Method for extracting total protein from loranthus parasiticus seeds

Abstract

The invention discloses a method for extracting total protein from loranthus parasitic seeds, which comprises the following steps: 1) loranthus parasitic fresh seeds are cleaned with sterile water, placed in a mortar precooled with liquid nitrogen, polyvinyl pyrrolidone is added, ground and pulverized in liquid nitrogen to obtain a crushed material; 2) the crushed material is placed in a precooled EP tube, RIPA protein lysate and PMSF solution are added, and the mixture is placed on ice for cracking after shaking; 3) after cracking, the mixture is crushed, centrifuged, and the first supernatant is obtained; 4) RIPA protein lysate is added again to the first supernatant, cracked on the ice, centrifuged, and the second supernatant is obtained; 5) precooled acetone is added to the second supernatant, precipitated overnight at 20°C, centrifuged, and protein precipitation is obtained. The protein precipitation is dried under a natural state and dissolved with protein lysate; 6) after the protein solution is heated in a water bath, ice bath is cooled, centrifuged, and supernatant is taken. The present invention has the characteristics of improving the extraction efficiency of protein, and the protein extraction process is not easily degraded.

Description

Technical Field

[0001] The present invention belongs to the field of seed protein extraction, and more specifically relates to a method for extracting total protein from loranthus parasiticus seeds. Background Art

[0002] The botanical name of the mistletoe plant, Mistletoe Parasite, is a traditional and commonly used traditional Chinese medicinal herb with benefits such as nourishing the liver and kidneys, dispelling rheumatism, strengthening bones and muscles, and stabilizing pregnancy. It has been included in all editions of the Chinese Pharmacopoeia. Mistletoe can only reproduce by seeds. In the wild, its spread and reproduction are primarily achieved by frugivorous birds that consume the fruit, digest the flesh, and excrete the seeds. With the dwindling bird population, natural reproduction has become increasingly difficult, leading to a significant decrease in wild resources. Fresh mistletoe seeds have a high germination rate but quickly lose viability, making them recalcitrant. This recalcitrant nature severely restricts mistletoe breeding and will undoubtedly become a technical bottleneck for its standardized cultivation. Desiccation sensitivity and low-temperature sensitivity are the main characteristics of recalcitrant mistletoe seeds, and the underlying mechanisms require further investigation. Proteomic analysis is highly advantageous for revealing the molecular mechanisms underlying seed development from a new perspective. Proteomic studies of mistletoe seeds require total protein extraction.

[0003] There are many methods for extracting total protein from seeds, but the yields vary and are significantly lower than those from leaves. As mistletoe seeds are stubborn seeds, they dehydrate and become inactivated quickly, and the seeds are difficult to grind, resulting in a lower total protein yield. In addition, the traditional phenol extraction method cannot effectively remove polyphenols and salts when extracting seed protein. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] An object of the present invention is to provide a method for extracting total protein from loranthus parasiticus seeds, which can improve protein extraction efficiency and prevent protein degradation from occurring during the protein extraction process.

[0006] In order to achieve these objects and other advantages of the present invention, a method for extracting total protein from loranthus parasiticus seeds is provided, comprising the following steps:

[0007] 1) After removing the peel and pectin, fresh Morus alba seeds were washed with sterile water, placed in a mortar pre-cooled with liquid nitrogen, added with polyvinyl pyrrolidone, and ground in liquid nitrogen to obtain a pulverized product;

[0008] 2) Place the crushed material into a pre-chilled EP tube, add RIPA protein lysis buffer and PMSF solution, shake well, and place the EP tube on ice for lysis;

[0009] 3) After the lysis is completed, the mixture is crushed with a portable high-speed disperser and centrifuged to obtain a first supernatant;

[0010] 4) Add RIPA protein lysis buffer to the first supernatant again, lyse on ice, and centrifuge to obtain the second supernatant;

[0011] 5) Add pre-chilled acetone to the second supernatant and precipitate at -20°C overnight. Centrifuge to obtain a protein precipitate. Allow the protein precipitate to dry naturally and dissolve it in RIPA protein lysis buffer to obtain a protein solution.

[0012] 6) After heating the protein solution in a water bath, cool it in an ice bath, centrifuge it, and collect the supernatant.

[0013] Preferably, in step 1), the mass of fresh loranthus parasiticus seeds is 2-4 g, and the mass of polyvinyl pyrrolidone is 1-3 g.

