Breeding method of selenium-rich poecilobdella manillensis and preparation method and application of selenium-rich poecilobdella manillensis freeze-dried powder

Abstract

The invention relates to a breeding method of selenium-rich poecilobdella manillensis and a preparation method and application of selenium-rich poecilobdella manillensis freeze-dried powder, and belongs to the technical field of aquaculture. The invention provides a selenium-rich poecilobdella manillensis breeding method which comprises the following steps: (1) mixing selenium polypeptide with serum to obtain selenium-containing serum; (2) feeding the poecilobdella manillensis with the selenium-containing serum obtained in the step (1) once every 7-15 days, and feeding for 4-6 times to obtain selenium-rich poecilobdella manillensis; the final concentration of selenium in the selenium-containing serum in the step (1) is 1-4mg / L. The breeding method of the selenium-rich poecilobdella manillensis has no adverse effect on growth and death of the poecilobdella manillensis, the antithrombin activity of the prepared selenium-rich poecilobdella manillensis freeze-dried powder reaches up to 1173.02 U / g, the selenium content can reach up to 10.91 mg / kg, and the selenium content is remarkably higher than that of common poecilobdella manillensis freeze-dried powder.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a method for cultivating selenium-enriched leeches and a method for preparing selenium-enriched leech freeze-dried powder and application thereof. Background Art

[0002] Leech is a traditional Chinese medicine, first recorded in Shennong's Herbal Classic, with the functions of breaking blood, promoting menstruation, and dispersing blood stasis; it is mainly used to treat blood stasis, amenorrhea, abdominal pain, accumulation of lumps, and traumatic injuries. The main active ingredient in leeches is hirudin, which is the strongest specific anticoagulant substance discovered in nature so far. It is widely present in the salivary glands and saliva of blood-sucking leeches. Its efficacy is better than recombinant hirudin (recombinant hirudin, rH) and heparin, and its side effects are lower than recombinant hirudin and heparin. Hirudin is a kind of leech, which contains hirudin, bufrudin, plasmin, hyaluronidase, platelet aggregation inhibitor and other active ingredients in its body. It has anticoagulant, anti-tumor, anti-inflammatory, and lipid-lowering effects. The antithrombin activity of leech extract is higher than that of other leeches, and it has broad application prospects in medicine. In recent years, due to the urgent need for cardiovascular drugs and the irreplaceable nature of natural hirudin, people have begun to pay attention to the research on the effective ingredients and breeding techniques of Philippine cattle leeches.

[0003] Based on this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for cultivating selenium-enriched leeches and a method for preparing selenium-enriched leeches freeze-dried powder and application thereof, so as to solve the problem of how to cultivate leeches in the prior art to improve their efficacy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for cultivating selenium-enriched leeches, comprising the following steps:

[0007] (1) mixing the selenium polypeptide with serum to obtain selenium-containing serum;

[0008] (2) feeding leeches with the selenium-containing serum obtained in step (1), once every 7 to 15 days, for 4 to 6 times, to obtain selenium-enriched leeches;

[0009] The final concentration of selenium in the selenium-containing serum in step (1) is 1 to 4 mg / L.

[0010] Preferably, the selenopolypeptide in step (1) is Cardamine violaceum selenopolypeptide, and the serum is animal serum.

[0011] Preferably, the amount of selenium-containing serum fed each time in step (2) is 0.8 to 1.2 times the mass of the leech.

[0012] The invention provides selenium-enriched phenanthrene leeches obtained by the breeding method.

[0013] The present invention provides a method for preparing selenium-enriched phenanthrene leech freeze-dried powder, comprising the following steps:

[0014] (1) freezing the selenium-enriched phenanthrene leech for 3 to 5 hours to obtain frozen selenium-enriched phenanthrene leech;

[0015] (2) thawing the frozen selenium-enriched phenanthrene leech obtained in step (1) for 0.3 to 0.7 hours, and slicing and freeze-drying to obtain freeze-dried slices;

[0016] (3) crushing the freeze-dried slices obtained in step (2) to a particle size of 38 to 42 meshes to obtain selenium-enriched phenanthrene leech freeze-dried powder;

[0017] The selenium-rich phenanthrene leech in step (1) is the selenium-rich phenanthrene leech obtained by the breeding method or the selenium-rich phenanthrene leech.

