CN111440835B - Method for extracting active peptide from low-value marine organism leftovers - Google Patents
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Abstract
The invention discloses a method for extracting active peptide from low-value marine organism leftovers, which comprises the following steps: (1) washing the low-value marine organism leftovers, adding water, and pulping to prepare a raw material liquid; (2) inoculating lactobacillus casei to the raw material liquid prepared in the step (1), and carrying out aerobic fermentation to prepare fermentation liquid; (3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2); (4) repeating the operation step (3), and mixing the supernatant obtained by each centrifugation to obtain a final fermented product; (5) and (4) separating the final fermentation product obtained in the step (4) by an ultrafiltration membrane, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide. The active peptide prepared by the extraction method has high yield, not only has the function of reducing uric acid, but also can effectively treat hyperplasia of mammary glands.
Description
Technical Field
The invention relates to the technical field of recycling and extracting active peptide from low-value marine organism leftovers, in particular to a method for extracting active peptide from low-value marine organism leftovers.
Background
In the marine organism processing process, marine organism leftovers such as fish, squid viscera and the like are generally not easy to be utilized and are directly discarded, so that on one hand, the waste of resources is caused, and on the other hand, the environmental pollution is caused.
The hyperplasia of mammary glands is the retrograde and progressive change of the structure of a mammary duct and lobules, takes breast pain and breast lumps as main clinical manifestations, is the most common mammary gland disease of women of childbearing age, seriously influences the life quality of patients, and is generally treated by hormones and vitamins in the existing treatment mode, but the curative effect is not exact, the side effect is large, and the treatment effect is poor. At present, no low-value marine organism leftovers are applied to relevant research on prevention and treatment of hyperplasia of mammary glands, and the active peptide extracted by the method has an important effect on prevention and treatment of hyperplasia of mammary glands after research.
Disclosure of Invention
In view of the prior art, the invention aims to provide a method for extracting active peptide from low-value marine organism leftovers. The active peptide is extracted according to the method, the yield of the active peptide is high, and the extracted active peptide not only has the function of reducing uric acid, but also can effectively treat hyperplasia of mammary glands; the invention reuses the low-value marine organism leftovers, avoids the waste of the leftovers and develops a new way for utilizing the low-value marine organism leftovers.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a method for extracting active peptide from low-value marine organism leftovers is provided, which comprises the following steps:
(1) washing the low-value marine organism leftovers, adding water, and pulping to prepare a raw material liquid with the mass concentration of 10% -15%;
(2) inoculating lactobacillus casei into the raw material liquid prepared in the step (1), and performing aerobic fermentation for 2-3 days at the temperature of 20-30 ℃ to prepare fermentation liquid;
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2);
(4) repeating the step (3), realizing circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) And (5) separating the final fermentation product obtained in the step (4) by an ultrafiltration membrane to obtain a permeate, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide.
Preferably, the low-value marine organism leftovers are viscera of fishes and squids.
Preferably, in the step (2), the inoculation amount of the lactobacillus paracasei is 105-106CFU/mL。
Preferably, the lactobacillus paracasei is deposited by China Industrial microorganism culture Collection, the strain numbers are CICC20241 and CICC6270, and the inoculation ratio of the CICC20241 and the CICC6270 is 1 (2-4).
Preferably, in the step (3), the solid-to-liquid ratio of the precipitate to the water is 1g (4-5) ml.
Preferably, in the step (4), the operation of the step (3) is repeated 3 to 4 times.
Preferably, in step (5), the membrane flux of the ultrafiltration membrane is 5000 Da.
Preferably, in the step (5), centrifugal impurity removal is performed before membrane separation, and the centrifugal force is 8000-10000 g.
In a second aspect of the present invention, the active peptide is extracted by the above method.
In a third aspect of the invention, the application of the active peptide in preparing a medicament for treating hyperplasia of mammary glands is provided.
In a fourth aspect of the present invention, there is provided a pharmaceutical agent for treating mammary gland hyperplasia, which comprises the above-mentioned active peptide as an active ingredient.
The invention has the beneficial effects that:
the active peptide is extracted according to the method, the yield of the active peptide is high, and the extracted active peptide not only has the function of reducing uric acid, but also can effectively treat hyperplasia of mammary glands; the invention reuses the low-value marine organism leftovers, avoids the waste of the leftovers and develops a new way for utilizing the low-value marine organism leftovers; wide raw material source, low price, low production cost, simple production process and suitability for various scale production.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, the utilization effect of the marine organism leftovers is poor and the yield of the active peptides is low. Based on the above, the active peptide is extracted by the extraction method, the yield of the active peptide is high, and the extracted active peptide not only has the effect of reducing uric acid, but also can effectively treat hyperplasia of mammary glands; the invention reuses the low-value marine organism leftovers, avoids the waste of the leftovers and develops a new way for utilizing the low-value marine organism leftovers.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples and comparative examples of the present invention are conventional in the art and are commercially available.