[0014] Preferably, in step 2), the volume of the RIPA protein lysis buffer is 5-10 mL, and the volume ratio of the RIPA protein lysis buffer to the PMSF solution is 1:100.

[0015] Preferably, in step 3), the crushing time of the portable high-speed disperser is 1-5 minutes, the centrifugal speed is 5000 rpm, and the centrifugation is carried out for 15 minutes at 4°C.

[0016] Preferably, in step 4), the volume of RIPA protein lysis buffer is 3-5 mL, lysis is carried out on ice for 15-35 min, and centrifugation is carried out at 5000 rpm for 15 min at 4°C.

[0017] Preferably, in step 5), the volume of pre-cooled acetone is 3-5 times the volume of the second supernatant, and the mixture is centrifuged at 5000 rpm for 15 min at 4°C.

[0018] Preferably, in step 6), the mixture is placed in a water bath at 95° C. for 5 to 10 minutes, then quickly placed in an ice bath for 5 minutes, and centrifuged at 5000 rpm for 10 minutes.

[0019] Preferably, the peeling operation is performed in a peeling device to obtain seeds with the peel removed and pectin adhered thereto, and then the seeds with the peel removed and pectin adhered thereto are cleaned in a cleaning mechanism to remove the pectin on the surface of the seeds, thereby obtaining fresh mistletoe seeds with the peel and pectin removed.

[0020] Preferably, the precipitate A obtained by centrifugation in step 3) is vacuum dried at a vacuum degree of -0.099 MPa and a drying temperature of 7°C. After drying, pre-cooled sodium salicylate solution, RIPA protein lysis buffer and PMSF solution are added, and the mixture is frozen at -20°C for 2-5 hours, taken out and thawed at room temperature, crushed with a portable high-speed disperser, and centrifuged to obtain a re-extraction supernatant, which is combined with the first supernatant and proceeds to step 4).

[0021] Preferably, the volume of the sodium salicylate solution is 1-3 mL, the volume of the RIPA protein lysate solution is 5-10 mL, the volume ratio of the RIPA protein lysate solution to the PMSF solution is 1:100, and the concentration of the sodium salicylate solution is 2-5 mol / L.

[0022] The present invention has at least the following beneficial effects:

[0023] First, the method of the present invention solves the problem that the traditional phenol extraction method cannot effectively remove polyphenols and salt substances when extracting seed protein, and improves the protein extraction efficiency, and is less likely to cause protein degradation during the protein extraction process.

[0024] Second, the present invention vacuum-dries the precipitate obtained by centrifugation in step 3), thereby destroying the structure of granular matter in the precipitate, such as starch granules, and adding pores inside the precipitate to facilitate the entry of sodium salicylate solution, RIPA protein lysis buffer, and PMSF solution into the precipitate. During freeze-thaw, the granular matter can be further broken up to extract protein from the precipitate, thereby improving the protein extraction rate.

[0025] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The protein gel electrophoresis diagram of the protein extracted by the present invention;

[0027] Figure 2 A schematic top view of a cleaning mechanism according to an embodiment of the present invention;

[0028] Figure 3 A side view of a peeling device according to an embodiment of the present invention;

[0029] Figure 4 This is a side view of a peeling device used in combination with a cleaning mechanism according to an embodiment of the present invention.

[0030] 1. Outer barrel; 2. First inner barrel; 3. Second inner barrel; 4. First stirring device; 5. Second stirring device; 6. Squeeze box; 7. Squeeze roller; 8. Feed port; 9. Scraper; 10. Conical discharge port; 11. Filter bag; 12. Sealing cover; 13. Vacuum pump. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0032] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0033] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified. In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected or set, or detachably connected or set, or integrally connected or set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. The orientations or positional relationships indicated by the terms "transverse", "longitudinal", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc. are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention.

