Anoectochilus formosanus rich in organic selenium polysaccharide, cultivation method thereof and preparation method of anoectochilus formosanus selenium polysaccharide

Abstract

The invention provides anoectochilus formosanus rich in organic selenium polysaccharide, a cultivation method of the anoectochilus formosanus rich in organic selenium polysaccharide and a preparation method of anoectochilus formosanus selenium polysaccharide, and the cultivation method comprises the following steps: inoculating an improved MS culture medium with a single-bud stem segment of an anoectochilus formosanus tissue culture seedling plant, germinating axillary buds by the stem segment and carrying out strong seedling culture and seedling propagation, ammonium nitrate is not adopted, and inositol is avoided; anoectochilus formosanus seedlings are obtained through seedling hardening and cleaning, then the anoectochilus formosanus seedlings are planted into a transplanting matrix containing selenium, and organic selenium polysaccharide enriched anoectochilus formosanus raw material plants are planted and produced in combination with foliage selenium application. The anoectochilus formosanus plants rich in selenium polysaccharide are produced through anoectochilus formosanus seedling propagation and combination of various cultivation ways and serve as anoectochilus formosanus selenium polysaccharide extraction raw materials, the high biotransformation rate of anoectochilus formosanus to selenium is fully played, natural anoectochilus formosanus selenium polysaccharide is produced through biosynthesis, the anoectochilus formosanus selenium polysaccharide is prepared through extraction and purification, the preparation rate is improved, and the production cost is reduced. The edible and medicinal values of the anoectochilus formosanus are improved.

Description

Technical Field

[0001] The invention relates to the technical field of roxburghii selenium polysaccharides, and in particular to a roxburghii rich in organic selenium polysaccharides and a cultivation method thereof, and a preparation method of roxburghii selenium polysaccharides. Background Art

[0002] There are a few types of selenium polysaccharides on the market, including garlic selenium polysaccharides, ganoderma lucidum selenium polysaccharides, astragalus selenium polysaccharides and seaweed selenium polysaccharides. There are two ways to obtain selenium polysaccharides: one is to extract natural biological selenium polysaccharides, and the other is to synthesize artificial selenium polysaccharides. Because polysaccharides have hydroxyl, ketone and aldehyde groups, after the organism absorbs selenium, the functional groups are shrunk and selenized in the body, and then converted into natural biological selenium polysaccharides, which are extracted by water or enzyme solution combined with ultrasonic or microwave extraction methods. Artificially synthesized selenium polysaccharides are generated by the direct reaction of polysaccharides with selenium compounds.

[0003] At present, synthetic selenium polysaccharides are not only limited in types, but also lack safety guarantees. Anoectochilus roxburghii selenium polysaccharide is a natural selenium polysaccharide. Among them, anoectochilus glycoside is the main characteristic component of Anoectochilus roxburghii polysaccharide, which has unique medicinal and edible functions. Therefore, Anoectochilus roxburghii selenium polysaccharide is significantly different from existing natural selenium polysaccharides. Anoectochilus roxburghii selenium polysaccharide is an ideal organic selenium polysaccharide with high safety. Even if consumed in excess, it has basically no toxic side effects. It is one of the most ideal selenium supplement products for the human body.

[0004] A method for enriching Anoectochilus with selenium includes applying selenium-containing substances such as selenium-rich tea powder as exogenous selenium fertilizer to the roots or leaves of Anoectochilus, so that it can be absorbed and converted into organic selenium (including Anoectochilus selenium polysaccharides) in the body. However, selenium-rich biological residues (or scraps) such as selenium-rich tea powder must be planted on selenium-rich soil. Planting Anoectochilus with them as selenium sources to obtain selenium-rich Anoectochilus has problems such as unstable selenium content and limited resources. In addition, the extraction rate of Anoectochilus selenium polysaccharides needs to be improved. Summary of the invention

[0005] The purpose of the present invention is to provide a golden thread vine rich in organic selenium polysaccharide and a cultivation method thereof, and a method for preparing golden thread vine selenium polysaccharide. The golden thread vine seedlings are expanded and cultured by adopting a modified MS culture medium containing selenium element, and the golden thread vine seedlings are transplanted and planted by adopting a transplanting matrix added with selenium fertilizer and spraying selenium element on the leaves, so as to obtain a golden thread vine plant rich in selenium polysaccharide. Finally, the extraction and purification steps of golden thread vine selenium polysaccharide are optimized, thereby improving the preparation rate of golden thread vine selenium polysaccharide.

