuum-pumping filtration of the embodiment 1 takes longer time, and the protein recovery rate is higher; compared with the control groups 7-9 without adding ethyl acetate and/or microcrystalline cellulose in the step (4), the vacuum filtration of the example 1 takes more time and the protein recovery rate is higher.

TABLE 3

	Vacuum pumping filtration time (min)	Protein recovery (%)
			Example 1	10	85.2
Control group 6	92	63.0
			Control group 7	53	70.2
Control group 8	88	68.1
			Control group 9	82	66.4

The above are only typical examples of the present invention, and besides, the present invention may have other embodiments, and all the technical solutions formed by equivalent substitutions or equivalent changes are within the scope of the present invention as claimed.