[0034] A method for extracting total protein from loranthus parasiticus seeds comprises the following steps:

[0035] 1) After removing the peel and pectin, fresh Morus alba seeds were washed with sterile water, placed in a mortar pre-cooled with liquid nitrogen, added with polyvinyl pyrrolidone, and ground in liquid nitrogen to obtain a pulverized product;

[0036] 2) Place the crushed material into a pre-chilled EP tube, add RIPA protein lysis buffer and PMSF solution, shake well, and place the EP tube on ice for lysis;

[0037] 3) After the lysis is completed, the mixture is crushed with a portable high-speed disperser and centrifuged to obtain a first supernatant;

[0038] 4) Add RIPA protein lysis buffer to the first supernatant again, lyse on ice, and centrifuge to obtain the second supernatant;

[0039] 5) Add pre-chilled acetone to the second supernatant and precipitate at -20°C overnight. Centrifuge to obtain a protein precipitate. Allow the protein precipitate to dry naturally and dissolve it in RIPA protein lysis buffer to obtain a protein solution.

[0040] 6) After heating the protein solution in a water bath, cool it in an ice bath, centrifuge it, and collect the supernatant.

[0041] In another technical solution, in step 1), the mass of fresh loranthus parasiticus seeds is 2-4 g, and the mass of polyvinyl pyrrolidone is 1-3 g.

[0042] In another technical solution, in step 2), the volume of RIPA protein lysis buffer is 5-10 mL, and the volume ratio of RIPA protein lysis buffer to PMSF solution is 1:100; the RIPA protein lysis buffer includes the following concentration components: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 2.5 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na3VO4, 0.5 ug / ml leupeptin; the concentration of PMSF solution is 0.5 mM.

[0043] In another technical solution, in step 3), the crushing time of the portable high-speed disperser is 1-5 minutes, and the centrifugal speed is 5000 rpm at 4° C. for 15 minutes.

[0044] In another technical solution, in step 4), the volume of RIPA protein lysis buffer is 3-5 mL, lysis is carried out on ice for 15-35 min, and centrifugation is carried out at 5000 rpm for 15 min at 4°C.

[0045] In another technical solution, in step 5), the volume of pre-cooled acetone is 3-5 times the volume of the second supernatant, and the mixture is centrifuged at 5000 rpm for 15 min at 4°C.

[0046] In another technical solution, in step 6), the mixture is placed in a water bath at 95° C. for 5 to 10 minutes, then quickly placed in an ice bath for 5 minutes, and centrifuged at 5000 rpm for 10 minutes.

[0047] In another technical solution, a peeling operation is performed in a peeling device to obtain seeds with the peel removed and pectin adhered thereto, and then the seeds with the peel removed and pectin adhered thereto are cleaned in a cleaning mechanism to remove the pectin on the surface of the seeds, thereby obtaining fresh mistletoe seeds with the peel and pectin removed.

[0048] In another technical solution, the precipitate A obtained by centrifugation in step 3) is vacuum dried at a vacuum degree of -0.099 MPa and a drying temperature of 7°C. After drying, pre-cooled sodium salicylate solution, RIPA protein lysis buffer and PMSF solution are added, and the mixture is frozen at -20°C for 2-5 hours, taken out and thawed at room temperature, crushed with a portable high-speed disperser, and centrifuged to obtain a re-extraction supernatant, which is combined with the first supernatant and proceeds to step 4).

[0049] In another technical solution, the volume of the sodium salicylate solution is 1-3 mL, the volume of the RIPA protein lysate solution is 5-10 mL, the volume ratio of the RIPA protein lysate solution to the PMSF solution is 1:100, and the concentration of the sodium salicylate solution is 2-5 mol / L.

[0050] <Example 1>

[0051] A method for extracting total protein from loranthus parasiticus seeds comprises the following steps:

[0052] 1) After removing the peel and pectin, fresh Morus alba seeds were washed with sterile water. 2 g of the seeds were placed in a liquid nitrogen-precooled mortar and pestle. 1 g of polyvinyl pyrrolidone (PVP) was added and the mixture was ground in liquid nitrogen.

[0053] 2) Take the ground sample and place it in a pre-chilled 15 mL EP tube. Add 5 mL of RIPA protein lysis buffer and PMSF solution (1:100), shake well, and lyse on ice for 30-60 minutes.

[0054] 3) The lysed sample was pulverized using a portable high-speed disperser to achieve more complete tissue lysis, and then centrifuged at 5000 rpm for 15 minutes at 4°C to obtain the first supernatant;

[0055] 4) Add 3 mL of RIPA protein lysis buffer to the supernatant again, continue lysis on ice for 15-35 minutes, and centrifuge at 5000 rpm for 15 minutes at 4°C to obtain the second supernatant;

[0056] 5) Quickly add 3 volumes of pre-chilled acetone and precipitate at -20°C overnight. Centrifuge at 5000 rpm for 15 minutes to obtain a protein precipitate. Air-dry for approximately 10 minutes and dissolve in RIPA protein lysis buffer to obtain a protein solution.