[0006] The present invention solves the technical problem by adopting the following technical solutions.

[0007] The present invention provides a method for cultivating aroximately 240 grams of organic selenium polysaccharide, comprising the following steps:

[0008] S1. Inoculating a single bud stem segment of a tissue culture seedling of Anoectochilus roxburghii on a modified MS medium, and performing seedling propagation and cultivation in a manner that axillary buds germinate from the stem segment and grow roots into seedlings, and obtaining Anoectochilus roxburghii seedlings after training the seedlings, washing and drying, wherein the modified MS medium includes selenium, and uses (NH4)2SO4, NH4Cl and Ca(NO3)2 instead of ammonium nitrate, and no inositol;

[0009] S2. Transplanting the roxburghii seedlings into a transplanting medium containing selenium to obtain roxburghii rich in organic selenium polysaccharides.

[0010] The present invention provides a golden thread vine rich in organic selenium polysaccharide, which is cultivated according to the cultivation method.

[0011] The present invention provides a method for preparing a roxburghii roxburghii selenium polysaccharide, comprising the following steps:

[0012] The organic selenium-rich polysaccharide roxburghii is harvested, washed, and dried to obtain a roxburghii plant dry product;

[0013] The dried product of the roxburghii plant is crushed and sieved to obtain sieved powder, and then the sieved powder is subjected to selenium polysaccharide extraction. The obtained crude selenium polysaccharide filtrate is purified to obtain roxburghii selenium polysaccharide.

[0014] The beneficial effects of the organic selenium-rich polysaccharide Anoectochilus roxburghii and its cultivation method, and the preparation method of Anoectochilus roxburghii selenium polysaccharide according to the embodiments of the present invention are:

[0015] The present invention proposes a complete solution for the extraction and purification of selenium polysaccharide from seedling propagation, transplanting and planting to the extraction and purification of selenium polysaccharide from Anoectochilus roxburghii, that is, the propagation medium of Anoectochilus roxburghii (improved with MS formula, and ammonium nitrate which is easy to make explosive is abandoned) is added with selenium elements (sodium selenite, Se 4+ ) Cultivate and expand the seedlings of Anoectochilus roxburghii, transplant and plant (substrate addition, spraying of selenium during planting period), harvest, dry, screen, extract, microwave + ultrasonic, rotary evaporation, protein removal and other propagation, planting, extraction and purification, and increase the content of selenium polysaccharides by extending the planting spraying absorption and conversion period, applying small amounts of leaves multiple times and other measures. The present invention can continuously and uninterruptedly produce and obtain Anoectochilus roxburghii selenium polysaccharides all year round, and can produce Anoectochilus roxburghii selenium polysaccharides with different selenium contents according to the requirements of grade (mainly organic selenium content), adding a new natural selenium polysaccharide product to the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a diagram illustrating the production method of the raw material plant of Anoectochilus roxburghii rich in organic selenium polysaccharide;

[0018] Figure 2 This is a schematic diagram of transplanting and planting in a seedling tray substrate;

[0019] Figure 3 This is a schematic diagram of understory planting;

[0020] Figure 4 This is a schematic diagram of sampling for understory planting;

[0021] Figure 5 This is a graph showing the test results of inorganic selenium and total selenium content in the roxburghii roxburghii plants cultured and propagated with organic selenium-rich polysaccharides in Example 1 and the roxburghii roxburghii plants in the control group. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0023] The following specifically describes the organic selenium-rich polysaccharide Anoectochilus roxburghii and its cultivation method, as well as the preparation method of Anoectochilus roxburghii selenium polysaccharide according to the embodiments of the present invention.