[0018] Preferably, the temperature of the freezing treatment in step (1) is -75°C to -85°C.

[0019] Preferably, the thickness of the slices in step (2) is 0.1 to 0.2 cm.

[0020] Preferably, the freeze-drying temperature in step (2) is -55°C to -65°C, the freeze-drying vacuum is -1Pa to 1Pa, and the freeze-drying time is 22 to 26 hours.

[0021] The present invention also provides selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method, wherein the selenium content in the selenium-enriched phenanthrene leech is 4.78-10.91 mg / kg.

[0022] The present invention also provides the selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method or the use of the selenium-enriched phenanthrene leech freeze-dried powder in the preparation of anticoagulant products.

[0023] The present invention has the following technical effects and advantages:

[0024] The invention provides a method for cultivating selenium-rich leeches and a method for preparing and applying selenium-rich leech freeze-dried powder. The method for cultivating selenium-rich leeches has no adverse effect on the growth and death of leeches. The prepared selenium-rich leech freeze-dried powder has a thrombin activity of up to 1173.02 U / g and a selenium content of up to 10.91 mg / kg, which is significantly higher than that of common leech freeze-dried powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The figure is the average weight of a single leech fed with serum of different selenium concentrations. The abscissa in the figure represents the selenium concentration in the serum, and the ordinate represents the average weight of a single leech (g). The black color represents the average weight of a single leech when fed with serum for the first time, the red color represents the average weight of a single leech when fed with serum for the second time, the blue color represents the average weight of a single leech when fed with serum for the third time, the pink color represents the average weight of a single leech when fed with serum for the fourth time, and the green color represents the average weight of a single leech when fed with serum for the fifth time.

[0026] Figure 2 The antithrombin activity of leech freeze-dried powder with different selenium concentrations, the horizontal axis represents the selenium concentration in serum, and the vertical axis represents the antithrombin activity of leech freeze-dried powder (U / g);

[0027] Figure 3 The selenium content of leech freeze-dried powder with different selenium concentrations. The horizontal axis represents the selenium concentration in serum, and the vertical axis represents the selenium content of leech dry powder (mg / kg). DETAILED DESCRIPTION

[0028] The present invention provides a method for cultivating selenium-enriched leeches, comprising the following steps:

[0029] (1) mixing the selenium polypeptide with serum to obtain selenium-containing serum;

[0030] (2) feeding the selenium-containing serum obtained in step (1) once every 7 to 15 days for 4 to 6 times to obtain selenium-enriched leeches, preferably once every 12 days for 5 times;

[0031] The final concentration of selenium in the selenium-containing serum in step (1) is 1-4 mg / L, preferably 4 mg / L.

[0032] In the present invention, the selenium polypeptide in step (1) is Cardamine violaceum selenium polypeptide, and the serum is animal serum, and the animal serum is preferably porcine blood serum.

[0033] In the present invention, before feeding the leeches with the selenium-containing serum obtained in step (1) in step (2), the leeches need to be weighed.

[0034] In the present invention, the amount of selenium-containing serum fed each time in step (2) is 0.8 to 1.2 times the mass of the leech, preferably 1 time.

[0035] The invention provides selenium-enriched phenanthrene leeches obtained by the breeding method.

[0036] The present invention provides a method for preparing selenium-enriched phenanthrene leech freeze-dried powder, comprising the following steps:

[0037] (1) freezing the selenium-enriched phenanthrene leech for 3 to 5 hours to obtain frozen selenium-enriched phenanthrene leech, wherein the freezing treatment time is preferably 4 hours;

[0038] (2) thawing the frozen selenium-enriched phenanthrene leech obtained in step (1) for 0.3 to 0.7 h, slicing and freeze-drying to obtain freeze-dried slices, wherein the thawing time is preferably 0.5 h;

[0039] (3) crushing the freeze-dried slices obtained in step (2) to a particle size of 38 to 42 meshes to obtain selenium-enriched leech freeze-dried powder, wherein the particle size is preferably 40 meshes;

[0040] The selenium-rich phenanthrene leech in step (1) is the selenium-rich phenanthrene leech obtained by the breeding method or the selenium-rich phenanthrene leech.