[0057] 6) Take the protein solution obtained in the previous step, incubate it in a 95°C water bath for 5-10 minutes, then quickly in an ice bath for 5 minutes, centrifuge it at 5000 rpm for 10 minutes, and collect the supernatant, which is the final total protein solution, which can be used for the next proteomic study.

[0058] <Example 2>

[0059] A method for extracting total protein from loranthus parasiticus seeds comprises the following steps:

[0060] 1) After removing the peel and pectin, fresh Morus alba seeds were washed with sterile water. 2 g of the seeds were placed in a liquid nitrogen-precooled mortar and pestle. 1 g of polyvinyl pyrrolidone (PVP) was added and the mixture was ground in liquid nitrogen.

[0061] 2) Take the ground sample and place it in a pre-chilled 15 mL EP tube. Add 5 mL of RIPA protein lysis buffer and PMSF solution (1:100), shake well, and lyse on ice for 30-60 minutes.

[0062] 3) The lysed sample was pulverized using a portable high-speed disperser to achieve more complete tissue lysis. The sample was centrifuged at 5000 rpm for 15 minutes at 4°C to obtain a first supernatant and precipitate A. The precipitate A was vacuum-dried at a vacuum degree of -0.099 MPa and a drying temperature of 7°C. After drying, pre-cooled sodium salicylate solution, RIPA protein lysis buffer, and PMSF solution were added and frozen at -20°C for 2-5 hours. The sample was removed and thawed at room temperature. The sample was pulverized using a portable high-speed disperser and centrifuged to obtain a re-extracted supernatant. The re-extracted supernatant was combined with the first supernatant to obtain a combined solution.

[0063] 4) Add 5 mL of RIPA protein lysis buffer to the combined solution and continue lysis on ice for 15-35 minutes. Centrifuge at 5000 rpm for 15 minutes at 4°C to obtain the second supernatant.

[0064] 5) Quickly add 3 volumes of pre-chilled acetone and precipitate at -20°C overnight. Centrifuge at 5000 rpm for 15 minutes to obtain a protein precipitate. Air-dry for approximately 10 minutes and dissolve in RIPA protein lysis buffer to obtain a protein solution.

[0065] 6) Take the protein solution obtained in the previous step, incubate it in a 95°C water bath for 5-10 minutes, then quickly in an ice bath for 5 minutes, centrifuge it at 5000 rpm for 10 minutes, and collect the supernatant, which is the final total protein solution, which can be used for the next proteomic study.

[0066] <Example 3>

[0067] In Example 1 and Example 2, the peel and pectin of fresh mistletoe seeds can be manually removed to obtain fresh mistletoe seeds. Alternatively, the seeds with pectin attached after the peel is removed can be cleaned using the following cleaning mechanism, that is, the seeds are manually squeezed out of the mistletoe fruit, and the seeds with pectin attached are placed in the cleaning mechanism for cleaning to obtain seeds with peel and pectin removed, such as Figure 2As shown, the cleaning mechanism includes an outer barrel 1, a first inner barrel 2, a second inner barrel 3, a first stirring device 4, a second stirring device 5 and a water supply pipe. The first inner barrel 2 and the second inner barrel 3 are arranged in the outer barrel 1, the first inner barrel 2 and the second inner barrel 3 are connected, and the first inner barrel 2 and the second inner barrel 3 are communicated. The first stirring device 4 is arranged in the first inner barrel 2, and the second stirring device 5 is arranged in the second inner barrel 3. The barrel walls and barrel bottoms of the first inner barrel 2 and the second inner barrel 3 are provided with filter holes, the aperture of the filter holes is smaller than the diameter of the fresh mistletoe seeds to prevent the seeds from passing through the filter holes, the bottom of the first inner barrel 2 and the second inner barrel 3 have a gap with the bottom of the outer barrel 1, and the bottom of the outer barrel 1 is provided with a drain outlet, which is connected to the drain pipe to discharge the water in the outer barrel 1, and the water supply pipe runs through the outer barrel 1 to supply water to the first inner barrel 2 and the second inner barrel 3.