[0024] Reference Figure 1 As shown, a method for cultivating a golden thread vine rich in organic selenium polysaccharide provided in an embodiment of the present invention comprises the following steps:

[0025] S1. Inoculate a single bud stem segment of a tissue culture seedling of Anoectochilus roxburghii on a modified MS medium, and perform seedling propagation in a manner that the stem segment germinates axillary buds and grows roots into seedlings. After training the seedlings, washing and drying, obtain Anoectochilus roxburghii seedlings, wherein the modified MS medium includes selenium, and (NH4)2SO4, NH4Cl and Ca(NO3)2 are used instead of ammonium nitrate, and inositol is absent. Preferably, the present invention uses equivalent concentrations of (NH4)2SO4, NH4Cl and Ca(NO3)2 to replace NH4 in ammonium nitrate (NH4NO3). + -N and NO3 - -N, to provide the same equivalent concentration of NH4 + -N and NO3-N, omitting the addition of inositol. The present invention adopts a modified MS medium for the propagation of golden thread vine seedlings. The modified MS medium discards the explosive ammonium nitrate and inositol, and adds selenium (sodium selenite, Se 4+ ) Cultivate and propagate Anoectochilus roxburghii seedlings, thereby increasing the content of selenium polysaccharides in Anoectochilus roxburghii.

[0026] Further, in a preferred embodiment of the present invention, the improved MS medium includes (NH4)2SO4 190-220g / L, NH4Cl 100-120g / L, Ca(NO3)2·4H2O 620g / L, KNO31900-2200g / L, KH2PO3 170-250g / L, Mg(NO3)2·6H2O 380-420g / L, FeSO4·7H2O 27.8mg / L, Na2EDTA 37.3mg / L, H3BO3 6.2mg / L, MnSO4·4H2O22.3mg / L, ZnSO4·7H2O 8.6mg / L, KI 0.83mg / L, Na2MoO4·2H2O 0.25 mg / L, glycine 2 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.5 mg / L, naphthaleneacetic acid (NAA) 0.5-1.2 mg / L, banana puree 80-100 g / L, potato 30-40 g / L, peptone 1.0-1.5 g / L, sucrose 30 g / L, agar powder 5.2-5.6 g / L, activated carbon 0.6-1.0 g / L and sodium selenite (Na2SeO3) 0.2-15 mg / L, wherein the pH value of the improved MS culture medium is 5.8.

[0027] Furthermore, in a preferred embodiment of the present invention, the light intensity of the culture is 3500Lx-5000Lx, the culture time is 120-128 days, and the time of the seedling training is 52-60 days. The present invention regenerates new roxburghii plants by germinating axillary buds from a single bud stem segment of a roxburghii tissue culture seedling plant and growing roots to form seedlings. The plants are cultured for about 125 days under a culture light intensity of 3500Lx-5000Lx, and then moved into a seedling training greenhouse for 52-60 days before transplanting and planting.

[0028] S2. Transplanting the roxburghii seedlings into a transplanting medium containing selenium to obtain roxburghii plants rich in organic selenium polysaccharides.

[0029] Furthermore, in a preferred embodiment of the present invention, the planting is carried out by bag-type matrix transplanting or seedling tray-type matrix transplanting, and the planting time is 4 to 6 months.

[0030] Specifically, the specifications of the cultivation bag for transplanting with bag-type substrate are length × width = 60cm × 25cm, and the length of the iron wire is 83.5cm. Imported peat (EU, Nordherun) is used, with peat: river sand = 3.0 ~ 4.2: 1 (v / v) as the mixed matrix, 1.7L per bag, 0.8 ~ 1.2g / L of Huabao No. 2, and 4.8 ~ 5.2g / L of microbial agents are added. Among them, the biological agent is selected from Bacillus licheniformis or Bacillus laterosporus. Take the golden thread lotus seedlings after training, clip them out with a tweezer, wash the root culture medium with clean water, plant 1 bottle of seedlings (about 30 plants) in each bag after drying, and place them in a sunshade shed or greenhouse for post-transplant planting management. Regulate environmental conditions, with temperature as the main regulating factor, keep the ambient temperature no more than 30℃, especially in the hot summer season, the ambient temperature around the transplanting bag can be reduced by increasing the ambient humidity and cooling by water evaporation. When the temperature drops, adjust the light intensity with a double-layer sunshade net, and the light intensity should be 3500Lx~6000Lx. It can be harvested and washed 4 to 6 months after transplanting, and dried at 85℃±5℃ for 10 hours and then stored at 1~5℃.