[0041] In the present invention, the temperature of the freezing treatment in step (1) is -75°C to -85°C, preferably -80°C.

[0042] In the present invention, the thickness of the slices in step (2) is 0.1 to 0.2 cm, preferably 0.1 cm.

[0043] In the present invention, the freeze-drying temperature in step (2) is -55°C to -65°C, preferably -60°C, the freeze-drying vacuum is -1Pa to 1Pa, preferably 0Pa, and the freeze-drying time is 22 to 26h, preferably 24h.

[0044] The present invention also provides selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method, wherein the selenium content in the selenium-enriched phenanthrene leech is 4.78-10.91 mg / kg.

[0045] The present invention also provides the selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method or the use of the selenium-enriched phenanthrene leech freeze-dried powder in the preparation of anticoagulant products.

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The preparation method of Cardamine violaceum selenium polypeptide in the embodiment of the present invention is:

[0048] The Cardamine violaceae was crushed, and the crude protein of Cardamine violaceae was obtained after alkali extraction, acid precipitation, desalting and drying. A certain amount of crude protein of Cardamine violaceae was weighed and dissolved in distilled water at a mass concentration of 4%, and the temperature was raised to 50°C, the pH value was adjusted to 9, 160 mg of alkaline protease was added, and enzymolysis was performed for 2 hours. During the enzymolysis process, sodium hydroxide solution was used to maintain the pH value at 9 until the enzymolysis was completed. The supernatant was passed through 30, 5, and 1 kDa ultrafiltration membranes in sequence, and the filtrate of the 1 kDa membrane was collected. After reduced pressure concentration and freeze-drying, the Cardamine violaceae selenium polypeptide was obtained.

[0049] Example 1

[0050] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge to take the supernatant, add Cardamine violet selenium polypeptide and mix so that the final concentration of selenium in the serum is 1 mg / L to obtain selenium-containing serum for later use.

[0051] 30 Philippine leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 75.0 g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation, and the water was changed every 2 days.

[0052] Serum feeding method: Inject selenium-containing serum with a final selenium concentration of 1 mg / L into the pig small intestine, seal the two ends with ropes, and feed it to the Philippine leech breeding box. Feed 75.0 g of selenium-containing serum. Before each subsequent feeding of selenium-containing serum, weigh the Philippine leeches. The feeding amount of selenium-containing serum is 1 times the total mass of the Philippine leeches in the breeding box. Feed once every 7 days, and feed 5 times to obtain selenium-rich Philippine leeches.

[0053] Example 2

[0054] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge to take the supernatant, add Cardamine violet selenium polypeptide and mix so that the final concentration of selenium in the serum is 2 mg / L to obtain selenium-containing serum for later use.

[0055] 30 Philippine leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 62.0 g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation, and the water was changed every 2 days.

[0056] Serum feeding method: inject selenium-containing serum with a final selenium concentration of 2 mg / L into the pig small intestine, seal the two ends with ropes, and feed it to the Philippine leech breeding box. Feed 62.0 g of selenium-containing serum. Before each subsequent feeding of selenium-containing serum, weigh the Philippine leeches. The feeding amount of selenium-containing serum is 1 times the total mass of the Philippine leeches in the breeding box. Feed once every 7 days, and feed 5 times to obtain selenium-rich Philippine leeches.

[0057] Example 3

[0058] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge to take the supernatant, add Cardamine violet selenium polypeptide and mix so that the final concentration of selenium in the serum is 4 mg / L to obtain selenium-containing serum for later use.

[0059] 30 Philippine leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 74.7g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation, and the water was changed every 2 days.

[0060] Serum feeding method: Inject selenium-containing serum with a final selenium concentration of 4 mg / L into the pig small intestine, tie the two ends with ropes to seal it, and feed it to the Philippine leech breeding box. Feed 74.7 g of selenium-containing serum. Before each subsequent feeding of selenium-containing serum, weigh the Philippine leeches. The feeding amount of selenium-containing serum is 1 times the total mass of the Philippine leeches in the breeding box. Feed once every 7 days, and feed 5 times to obtain selenium-rich Philippine leeches.