Example 1: a method for extracting active peptide from low-value marine organism leftovers comprises the following steps:
(1) cleaning the internal organs of the pomfret, adding water, and pulping to prepare a raw material liquid with the mass concentration of 15%;
(2) inoculating lactobacillus casei in the raw material liquid prepared in the step (1) with the inoculation amount of 105CFU/mL, the lactobacillus casei is preserved by China Industrial microbial culture Collection management center, the strain numbers are CICC20241 and CICC6270, and the inoculation ratio of the CICC20241 to the CICC6270 is 1: 2; performing aerobic fermentation for 3 days at 20 deg.C to obtain fermentation broth;
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2), wherein the solid-to-liquid ratio of the precipitate to the water is 1g:4 ml;
(4) repeating the step (3) for 3 times to realize circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) And (5) carrying out centrifugation and impurity removal on the final fermentation product obtained in the step (4), wherein the centrifugal force is 8000g, then carrying out ultrafiltration membrane separation, wherein the membrane flux of an ultrafiltration membrane is 5000Da, evaporating and concentrating the permeate, and carrying out freeze drying to obtain the active peptide.
Determination of active peptide content by biuret method
1. Preparation of Standard Curve
Taking ten 10ml volumetric flasks, preparing 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8mg/ml Gly-Gly-Tyr-Arg tetrapeptide standard solutions by using 5% TCA in sequence, then respectively taking 6.0ml standard solutions, adding 4.0ml biuret reagent, uniformly mixing, standing for 10min, centrifuging at 2000r/min for 10min, taking supernate and determining the OD value at 540 nm. A standard curve was prepared with the concentration of the active peptide as X (mg/ml) abscissa and the OD value as Y ordinate.
2. Determination of active peptide content
Determination procedure of active peptide content: taking 2.5ml of sample liquid, adding 2.5ml of 10% (W/V) trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, centrifuging for 15min at 4000r/min, transferring all supernate into a 50ml volumetric flask, fixing the volume to the scale by using 5% TCA, and uniformly shaking. And then 6.0ml of the solution is put into another test tube, 4.0ml of biuret reagent (sample solution: biuret reagent is 3:2, V/V) is added into the test tube, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, the mixture is centrifuged at 2000r/min for 10min, the supernatant is taken and the OD value is measured at 540nm, the concentration of the active peptide in the sample solution is obtained by contrasting a standard curve, and the content of the active peptide in the sample can be further obtained.
The preparation method of biuret comprises the following steps: adding the biuret reagent A into the tissue sample solution, oscillating uniformly (an alkaline environment must be created), adding the biuret reagent B, and oscillating uniformly; the ingredient of the biuret reagent A is an aqueous solution with the mass fraction of sodium hydroxide of 0.1 g/mL; the ingredient of the biuret reagent B is an aqueous solution with the mass fraction of copper sulfate of 0.01 g/mL.
Calculating the yield of the active peptide:
yield (%) of active peptide (polypeptide content in ultrafiltration membrane permeate-polypeptide content in raw material solution)/total protein content
The total protein content is determined by the existing phenol reagent method, and the determination of the total protein content by the phenol reagent method belongs to the existing conventional technology and is not described in detail herein.
The yield of active peptide was calculated to be 89.5%.
Example 2: a method for extracting active peptide from low-value marine organism leftovers comprises the following steps:
(1) cleaning internal organs of the large yellow croaker, adding water, and pulping to prepare a raw material liquid with the concentration of 10%;
(2) inoculating lactobacillus casei in the raw material liquid prepared in the step (1) with the inoculation amount of 106CFU/mL, wherein the lactobacillus casei is preserved by China Industrial microorganism culture Collection, the strain numbers are CICC20241 and CICC6270, the inoculation ratio of the CICC20241 to the CICC6270 is 1:4, and aerobic fermentation is carried out for 2 days at the temperature of 30 ℃ to prepare fermentation liquor;
(3) Centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2), wherein the solid-to-liquid ratio of the precipitate to the water is 1g:5 ml;
(4) repeating the step (3) for 3 times to realize circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) and (4) carrying out centrifugal impurity removal on the final fermentation product obtained in the step (4), wherein the centrifugal force is 10000g, then carrying out ultrafiltration membrane separation, wherein the membrane flux of an ultrafiltration membrane is 5000Da, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide. The yield of active peptides was 90%.
