阅读说明：本技术 一种水母提取物的制备方法 (Preparation method of jellyfish extract ) 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：本申请公开一种水母提取物的制备方法,包括以下步骤：(1)取洗净后的水母触手,组织匀浆,冷冻干燥得到冻干粉；(2)将冻干粉和水混合,搅拌,分散均匀得到分散液；(3)调节分散液的pH至7.5～8.0,升温至45～50℃,加入胰蛋白酶,搅拌使之酶解,酶解后使酶失活；(4)向酶解后的分散液中乙醇和氯化钠,搅拌,在20～25℃下保温60～120s,离心取滤液；(5)取离心后的滤液,加入EDTA二钠,搅拌反应除重金属；(6)过滤：将去重金属后的滤液用0.5μm滤膜真空抽滤,取滤液；(7)浓缩、干燥,过程控温、控量等,具有抑制ACE的优点。(The application discloses a preparation method of a jellyfish extract, which comprises the following steps: (1) taking the cleaned jellyfish to touch hands, homogenizing tissues, and freeze-drying to obtain freeze-dried powder; (2) mixing the freeze-dried powder and water, stirring, and uniformly dispersing to obtain a dispersion liquid; (3) adjusting the pH value of the dispersion liquid to 7.5-8.0, heating to 45-50 ℃, adding trypsin, stirring for enzymolysis, and inactivating enzyme after enzymolysis; (4) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, preserving the heat for 60-120 s at 20-25 ℃, and centrifuging to obtain filtrate; (5) adding EDTA disodium into the centrifuged filtrate, and stirring to react to remove heavy metals; (6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate; (7) concentration, drying, temperature control and quantity control in the process and the like, and has the advantage of inhibiting ACE.)

1. The preparation method of the jellyfish extract is characterized by comprising the following steps:

(1) taking the cleaned jellyfish to touch hands, homogenizing tissues, and freeze-drying to obtain freeze-dried powder;

(2) mixing the freeze-dried powder and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the freeze-dried powder to water is 1: 8-12;

(3) adjusting the pH value of the dispersion liquid to 7.5-8.0, heating to 45-50 ℃, adding trypsin, stirring for enzymolysis for 1.5-2.5 hr, heating to 95-100 ℃ after enzymolysis, and keeping the temperature for 12-20 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.4-0.6;

(4) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, preserving the heat for 60-120 s at 20-25 ℃, and centrifuging to obtain filtrate; the mass ratio of the dispersion liquid to the ethanol to the sodium chloride is 1: 0.04-0.06: 0.002-0.004;

(5) adding EDTA disodium into the centrifuged filtrate, and stirring to react to remove heavy metals; the mass ratio of the centrifuged filtrate to the EDTA disodium is 100: 0.01-0.03;

(6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate;

(7) concentrating and drying.

2. The method for preparing jellyfish extract as claimed in claim 1, wherein jellyfish is selected from Cyanea alba; the step (3) comprises the following two parts:

regulating the pH value of the dispersion liquid to 7.5-8.0, heating to 45-50 ℃, adding trypsin, stirring for enzymolysis for 1.5-2.5 hr, heating to 95-100 ℃ after enzymolysis, and keeping the temperature for 12-20 min to inactivate enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.4-0.6;

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 preparing the jellyfish extract as claimed in claim 2, wherein the calcium bicarbonate, the sodium bicarbonate and the microcrystalline cellulose are added in the step (2), and the mass ratio of the freeze-dried powder, the calcium bicarbonate, the sodium bicarbonate, the microcrystalline cellulose and the water is 1: 0.02-0.03: 0.04-0.05: 0.01-0.03: 8-12.

4. The method for preparing an aequoria victoria extract according to claim 3, wherein in step (5), in addition to the disodium EDTA is added to the filtrate, microcrystalline cellulose and ethyl acetate are added in step (5); in the step (5), the mass ratio of the centrifuged filtrate to the EDTA disodium salt to the microcrystalline cellulose to the ethyl acetate is 100: 0.01-0.03: 0.07-0.09: 10-12.

5. The method for preparing an extract of jellyfish as claimed in claim 4, wherein the filtrate obtained in the step (6) is subjected to the following steps in addition to suction filtration: mixing the filtrate obtained after suction filtration with calcium chloride according to the mass ratio of 1: 0.06-0.10, adjusting the pH value to 6.5-7.0 by using acetic acid, and stirring and reacting at 40-45 ℃ for 40-50 min.