[0061] Comparative Example 1

[0062] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge and take the supernatant to obtain selenium-free serum for later use.

[0063] 30 Philippine leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 74.7g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation, and the water was changed every 2 days.

[0064] Serum feeding method: inject selenium-free serum into the pig small intestine, seal the two ends with ropes, and feed it to the Philippine cattle leech breeding box. Feed 74.7g of selenium-free serum. Before each subsequent feeding of selenium-free serum, weigh the Philippine cattle leeches. The feeding amount of selenium-free serum is 1 times the total mass of the Philippine cattle leeches in the breeding box. Feed once every 7 days, and feed 5 times to obtain ordinary Philippine cattle leeches.

[0065] Comparative Example 2

[0066] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge to take the supernatant, add Cardamine violaceum selenium polypeptide and mix so that the final concentration of selenium in the serum is 0.5 mg / L to obtain selenium-containing serum for use.

[0067] 30 Philippine leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 73.5g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation, and the water was changed every 2 days.

[0068] Serum feeding method: Inject selenium-containing serum with a final selenium concentration of 0.5 mg / L into the pig small intestine, tie the two ends with ropes to seal, and feed it to the Philippine leech breeding box. Feed 73.5 g of selenium-containing serum. Before each subsequent feeding of selenium-containing serum, weigh the Philippine leeches. The feeding amount of selenium-containing serum is 1 times the total mass of the Philippine leeches in the breeding box. Feed once every 7 days, and feed 5 times to obtain low-selenium leeches.

[0069] Comparative Example 3

[0070] Take pig blood serum without adding anticoagulant, let it stand for 5 hours, centrifuge to take the supernatant, add Cardamine violet selenium polypeptide and mix, so that the final concentration of selenium in the serum is 8 mg / L to obtain selenium-containing serum for use.

[0071] 30 Philippine cattle leech seedlings with uniform size, strong physique and no disease on the body surface, with a total mass of 72.9g, were randomly selected and placed in a breeding box. Pure water at room temperature was added for cultivation. Selenium-containing serum with a final selenium concentration of 8mg / L was poured into the small intestine of the pigs, and the two ends were tied with ropes to seal the mouth and fed into the Philippine cattle leech breeding box. 72.9g of selenium-containing serum was fed. After the Philippine cattle leeches ate half of the serum, the serum coagulated and could not be sucked.

[0072] Experimental example

[0073] 1. Effect of selenium concentration on the growth of Philippine leech

[0074] By comparing Examples 1 to 3 and Comparative Examples 2 to 3, it is known that when the selenium concentration in the serum of the leeches fed is 0.5 to 4 mg / L, the leeches can normally inhale, and when the selenium concentration in the serum is 8 mg / L, the serum will coagulate, resulting in the inability of the leeches to inhale. This indicates that when too much Cardamine violaceum selenium polypeptide is added to the serum to a selenium concentration exceeding 4 mg / L, the serum is prone to coagulation, resulting in the inability of the leeches to inhale normally, so the experiment was conducted with a selenium concentration of 0.5 to 4 mg / L in the serum.

[0075] Examples 1 to 3 and comparative example 2 were used as treatment groups, and comparative example 1 was used as a control group. Each group was repeated three times to determine the effect of selenium concentration on the growth of Philippine leech. The specific method was as follows: the number of deaths of Philippine leech seedlings in the breeding boxes of Examples 1 to 3 and Comparative Examples 1 to 2 was recorded, and the effect of selenium concentration in serum on the mortality rate of Philippine leech was calculated. The mortality rate was calculated as follows:

[0076] Mortality rate = A / B × 100%

[0077] Where A represents the number of dead Philippine cattle leeches, and B represents the total number of Philippine cattle leeches.

[0078] The mortality rate of each replicate in the treatment group and the control group was calculated, and the average mortality rate of each group was calculated. The results are shown in Table 1.