[0068] The method of using the cleaning structure of the present invention is as follows: squeeze the seeds of mistletoe from the fruit, and the seeds are attached with pectin. The seeds are placed in the first inner barrel 2 or the second inner barrel 3, and clean water is injected into the first inner barrel 2 or the second inner barrel 3. The first stirring device 4 and the second stirring device 5 are started. The rotation directions of the first stirring device 4 and the second stirring device 5 are opposite. Clean water is continuously injected into the barrel and the water is discharged through the drain port, so that water is injected, drained and stirred at the same time, thereby cleaning the seeds attached to the seed surface.

[0069] The present invention uses the water flow in the second inner barrel 3 to clean the back of the seeds in the first inner barrel 2, and uses the water flow in the first inner barrel 2 to clean the back of the seeds in the second inner barrel 3, so that both the front and back of the seeds can be effectively cleaned.

[0070] <Example 4>

[0071] Example 3 The peel of the fresh seeds of the mistletoe can be removed manually to obtain fresh seeds of the mistletoe with pectin attached. The peel of the fresh seeds of the mistletoe can also be removed by the following peeling device, such as Figure 3 As shown, the peeling device includes:

[0072] The squeezing structure includes a squeezing box 6, a squeezing roller 7 and a driving assembly, wherein the squeezing box 6 is a rectangular parallelepiped structure, and the two squeezing rollers 7 are rotatably arranged in parallel in the squeezing box 6. The driving assembly is connected to the two squeezing rollers 7 to drive the two squeezing rollers 7 to rotate toward each other, and the distance between the two squeezing rollers 7 is less than the diameter of the fresh fruit of the mistletoe and greater than the diameter of the fresh seeds of the mistletoe, so that the fresh seeds of the mistletoe can pass between the two squeezing rollers 7. The surface of the squeezing roller 7 is provided with at least one group of hemispherical protrusions, each group of protrusions is arranged along the axial direction of the squeezing roller 7, and the height of the protrusions is less than the distance between the two squeezing rollers 7. A feed port 8 is provided on the top of the squeezing box 6, and the feed port 8 is a conical structure. Both sides of the lower end of the feed port 8 are provided with elastic plates. The free ends of the elastic plates abut against the upper surfaces of the squeezing rollers 7 to limit the mistletoe fruits to be squeezed above the squeezing rollers 7. A sealing cover 12 is provided at the feed port 8, and a conical discharge port 10 is provided at the bottom of the squeezing box 6;

[0073] A scraper plate 9 is rotatably provided on the box walls on both sides of the squeezing box 6 and is located below the squeezing roller 7. The free end of the scraper plate 9 extends upward and abuts against the squeezing roller 7. A return spring is provided at the intersection of the scraper plate 9 and the box wall to ensure that the scraper plate 9 is tightly abutted against the squeezing roller 7.

[0074] A separation structure includes a vacuum box, a filter bag 11, and a vacuum pump 13. The vacuum box is arranged below the extrusion box 6. The free end of the tapered discharge port 10 extends into the vacuum box. The vacuum pump 13 is connected to the vacuum box to provide a vacuum environment for the vacuum box. The filter bag 11 is arranged at the tapered discharge port 10. The tapered discharge port 10 is made of a filter material. The mesh apertures of the filter bag 11 and the tapered discharge port 10 are larger than the diameter of fresh mistletoe seeds and smaller than fresh mistletoe fruits.

[0075] The infiltration structure includes a plurality of nozzles and a liquid storage tank. The plurality of nozzles are arranged in the extrusion box 6 to spray the infiltration liquid on the extrusion roller 7, the scraper plate 9 and the tapered discharge port 10. The infiltration liquid includes ethanol, glycerin and sugar syrup.

[0076] The method of using the peeling device of the present invention is as follows: a spray nozzle is used to spray a wetting liquid onto the squeezing roller 7, the squeezing box 6, and the conical discharge port 10, and the mistletoe fruit is placed into the feed port 8. While the spray nozzle sprays the wetting agent, the vacuum pump 13 and the drive component, such as a drive motor, are started to drive the two squeezing rollers 7 to rotate in opposite directions. The seed coat of the mistletoe fruit is squeezed to squeeze out the fresh mistletoe seeds, and the fresh mistletoe seeds fall into the conical discharge port 10. Under the action of pressure, the fresh mistletoe seeds enter the bottom of the vacuum box through the conical discharge port 10. Since the diameter of the fruit peel is larger than the aperture of the conical discharge port 10, the seeds and the fruit peel are separated. The separation of the fruit peel and the seeds is completed, and the vacuum pump 13 stops working.