6. The method for preparing the jellyfish extract as claimed in claim 5, comprising the steps of:

(1) taking the cleaned white cyanea nozakii tentacles, homogenizing tissues, and freeze-drying to obtain freeze-dried powder;

(2) mixing the freeze-dried 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 freeze-dried powder, the monocalcium carbonate, the sodium bicarbonate, the microcrystalline cellulose and the water is 1:0.02:0.04:0.02: 10;

(3) enzymolysis:

regulating pH of the dispersion to 7.8, heating to 45 deg.C, adding trypsin, stirring for enzymolysis for 2.0hr, heating to 100 deg.C after enzymolysis, and keeping the temperature for 15min to inactivate enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.5;

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) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, keeping the temperature at 25 ℃ for 90s, and centrifuging to obtain filtrate; the mass ratio of the dispersion liquid to the ethanol to the sodium chloride is 1:0.05: 0.003;

(5) adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the centrifuged filtrate, and stirring for reaction to remove heavy metals; the mass ratio of the centrifuged filtrate to the EDTA disodium salt to the microcrystalline cellulose to the ethyl acetate is 100:0.02:0.08: 10;

(6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate; mixing the filtrate obtained after suction filtration with calcium chloride according to the mass ratio of 1:0.08, adjusting the pH to 6.8 with acetic acid, and stirring and reacting at 42 ℃ for 45 min;

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

Technical Field

The invention relates to a preparation method of jellyfish extract.

Background

White cyanea nozakii is commonly known as: jellyfish and jellyfish. White Cyanea nozakii Kishinouye belongs to the phylum coelenterate, the subdivision Cyrtonema (Coelenterata), the Class of Pot jellyfish (Class Scyphomedusae), the order of Siphonosteales (Semaeostomeae), the family of Cyaneae (Cyaneidae), the genus Cyanea, and is a large group of marine zooplankton. The white cyanea nozakii has abundant resources and wide distribution range, and is the dominant population in large jellyfishes in China coastal continental shelf areas.

At present, researches on cyanea nozakii at home and abroad mainly focus on the aspects of morphological structure, growth rule, fishery ecology, harm to marine fishery resources and the like of cyanea nozakii. The research on the medicinal value of the cyanea nozakii mainly focuses on the aspect of jellyfish toxin, and the cyanea nozakii is rich in collagen, but only primary processed food is taken as the main product, only food-class instant products are seen in the market, and the development of downstream deep-processed products is not seen.

Therefore, the development of a jellyfish extract is of great significance.

Disclosure of Invention

The invention aims to provide a preparation method of jellyfish extract, which has the advantage of inhibiting ACE.

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

a method for preparing jellyfish extract comprises the following steps:

(1) taking the cleaned jellyfish to touch hands, homogenizing tissues, and freeze-drying to obtain freeze-dried powder;

(2) mixing the freeze-dried powder and water, stirring, and uniformly dispersing to obtain a dispersion liquid; the adding mass ratio of the freeze-dried powder to water is 1: 8-12;

(3) adjusting the pH value of the dispersion liquid to 7.5-8.0, heating to 45-50 ℃, adding trypsin, stirring for enzymolysis for 1.5-2.5 hr, heating to 95-100 ℃ after enzymolysis, and keeping the temperature for 12-20 min to inactivate the enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.4-0.6;

(4) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, preserving the heat for 60-120 s at 20-25 ℃, and centrifuging to obtain filtrate; the mass ratio of the dispersion liquid to the ethanol to the sodium chloride is 1: 0.04-0.06: 0.002-0.004;

(5) adding EDTA disodium into the centrifuged filtrate, and stirring to react to remove heavy metals; the mass ratio of the centrifuged filtrate to the EDTA disodium is 100: 0.01-0.03;

(6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate;

(7) concentrating and drying.

Preferably, the jellyfish is Cyanea nozakii with white color; the step (3) comprises the following two parts:

regulating the pH value of the dispersion liquid to 7.5-8.0, heating to 45-50 ℃, adding trypsin, stirring for enzymolysis for 1.5-2.5 hr, heating to 95-100 ℃ after enzymolysis, and keeping the temperature for 12-20 min to inactivate enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.4-0.6;

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.

Preferably, calcium bicarbonate, sodium bicarbonate and microcrystalline cellulose are added in the step (2), and the adding mass ratio of the freeze-dried powder, the calcium bicarbonate, the sodium bicarbonate, the microcrystalline cellulose and the water is 1: 0.02-0.03: 0.04-0.05: 0.01-0.03: 8-12.

Preferably, in the step (5), besides adding disodium EDTA to the filtrate, microcrystalline cellulose and ethyl acetate are added in the step (5); in the step (5), the mass ratio of the centrifuged filtrate to the EDTA disodium salt to the microcrystalline cellulose to the ethyl acetate is 100: 0.01-0.03: 0.07-0.09: 10-12.