[0079] Table 1 Mortality of leeches fed with serum of different selenium concentrations

[0080]

[0081]

[0082] According to the results in Table 1, when the leeches were fed with serum containing selenium concentrations of 0.5, 1, 2, and 4 mg / L, the mortality rate of the leeches was not significantly affected, indicating that the selenium concentration in the serum of 0.5 to 4 mg / L had no effect on the growth of the leeches.

[0083] 2. Effect of selenium concentration on the body weight of Philippine leech

[0084] With Examples 1 to 3 and Comparative Example 2 as treatment groups and Comparative Example 1 as a control group, the effect of selenium concentration on the body weight of Philippine leeches was determined. The specific method was as follows: the total weight of Philippine leeches in Examples 1 to 3 and Comparative Examples 1 to 2 was recorded respectively at each feeding, and the average weight of a single Philippine leech in Examples 1 to 3 and Comparative Examples 1 to 2 was calculated at each feeding of serum. The calculation formula for the average weight of a single Philippine leech is as follows:

[0085] Average weight of a single piece = A / B

[0086] Wherein A is the total weight of the Philippine cattle leeches, and B is the total number of the Philippine cattle leeches.

[0087] The average weight of each leech fed with serum of different selenium concentrations is shown in Table 2 and Figure 1 As shown in the figure, the abscissa represents the selenium concentration in serum, the ordinate represents the average weight of a single leech (g), black represents the average weight of a single leech when fed with serum for the first time, red represents the average weight of a single leech when fed with serum for the second time, blue represents the average weight of a single leech when fed with serum for the third time, pink represents the average weight of a single leech when fed with serum for the fourth time, and green represents the average weight of a single leech when fed with serum for the fifth time.

[0088] Table 2 Average weight of a single leech fed with serum of different selenium concentrations

[0089] Feeding frequency 1st time 2nd 3rd 4th 5th 0.5mg / L 2.45g 3.55g 4.61g 6.33g 7.58g 1mg / L 2.50g 3.53g 4.56g 6.29g 7.50g 2mg / L 2.40g 3.67g 4.48g 6.31g 7.85g 4mg / L 2.49g 3.39g 4.38g 6.14g 7.67g Control group 2.49g 3.54g 4.23g 5.93g 7.29g

[0090] According to Table 2 and Figure 1It can be seen that when the leeches were fed with serum containing 0.5, 1, 2 and 4 mg / L of selenium, the average weight of each leech at the fifth feeding was 3.09, 3, 3.27 and 3.08 times the average weight of each leech at the first feeding, respectively, and the average weight of each leech in the control group at the fifth feeding was 2.92 times the average weight of each leech at the first feeding. It can be seen that when the leeches were fed with serum containing 0.5, 1, 2 and 4 mg / L of selenium, the weight of the leeches increased with the increase in the number of feedings, and there was no significant difference in weight gain; there was also no significant difference in weight gain between the leeches fed with serum containing 0.5, 1, 2 and 4 mg / L of selenium and the leeches in the control group. This indicates that feeding leeches with serum containing selenium concentrations of 0.5, 1, 2, and 4 mg / L has no adverse effect on their growth.

[0091] 3. Effect of selenium concentration on antithrombin activity and selenium content of leech freeze-dried powder

[0092] The selenium-enriched phenanthrene leech obtained in Examples 1 to 3 is prepared into selenium-enriched phenanthrene leech freeze-dried powder. The preparation method of the selenium-enriched phenanthrene leech freeze-dried powder is as follows:

[0093] The selenium-rich phenanthrene leech is frozen at -80°C for 4 hours to obtain frozen selenium-rich phenanthrene leech, and then the frozen selenium-rich phenanthrene leech is thawed for 0.5 hour, and the thawed selenium-rich phenanthrene leech is cut into 0.1 cm thick slices, and freeze-dried at -60°C and 0 Pa vacuum for 24 hours to obtain freeze-dried slices, and then the freeze-dried slices are crushed to a particle size of 40 mesh to obtain selenium-rich phenanthrene leech freeze-dried powder.

[0094] The common leech obtained in Comparative Example 1 was prepared into common leech freeze-dried powder, and the preparation method of the common leech freeze-dried powder was consistent with the preparation method of the selenium-enriched leech freeze-dried powder.