[0077] The present invention scrapes off seeds adhered to the squeeze roller 7 by means of a scraper plate 9. The hemispherical protrusions on the squeeze roller 7 cause the scraper plate 9 to vibrate, causing the seeds adhered to the scraper plate 9 to fall to the conical discharge port 10. The elastic plate vibrates under the action of the hemispherical protrusions on the squeeze roller 7, thereby preventing the problem of mistletoe fruits accumulating at the feed port 8 and being difficult to drop.

[0078] <Example 5>

[0079] The present invention can also use the cleaning mechanism provided in Example 3 and the peeling device provided in Example 4 in combination to increase the continuity of the seed peeling pectin process and increase the automated cleaning process, that is, the cleaning mechanism is arranged in the vacuum box and is located below the conical discharge port 10. Specifically, the outer barrel 1 is arranged in the vacuum box and is located below the conical discharge port 10, as shown in FIG. Figure 4 shown.

[0080] The method of using the cleaning mechanism and peeling device of the present invention in combination is as follows: use a nozzle to spray a moistening liquid on the squeezing roller 7, the squeezing box 6, and the tapered discharge port 10, put the mistletoe fruit into the feed port 8, and at the same time as the nozzle sprays the moistening liquid, start the vacuum pump 13 and the drive assembly to drive the two squeezing rollers 7 to rotate in opposite directions, squeeze the seed coat of the mistletoe fruit to squeeze out the fresh mistletoe seeds, and the fresh mistletoe seeds fall into the tapered discharge port 10. Under the action of pressure, the fresh mistletoe seeds enter the first feed port 8 through the tapered discharge port 10. Inner barrel 2 or the second inner barrel 3, and the peel is separated from the seeds and peel molecules because its diameter is larger than the aperture of the tapered discharge port 10, and the peel and seeds are separated. The vacuum pump 13 stops working, and clean water is injected into the first inner barrel 2 and the second inner barrel 3 through the water supply pipe. The first stirring device 4 and the second stirring device 5 are started and stirred at the same time. The water is discharged from the drain port to keep the water level in the first inner barrel 2 and the second inner barrel 3 unchanged. The stirring and cleaning is continued until the pectin on the surface of the seeds is cleaned, and the fresh mistletoe seeds after removing the peel and pectin can be obtained.

[0081] The present invention utilizes a squeezing roller 7 to break the mistletoe fruit, reduces the viscosity of the seeds by using a moistening liquid, and allows the seeds to pass through the filter holes of the tapered discharge port 10 under the action of vacuum and enter the first inner barrel 2 and the second inner barrel 3. The seeds are stirred in the first inner barrel 2 and the second inner barrel 3 to remove pectin attached to the seeds. When the seeds are stirred in the first inner barrel 2, the seeds rotate in one direction, and pectin will remain on the back. The water flow generated by the directional rotation of the second inner barrel 3 cuts the back of the seeds in the first inner barrel 2, thereby cleaning the pectin attached to the back of the seeds.

[0082] <Effect Test>

[0083] <Test 1>

[0084] The proteins extracted from Example 1 and Example 2 were subjected to electrophoresis, and the results were as follows. Figure 1 As shown in FIG. 1 is the protein sample extracted in Example 1, 2 is the protein sample extracted in Example 2, and M is the protein MARKER. Figure 1 It can be seen that the protein bands extracted in Example 1 and Example 2 are clear and can meet the requirements for further protein analysis.

[0085] <Test 2>

[0086] Comparative Example 1: Phenol extraction method was used to extract total protein. The specific operation included the following steps:

[0087] 1) After removing the peel and pectin, fresh Morus alba seeds were washed with sterile water. 2 g of the seeds were placed in a liquid nitrogen-precooled mortar and pestle. 1 g of polyvinyl pyrrolidone (PVP) was added and the mixture was ground in liquid nitrogen.