Preferably, in step (6), in addition to the suction filtration, the filtrate is subjected to the following treatment: mixing the filtrate obtained after suction filtration with calcium chloride according to the mass ratio of 1: 0.06-0.10, adjusting the pH value to 6.5-7.0 by using acetic acid, and stirring and reacting at 40-45 ℃ for 40-50 min.

Preferably, the method comprises the following steps:

(1) taking the cleaned white cyanea nozakii tentacles, homogenizing tissues, and freeze-drying to obtain freeze-dried powder;

(2) mixing the freeze-dried 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 freeze-dried powder, the monocalcium carbonate, the sodium bicarbonate, the microcrystalline cellulose and the water is 1:0.02:0.04:0.02: 10;

(3) enzymolysis:

regulating pH of the dispersion to 7.8, heating to 45 deg.C, adding trypsin, stirring for enzymolysis for 2.0hr, heating to 100 deg.C after enzymolysis, and keeping the temperature for 15min to inactivate enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.5;

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) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, keeping the temperature at 25 ℃ for 90s, and centrifuging to obtain filtrate; the mass ratio of the dispersion liquid to the ethanol to the sodium chloride is 1:0.05: 0.003;

(5) adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the centrifuged filtrate, and stirring for reaction to remove heavy metals; the mass ratio of the centrifuged filtrate to the EDTA disodium salt to the microcrystalline cellulose to the ethyl acetate is 100:0.02:0.08: 10;

(6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate; mixing the filtrate obtained after suction filtration with calcium chloride according to the mass ratio of 1:0.08, adjusting the pH to 6.8 with acetic acid, and stirring and reacting at 42 ℃ for 45 min;

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

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

the white cyanea nozakii is rich in collagen, and the small molecular peptide substance obtained by the method can effectively inhibit Angiotensin Converting Enzyme (ACE) so as to achieve the effect of reducing blood pressure, and is high in safety due to being derived from food;

in the treatment process, EDTA disodium is used for carrying out heavy metal treatment, so that the content of heavy metal 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 enzymolysis liquid obtained after enzymolysis is good in emulsification stability, unsatisfactory in separation and capable of influencing separation time and product quality, fat is subjected to enzymolysis through lipase, the interaction between protein and fat is destroyed, and the stability of 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;

after enzymolysis, the stability of the product is improved by ethanol sedimentation and chelation of the filtrate and calcium chloride, and the product is suitable for room temperature storage.

Detailed Description

Example 1: a method for preparing jellyfish extract comprises the following steps:

(1) taking the cleaned white cyanea nozakii tentacles, homogenizing tissues, and freeze-drying to obtain freeze-dried powder;

(2) mixing the freeze-dried 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 freeze-dried powder, the monocalcium carbonate, the sodium bicarbonate, the microcrystalline cellulose and the water is 1:0.02:0.04:0.02: 10;

(3) enzymolysis:

regulating pH of the dispersion to 7.8, heating to 45 deg.C, adding trypsin, stirring for enzymolysis for 2.0hr, heating to 100 deg.C after enzymolysis, and keeping the temperature for 15min to inactivate enzyme; the mass ratio of the dispersion liquid to the trypsin is 100: 0.5;

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) adding ethanol and sodium chloride into the dispersion liquid after enzymolysis, stirring, keeping the temperature at 25 ℃ for 90s, and centrifuging to obtain filtrate; the mass ratio of the dispersion liquid to the ethanol to the sodium chloride is 1:0.05: 0.003;

(5) adding EDTA disodium, microcrystalline cellulose and ethyl acetate into the centrifuged filtrate, and stirring for reaction to remove heavy metals; the mass ratio of the centrifuged filtrate to the EDTA disodium salt to the microcrystalline cellulose to the ethyl acetate is 100:0.02:0.08: 10;

(6) and (3) filtering: vacuum filtering the filtrate with 0.5 μm filter membrane to obtain filtrate; mixing the filtrate obtained after suction filtration with calcium chloride according to the mass ratio of 1:0.08, adjusting the pH to 6.8 with acetic acid, and stirring and reacting at 42 ℃ for 45 min;

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

Example 2: effect of step (2) on the method of preparing jellyfish extract

Test objects: example 1, control 1-4.

Control group 1: referring to example 1, the difference from example 1 is that, in step (2), the input mass ratio of the lyophilized powder, calcium bicarbonate, sodium bicarbonate, microcrystalline cellulose and water is 1:0.03:0.05:0.03: 10.