Example 3: a method for extracting active peptide from low-value marine organism leftovers comprises the following steps:
(1) cleaning internal organs of the squid, adding water, and pulping to prepare a raw material liquid with the concentration of 12%;
(2) inoculating lactobacillus casei in the raw material liquid prepared in the step (1) with the inoculation amount of 106CFU/mL, the lactobacillus paracasei is preserved by China center for the preservation and management of industrial microorganism strains, and the strain number is CICC20241 and CICC6270, wherein the inoculation ratio of the CICC20241 and the CICC6270 is 1:3, and the fermentation liquid is prepared by aerobic fermentation for 2 days at the temperature of 25 ℃;
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2), wherein the solid-to-liquid ratio of the precipitate to the water is 1g:4 ml;
(4) Repeating the step (3) for 4 times to realize circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) and (5) carrying out centrifugation and impurity removal on the final fermentation product obtained in the step (4), wherein the centrifugal force is 9000g, then carrying out ultrafiltration membrane separation, wherein the membrane flux of an ultrafiltration membrane is 5000Da, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide. The yield of active peptide was 91.6%.
Comparative example 1
A method for extracting active peptide from low-value marine organism leftovers comprises the following steps:
(1) cleaning internal organs of the squid, adding water, and pulping to prepare a raw material liquid with the concentration of 12%;
(2) inoculating 10 spherical lactobacillus into the raw material liquid prepared in the step (1)6Performing aerobic fermentation for 2 days at the temperature of 25 ℃ by CFU/mL to obtain fermentation liquor;
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2), wherein the solid-to-liquid ratio of the precipitate to the water is 1g:4 ml;
(4) repeating the step (3) for 4 times to realize circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) and (4) carrying out centrifugal impurity removal on the final fermentation product obtained in the step (4), wherein the centrifugal force is 9000g, then carrying out ultrafiltration membrane separation, wherein the membrane flux of an ultrafiltration membrane is 5000Da, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide. The yield of active peptide was 71.6%.
Comparative example 1 compared to example 3, the fermentation was carried out using a different species.
Comparative example 2
A method for extracting active peptide from low-value marine organism leftovers comprises the following steps:
(1) cleaning internal organs of the squid, adding water, and pulping to prepare a raw material solution with the concentration of 12%;
(2) inoculating lactobacillus casei into the raw material liquid prepared in the step (1) in an inoculation amount of 106CFU/mL, wherein the lactobacillus casei is preserved by China Industrial microorganism culture Collection, the strain numbers are CICC20241 and CICC6270, the inoculation ratio of the CICC20241 and the CICC6270 is 1:3, and the fermentation liquid is prepared by aerobic fermentation for 5 days at the temperature of 25 ℃;
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain supernatant;
(4) and (4) centrifuging the supernatant obtained in the step (3) to remove impurities, wherein the centrifugal force is 9000g, then separating by an ultrafiltration membrane with the membrane flux of 5000Da, evaporating and concentrating the permeate, and freeze-drying to obtain the active peptide. The yield of active peptide was 79.6%.
Comparative example 2 compared to example 3, the fermentation conditions were different and no cyclic fermentation was used.
The active peptides extracted in examples 1 to 3 were subjected to animal experiments to verify the effect of treating hyperplasia of mammary glands.
Animal experiments:
wistar rats (provided by Experimental animal center in Shandong province) with the weight of 220-240g are subjected to adaptive pre-feeding for 1 week before the test; the rats were randomly divided into 5 groups, the number of rats in each group was 10, and the 5 groups of rats were divided into a blank group, a model control group, an example 1 group, an example 2 group, and an example 3 group. Except for the blank group, rats in other groups are injected with 0.5mg/kg of estradiol benzoate injection subcutaneously once a day for 25 consecutive days, the blank group is injected with equal volume of normal saline subcutaneously, and the injection is changed to 4mg/kg of progesterone subcutaneously on the 26 th day for 5 consecutive days, and the blank group is injected with equal volume of normal saline subcutaneously. The modeling was successful after 30 days.
After the modeling is successful, 2ml of physiological saline is respectively infused into the blank group and the model control group, 2g/kg of active peptide prepared in the example 1 is infused into the example 1 group, 1 time per day and 20 days are continued; example 2 groups were drenched with 2g/kg of the active peptide prepared in example 2 1 time per day for 20 consecutive days; example 3 groups were drenched with 2g/kg of the active peptide prepared in example 3 1 time per day for 20 consecutive days.
The diameter and height of the second pair of nipples of each rat in each group were measured with a vernier caliper after the last administration, and the measurement results are shown in table 1.
Table 1:
| group of | Nipple diameter (mm) | Nipple height (mm) |
| Blank group | 1.05±0.28 | 1.08±0.22 |
| Model control group | 1.33±0.43 | 1.36±0.40 |
| EXAMPLE 1 group | 1.22±0.29 | 1.24±0.31 |
| EXAMPLE 2 group | 1.20±0.28 | 1.23±0.33 |
| EXAMPLE 3 group | 1.15±0.29 | 1.19±0.31 |
And (3) pathological detection: the rat 2 nd pair was removed and the intact mammary glands were fixed in 10% formalin, sectioned with paraffin embedding, and examined for pathological tissue of the mammary glands with an optical lens after HE staining. The mammary tissue was classified into four grades according to the pathological proliferation status and scored.