[0095] The low-selenium phenanthrene leech obtained in Comparative Example 2 was prepared into low-selenium phenanthrene leech freeze-dried powder. The preparation method of the low-selenium phenanthrene leech freeze-dried powder was consistent with the preparation method of the selenium-rich phenanthrene leech freeze-dried powder.

[0096] 3.1 Effect of selenium concentration on antithrombin activity of leech freeze-dried powder

[0097] The selenium-rich phenanthrene leech freeze-dried powder prepared in Examples 1 to 3 and the low-selenium phenanthrene leech freeze-dried powder obtained in Comparative Example 2 were used as the treatment group, and the ordinary phenanthrene leech freeze-dried powder obtained in Comparative Example 1 was used as the control group to determine the antithrombin activity. The method for determining the antithrombin activity is as follows:

[0098] Accurately weigh 0.25 g of the lyophilized powder of Hirudo philadelphica to be tested, add 15 mL of 0.9% sodium chloride solution, stir thoroughly, and extract for 30 minutes to obtain the active ingredient extract of Hirudo philadelphica, filter to obtain the active ingredient filtrate of Hirudo philadelphica, accurately measure 100 μL of the active ingredient filtrate of Hirudo philadelphica, put it in a test tube (8 mm × 38 mm), add 200 μL of tris(hydroxymethylaminomethane) hydrochloric acid buffer containing 0.5% bovine fibrinogen (calculated as coagulant), shake well, put it in a water bath (37°C ± 0.5°C) for 5 minutes, add 40 units of thrombin solution per 1 mL (add once every 1 minute, 5 μL each time, and gently shake well while adding) until coagulation, record the volume of the consumed thrombin solution, and calculate the antithrombin activity of the lyophilized powder of Hirudo philadelphica to be tested. The calculation formula is as follows:

[0099] Antithrombin activity of Philippine leech (U) = C1×V1 / C2×V2

[0100] U is the unit of thrombin activity per 1g of the test sample (U / g), C1 is the mass concentration of the thrombin solution (U / mL), C2 is the mass concentration of the test sample solution (g / L), V1 is the volume of the consumed thrombin solution (μL), and V2 is the amount of the test sample solution added (μL).

[0101] The antithrombin activity of lyophilized powder of leech with different selenium concentrations was determined. The results are shown in Table 3 and Figure 2 As shown in the figure, the horizontal axis represents the selenium concentration in serum, and the vertical axis represents the antithrombin activity (U / g) of the lyophilized powder of Hirudo philadelphica.

[0102] Table 3 Antithrombin activity of lyophilized leech powder with different selenium concentrations

[0103] Selenium concentration Antithrombin activity U / g 0.5mg / L 480.54 1mg / L 473.75 2mg / L 712.3 4mg / L 1173.02 Control group 471.88

[0104] According to Table 3 and Figure 2 It can be seen that there is no significant difference in antithrombin activity between the selenium-enriched leech freeze-dried powder obtained by feeding serum with a selenium concentration of 1 mg / L and the low-selenium leech freeze-dried powder obtained by feeding serum with a selenium concentration of 0.5 mg / L and the ordinary leech freeze-dried powder in the control group, but with the increase of selenium content in the feeding serum, the antithrombin activity of the selenium-enriched leech freeze-dried powder also increases, reaching a maximum of 1173.02 U / g; when the serum selenium content is 2-4 mg / L, the antithrombin activity of the selenium-enriched leech freeze-dried powder is significantly higher than that of the ordinary leech freeze-dried powder in the control group, which indicates that feeding serum with a selenium concentration of 2-4 mg / L can significantly improve the antithrombin activity of selenium-enriched leech freeze-dried powder.

[0105] 3.2 Effect of selenium concentration on selenium content in leech freeze-dried powder

[0106] The selenium-rich leech freeze-dried powder prepared in Examples 1 to 3 and the low-selenium leech freeze-dried powder obtained in Comparative Example 2 were used as the treatment group, and the ordinary leech freeze-dried powder obtained in Comparative Example 1 was used as the control group to determine the selenium content. The selenium content was determined by the first method in "GB 5009.93-2017 National Food Safety Standard Determination of Selenium in Food".