Control group 2: referring to example 1, the difference from example 1 is that calcium bicarbonate, sodium bicarbonate, or microcrystalline cellulose is not added to the lyophilized powder in step (2), only water is added, and the mass ratio of the lyophilized powder to the water is 1: 10.

Control group 3: referring to example 1, the difference from example 1 is that microcrystalline cellulose is not added to the lyophilized powder in step (2), and the mass ratio of the lyophilized powder, calcium bicarbonate, sodium bicarbonate and water is 1:0.02:0.04: 10.

Control group 4: referring to example 1, the difference from example 1 is that, in step (2), the input mass ratio of the lyophilized powder, calcium bicarbonate, sodium bicarbonate, microcrystalline cellulose and water is 1:0.05:0.07:0.06: 10.

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 × 100%/raw material protein content, i.e., the protein content in the product obtained after spray-drying in step (7), and the raw material protein content is the lyophilized protein content 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 (7).

The test results are shown in table 1. Table 1 shows: the procedure of step (2) of example 1 and control 1 was followed, and the protein recovery rate was high, the heavy metal content was low, and example 1 was the best.

TABLE 1

Example 3: influence of step (3) and step (5)

Test objects: example 1, control 5-7.

Control group 5: 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 7.8, heating to 45 deg.C, adding trypsin, stirring for enzymolysis for 2.0hr, heating to 100 deg.C after enzymolysis, and keeping the temperature for 15min to inactivate enzyme; the mass ratio of the dispersion to trypsin was 100: 0.5.

Control group 6: referring to example 1, the difference from example 1 is that step (3) includes:

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 to the lipase was 100: 0.2.

Control group 7: referring to example 1, the difference from example 1 is that only disodium EDTA was added to the filtrate after centrifugation in step (5), and microcrystalline cellulose or ethyl acetate 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 filtrate treated in the step (5), 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 ratio (%) — product protein content × 100%/raw material protein content, i.e., protein content in the product obtained after spray drying in step (7), and raw material protein content is protein content of the lyophilized powder obtained in step (1).

The test results are shown in table 2. Table 2 shows that, compared with control group 5 which was not subjected to the two-step treatment in step (3) and control group 6 which was treated with neutral protease in (r), vacuum filtration of example 1 of the present application took more time and protein recovery was higher; compared with the control group 7 without adding ethyl acetate or microcrystalline cellulose in the step (5), the vacuum filtration of the example 1 of the present application takes more time and the protein recovery rate is higher.

TABLE 2

	Vacuum pumping filtration time (min)	Protein recovery (%)
			Example 1	7	74.3
Control group 5	56	51.0
			Control group 6	31	50.3
Control group 7	36	47.5

Example 4: ACE activity assay

Test objects: example 1, control groups 8-9, control group 6.

Control group 8: referring to example 1, the difference from example 1 is that step (4) is not provided.

Control group 9: referring to example 1, the difference from example 1 is that step (6) includes only filtration: and (4) carrying out vacuum filtration on the filtrate from which the heavy metals are removed by using a 0.5-micron filter membrane, and taking the filtrate.

Determination of ACE inhibition: the test object was dissolved in 0.1mol/L boric acid buffer (pH 8.3, containing 0.3mol/L NaCl) to prepare a sample solution. HHL (equacy-histidyl-leucine) and ACE (angiotensin converting enzyme) were mixed with a 5mmol/L HHL solution and a 100Mu/mL ACE solution in 0.1mol/L boric acid buffer (pH 8.3, containing 0.3mol/L NaCl), respectively. Mixing 30 μ L HHL with 10 μ L sample test solution (blank group using boric acid buffer solution instead of sample test solution), preheating at 37 deg.C for 5min, adding 20 μ L ACE solution, mixing, reacting at 37 deg.C for 1hr, adding 70 μ L1 mol/L HCl to terminate the reaction, and analyzing the result with high performance liquid chromatography.

Chromatographic conditions are as follows: Xbridge-C18 column (4.6X 250mm, 5 μm, water company, USA); mobile phase: a is 0.05% trifluoroacetic acid aqueous solution, B is acetonitrile, and gradient elution conditions are as follows: 0-30 min, 10-60% (volume) of mobile phase B, and the flow rate of 0.8 mL/min; the detection wavelength is 228 nm; the column temperature is 30 ℃; the amount of the sample was 10. mu.l.

The samples were stored in glass bottles in a sealed manner and placed at 0 ℃ and 25 ℃ for 60 days, respectively, for the ACE inhibition test.

TABLE 3

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.