[0088] 2) Add 3 volumes of extraction buffer (500 mmol / L Tris-HCl pH 8.65, 50 mmol / L EDTA, 100 mmol / L KCl, 2 mmol / L DTT), 3 volumes of Tris-HCl (pH 8.0) buffer saturated with phenol, evenly saturate for 1 min, centrifuge at 12000g, 20°C for 10 min, collect the phenol phase and the interface, discard the aqueous phase and precipitate, add 10 volumes of cold methanol containing 0.1 mmol / L NH4Ac to the collected solution, centrifuge at -20°C overnight, resuspend the precipitate in methanol containing 0.1 mmol / L NH4Ac, wash three times, wash once with cold acetone, dry naturally, and obtain a powdered crude protein, which is sealed and stored at -70°C for later use.

[0089] In step 5) of Example 1 and Example 2, the protein precipitate was dried under natural conditions to obtain a protein dry matter. An equal volume of RIPA protein lysis buffer was added to the protein dry matter of Example 1 and Example 2 and the powdered crude protein of Comparative Example 1 to dissolve the protein solution. The protein content of the protein solution was determined using the Coomassie Brilliant Blue G-250 method. The results are shown in Table 1.

[0090] Table 1

[0091] Protein content (ug / uL) Comparative Example 1 1.415 Example 1 1.462 Example 2 1.579

[0092] As shown in Table 1, the protein content of Example 1 and Comparative Example 1 is slightly higher than that of Comparative Example 1, demonstrating that the extraction method of the present invention can achieve the same protein extraction level as traditional phenol extraction. Compared with traditional phenol extraction methods, the extraction process of the present invention does not require the use of phenols and salts, avoiding the introduction of phenolic and salt impurities into the extracted protein and reducing impurities in the extracted protein. The protein content of Example 2 is higher than that of Example 1, demonstrating that the treatment method of the present invention can increase the protein extraction rate.

[0093] <Test 3>

[0094] The peeling device of Example 4 was used to remove the peel of fresh mistletoe seeds. Specifically, the mistletoe fruit was placed in the feed port, the squeezing roller and the vacuum pump were started to squeeze the mistletoe fruit, and the fresh mistletoe seeds attached with pectin were separated from the peel by the vacuum pump. After the fruit at the feed port was completely squeezed, the vacuum pump continued to operate for 10 minutes, and the seeds remaining in the conical feed port and the filter bag were counted, and the peel entering the bottom of the vacuum box was observed. The results are shown in Table 2.

[0095] Table 2

[0096] Example 4 Seeds remaining in the conical discharge port and filter bag (%) 8 The peel enters the bottom of the vacuum box No peels were seen entering the bottom of the vacuum box

[0097] From the results in Table 2, it can be seen that in the peeling device provided by the present invention, most of the seeds of the fruits that are crushed by the squeezing roller can enter the bottom of the vacuum box under the action of the vacuum pump, thereby effectively separating the peel and the seeds.

[0098] <Test 4>

[0099] Comparative Example 2: After squeezing seeds from parasitic fruits, the seeds with pectin attached are placed in a cleaning device for cleaning, the cleaning device comprising:

[0100] A mixing barrel provided with a drain outlet;

[0101] The filter barrel has filter holes on its barrel wall and barrel bottom. The filter barrel is set in the mixing barrel, and there is a gap between the bottom of the filter barrel and the bottom of the mixing barrel.

[0102] The stirring device is arranged in the stirring barrel.

[0103] When in use, the seeds with pectin attached are placed in the cleaning device, clean water is poured into the filter barrel, and the stirring device is started to stir the seeds in the filter barrel. The stirring speed is 1000r / min. While stirring and cleaning, the water after washing is discharged from the drain outlet. Even if water is poured and drained, stirring is performed while stirring. Stir and clean for 30 minutes, take out, and observe the pectin attachment on the surface of the seeds. The results are shown in Table 3.

[0104] Example 3: Using the cleaning mechanism of Example 3, seeds with pectin attached and the peel manually removed were placed in the first stirring device and the second stirring device for pectin elution operation. The stirring speed of the first stirring device and the second stirring device was 1000 r / min. The seeds were stirred and cleaned for 30 minutes, and then taken out to observe the pectin adhesion on the surface of the seeds. The results are shown in Table 3.

[0105] Example 5: Using Example 5, that is, using the cleaning mechanism and the peeling device in combination, the mistletoe fruit was placed in the peeling device. After peeling, the seeds with pectin adhered to them fell into the first stirring device and the second stirring device under the action of the vacuum pump for pectin elution. The stirring speed of the first stirring device and the second stirring device was 1000 r / min. The seeds were stirred and cleaned for 30 minutes, and then taken out to observe the pectin adhesion on the surface of the seeds. The results are shown in Table 3.