[0107] The selenium content of leech freeze-dried powder with different selenium concentrations was determined, and the results are shown in Table 4 and Figure 3 As shown in the figure, the horizontal axis represents the selenium concentration in serum, and the vertical axis represents the selenium content (mg / kg) of the leech freeze-dried powder.

[0108] Table 4 Selenium content of leech freeze-dried powder with different selenium concentrations

[0109]

[0110]

[0111] According to Table 4 and Figure 3 It can be seen that the selenium content of the selenium-enriched leech freeze-dried powder obtained by feeding serum with selenium concentrations of 1, 2, and 4 mg / L was significantly higher than the selenium content of the ordinary leech freeze-dried powder in the control group and the low-selenium leech freeze-dried powder obtained by feeding serum with a selenium concentration of 0.5 mg / L, and with the increase of selenium content in the feeding serum, the selenium content of the selenium-enriched leech freeze-dried powder also increased accordingly. When feeding serum with a selenium concentration of 4 mg / L, the selenium content of the selenium-enriched leech freeze-dried powder was the highest, which was 10.91 mg / kg.

[0112] It can be seen from the above embodiments that the present invention provides a method for cultivating selenium-enriched leeches and a method for preparing and using selenium-enriched leeches freeze-dried powder. The method for cultivating selenium-enriched leeches according to the present invention has no adverse effect on the growth and death of leeches, and the prepared selenium-enriched leeches freeze-dried powder has a thrombin activity of up to 1173.02 U / g, and a selenium content of up to 10.91 mg / kg, which is significantly higher than that of ordinary leeches freeze-dried powder.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for cultivating selenium-enriched phenanthrene leeches, characterized in that: The steps include: (1) mixing the selenium polypeptide with serum to obtain selenium-containing serum; (2) feeding leeches with the selenium-containing serum obtained in step (1), once every 7 to 15 days, for 4 to 6 times, to obtain selenium-enriched leeches; The final concentration of selenium in the selenium-containing serum in step (1) is 1 to 4 mg / L.

2. The breeding method according to claim 1, characterized in that: The selenium polypeptide in step (1) is Cardamine violaceum selenium polypeptide, and the serum is animal serum.

3. The breeding method according to claim 1, characterized in that: The amount of selenium-containing serum fed each time in step (2) is 0.8 to 1.2 times the mass of the leech.

4. The selenium-enriched phenanthrene leech obtained by the breeding method according to any one of claims 1 to 3.

5. A method for preparing selenium-enriched phenanthrene leech freeze-dried powder, characterized in that: The steps include: (1) freezing the selenium-enriched phenanthrene leech for 3 to 5 hours to obtain frozen selenium-enriched phenanthrene leech; (2) thawing the frozen selenium-enriched phenanthrene leech obtained in step (1) for 0.3 to 0.7 hours, and slicing and freeze-drying to obtain freeze-dried slices; (3) crushing the freeze-dried slices obtained in step (2) to a particle size of 38 to 42 meshes to obtain selenium-enriched phenanthrene leech freeze-dried powder; The selenium-rich phenanthrene leech described in step (1) is the selenium-rich phenanthrene leech obtained by the breeding method described in any one of claims 1 to 3 or the selenium-rich phenanthrene leech described in claim 4.

6. The preparation method according to claim 5, characterized in that: The temperature of the freezing treatment in step (1) is -75°C to -85°C.

7. The preparation method according to claim 5, characterized in that: The thickness of the slices in step (2) is 0.1 to 0.2 cm.

8. The preparation method according to claim 5, characterized in that: The freeze-drying temperature in step (2) is -55°C to -65°C, the freeze-drying vacuum is -1Pa to 1Pa, and the freeze-drying time is 22 to 26 hours.

9. The selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method according to any one of claims 5 to 8, characterized in that: The selenium content in the selenium-enriched leech freeze-dried powder is 4.78-10.91 mg / kg.

10. Use of the selenium-enriched phenanthrene leech freeze-dried powder prepared by the preparation method according to any one of claims 5 to 8 or the selenium-enriched phenanthrene leech freeze-dried powder according to claim 9 in the preparation of anticoagulant products.

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