No hyperplasia; only the lobules are sparsely distributed, the number of acinus in each lobule is 5-10, no secretion exists in the catheter, and connective tissues exist among the acinus, and the score is 0;
mild hyperplasia: slightly increasing lobules, wherein 5-10 lobules can be seen in each low power visual field, increasing acinus number in the lobules to about 10, and some connective tissues are among the acinus, and some ducts are expanded and counted for 1 point;
moderate hyperplasia: under the low power microscope, 10 small leaves can be seen and are widely distributed, each small leaf has nearly 20 acini, the glands are in a back-to-back shape, and the duct is expanded or secreted, and the score is 2;
severe hyperplasia: the number of lobules is more, the lobules account for 1/2-3/4 under the low power visual field, the muscles and subcutaneous parts are distributed with the lobules, the lobules and acini are widely proliferated, and the ducts and the small ducts have more secretions, and the score is 3;
the measurement results are shown in table 2 below.
Table 2:
as can be seen from table 1 above, the height of the nipple and the diameter of the nipple are significantly reduced in the example 1 group, the example 2 group and the example 3 group, and as can be seen from table 2 above, the degree of hyperplasia of mammary glands is significantly reduced under the scope of the example 1 group, the example 2 group and the example 3 group, indicating that the active peptide extracted by the present invention has an inhibitory effect on hyperplasia of mammary glands.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (4)
1. A method for extracting active peptide from low-value marine organism leftovers is characterized by comprising the following steps:
(1) washing the low-value marine organism leftovers, adding water, and pulping to prepare a raw material liquid with the mass concentration of 10% -15%;
(2) inoculating lactobacillus casei into the raw material liquid prepared in the step (1), and performing aerobic fermentation for 2-3 days at the temperature of 20-30 ℃ to prepare fermentation liquid; the inoculation amount of the lactobacillus paracasei is 105-106CFU/mL; the lactobacillus paracasei is preserved by China industrial microorganism strain preservation management center, the strain numbers are CICC20241 and CICC6270, and the inoculation proportion of the CICC20241 and the CICC6270 is 1 (2-4);
(3) centrifuging the fermentation liquor prepared in the step (2) to obtain a precipitate and a supernatant, adding water into the precipitate, and repeating the step (2); the solid-liquid ratio of the precipitate to water is 1g (4-5) ml;
(4) Repeating the step (3) to realize circulating fermentation, and mixing the supernatant obtained by each centrifugation to obtain a final fermented product;
(5) separating the final fermentation product obtained in the step (4) by an ultrafiltration membrane, and performing centrifugal impurity removal before membrane separation, wherein the centrifugal force is 8000-10000 g; separating with membrane to obtain permeate, evaporating and concentrating the permeate, and lyophilizing to obtain active peptide; the membrane flux of the ultrafiltration membrane is 5000 Da.
2. The active peptide is extracted by the method of claim 1.
3. Use of the active peptide of claim 2 for the manufacture of a medicament for the treatment of mammary gland hyperplasia.
4. A medicament for the treatment of hyperplasia of mammary glands, characterized by: the agent comprises the active peptide according to claim 2 as an active ingredient.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1466529A1 (en) * | 2003-04-07 | 2004-10-13 | Chr. Hansen A/S | Composition with heart rate reducing properties |
| CN104694615A (en) * | 2015-03-03 | 2015-06-10 | 江南大学 | Method for preparing antihypertensive peptides by utilizing probiotic fermented marine organism processing leftovers |
| CN106036317A (en) * | 2016-06-12 | 2016-10-26 | 中国食品发酵工业研究院 | Asparagus fermented beverage with auxiliary hypolipidemic function and preparation method of asparagus fermented beverage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1466529A1 (en) * | 2003-04-07 | 2004-10-13 | Chr. Hansen A/S | Composition with heart rate reducing properties |
| CN104694615A (en) * | 2015-03-03 | 2015-06-10 | 江南大学 | Method for preparing antihypertensive peptides by utilizing probiotic fermented marine organism processing leftovers |
| CN106036317A (en) * | 2016-06-12 | 2016-10-26 | 中国食品发酵工业研究院 | Asparagus fermented beverage with auxiliary hypolipidemic function and preparation method of asparagus fermented beverage |
Non-Patent Citations (2)
| Title |
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| Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed;Jovanka Lukic 等;《plos one》;20190711;第1-18页 * |
| 海洋胶原肽长期喂养对SD 大鼠自发肿瘤的抑制作用;梁江 等;《食品与发酵工业》;20110531;第1-6页 * |
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