[0106] Table 3

[0107]

[0108] Note: The front side of the seed of the present invention refers to the side of the seed opposite to the rotating shaft during stirring and cleaning, and the back side of the seed refers to the side of the seed opposite to the rotating shaft during stirring and cleaning.

[0109] As can be seen from the results in Table 3, the results of Comparative Example 2 show that during unidirectional rotation, the shear force generated by the water flow rotation fails to act on the pectin on the back of the seeds, resulting in residual pectin on the back of the seeds. The results of Example 3 show that the cleaning mechanism of the present invention can clean and remove residual pectin on the back of the seeds, solving the problem of residual pectin on the back of the seeds during unidirectional stirring and cleaning, which affects subsequent protein extraction. The results of Example 5 show that when the peeling device and the cleaning mechanism are used in combination, the present invention can achieve automated peeling and automated pectin elution, increasing the automation of the peeling and degumming operations.

[0110] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for extracting total protein from loranthus parasiticus seeds, characterized in that: The following steps are involved: 1) fresh loquat seeds, after removing the peel and pectin, were washed with sterile water, placed in a liquid nitrogen-precooled mortar, polyvinyl pyrrolidone was added, and the mixture was ground in liquid nitrogen to obtain a pulverized product; wherein the mass of the fresh loquat seeds was 2-4 g and the mass of the polyvinyl pyrrolidone was 1-3 g; 2) Place the pulverized material into a pre-chilled EP tube, add RIPA protein lysis buffer and PMSF solution, shake well, and place the EP tube on ice for lysis. The volume of RIPA protein lysis buffer should be 5-10 mL, and the volume ratio of RIPA protein lysis buffer to PMSF solution should be 1:

100. 3) After the lysis is completed, the mixture is crushed using a portable high-speed disperser and centrifuged to obtain a first supernatant; wherein the crushing time of the portable high-speed disperser is 1-5 minutes, the centrifuge speed is 5000 rpm, and the centrifugation is carried out for 15 minutes at 4°C; 4) Add RIPA protein lysis buffer to the first supernatant again, lyse on ice, and centrifuge to obtain a second supernatant; the volume of RIPA protein lysis buffer is 3-5 mL, lyse on ice for 15-35 minutes, and centrifuge at 5000 rpm for 15 minutes at 4°C; 5) Add pre-cooled acetone to the second supernatant, precipitate at -20°C overnight, centrifuge to obtain protein precipitate, air dry the protein precipitate, and dissolve it with RIPA protein lysis buffer to obtain protein solution; the volume of pre-cooled acetone should be 3-5 times the volume of the second supernatant, centrifuge at 5000 rpm for 15 minutes at 4°C 6) The protein solution was placed in a 95°C water bath for 5-10 minutes, then quickly placed in an ice bath for 5 minutes, centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected.

2. The method for extracting total protein from loranthus parasiticus seeds according to claim 1, wherein The peeling operation is performed in a peeling device to obtain seeds with peels removed and pectin adhered thereto, and then the seeds with peels removed and pectin adhered thereto are cleaned in a cleaning mechanism to remove the pectin adhered to the seed surface, thereby obtaining fresh mistletoe seeds with peels and pectin removed.

3. The method for extracting total protein from loranthus parasiticus seeds according to claim 1, wherein Step 3) The precipitate A obtained by centrifugation is vacuum dried at a vacuum degree of -0.099 MPa and a drying temperature of 7°C. After drying, pre-cooled sodium salicylate solution, RIPA protein lysis buffer, and PMSF solution are added, and the mixture is frozen at -20°C for 2-5 hours, taken out and thawed at room temperature, crushed with a portable high-speed disperser, and centrifuged to obtain a re-extraction supernatant, which is combined with the first supernatant and proceeds to step 4).

4. The method for extracting total protein from loranthus parasiticus seeds according to claim 3, wherein The volume of sodium salicylate solution is 1-3 mL, the volume of RIPA protein lysis buffer is 5-10 mL, the volume ratio of RIPA protein lysis buffer to PMSF solution is 1:100, and the concentration of sodium salicylate solution is 2-5 mol / L.

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