[0031] Reference Figure 2 As shown, the transplanting matrix of seedling tray type substrate is peat: river sand = 3.0 ~ 4.2: 1 as the transplanting mixed matrix, adding Huabao No. 2 0.8 ~ 1.2g / L, biological agent 4.8 ~ 5.2g / L. After the matrix is ​​mixed, it is loaded into the seedling tray (50cm × 30cm × 5cm), and each tray is filled with 4L ~ 5L of mixed matrix. Then select the golden thread lotus tissue culture seedlings of relatively uniform size (remove the seedlings), wash the roots, plant 120 golden thread lotus seedlings in each tray, and place them on the brick (or sand) cushion layer or fence bed in the greenhouse for post-transplant management. The cultivation management environment temperature does not exceed 30℃, and the light is adjusted with a double-layer sunshade net. The light intensity is preferably 3500Lx ~ 6000Lx. It can be harvested and washed 4 to 6 months after transplanting, and dried at 85℃ ± 5℃ for 10h and stored at 1 ~ 5℃.

[0032] Furthermore, in a preferred embodiment of the present invention, the transplanting matrix containing selenium includes peat, river sand, Huabao No. 2, biological bacteria and sodium selenite (Na2SeO3), wherein the concentration of Huabao No. 2 is 0.8-1.2 g / L, the concentration of the biological bacteria is 4.8-5.2 g / L, and the concentration of sodium selenite is 0.5 mg / L-25 mg / L. The matrix mixing order is: the amount of Huabao No. 2 and sodium selenite to be added is calculated according to the volume of the matrix, and the two fertilizer sources are first weighed and dissolved into aqueous solutions, and then evenly sprayed on the surface of the river sand and then mixed with peat and biological bacteria fertilizer.

[0033] Further, in a preferred embodiment of the present invention, the transplanting of the golden thread vine seedlings into a transplanting matrix containing selenium also includes spraying the leaves of the golden thread vine seedlings with a first selenium fertilizer, which is a foliar fertilizer. The transplanting matrix containing selenium includes peat, river sand, Huabao No. 2, biological bacteria and sodium selenite, wherein the volume ratio of the peat to the river sand is 3.0-4.2:1, the concentration of Huabao No. 2 is 0.8-1.2g / L, the concentration of the biological bacteria is 4.8-5.2g / L, the concentration of sodium selenite is 0.5mg / L-25mg / L, and the first selenium fertilizer is a 100-200mg / L Na2SeO3 solution. The matrix mixing order is: Huabao No. 2 and selenium fertilizer need to be added according to the volume of the matrix, and they are weighed and dissolved into aqueous solutions respectively, and then evenly sprayed on the surface of the river sand and mixed with peat and biological bacteria fertilizer. The present invention first fixes the golden thread lotus seedlings in a planting tray, and sprays selenium fertilizer on the leaves after 25 to 30 days of fixing. Spray 100 to 200 mg / L of Na2SeO3 1 to 4 times (1 to 2 times for consumption, 3 to 4 times for selenium polysaccharide extraction), and the appropriate spraying amount is 0.5 mg / plant. Each spraying is spaced 15 days apart. Regulate environmental climate conditions, with temperature as the main regulating factor, that is, keep the ambient temperature ≤30°C. In the high temperature season in summer, the ambient humidity can be increased by spraying, and the ambient temperature around the transplanted seedlings can be reduced by the method of water evaporation cooling. When cooling, the light intensity is adjusted, and the light intensity is preferably 3500Lx to 6000Lx. It can be harvested 4 to 6 months after transplanting.

[0034] In the present invention, the amount of selenium applied (sodium selenite sprayed on the leaves) and the polysaccharide content are correlated with a polynomial curve, and the polynomial is: y=672.73x 2 +21.636x+42.436, R 2 =0.9932. Selenium content is correlated with polysaccharide content, with a correlation coefficient of r = -0.8967. Transplanting seedlings propagated and cultured with selenium in the propagation seedling medium combined with foliar spraying can increase the content of selenium polysaccharides (polysaccharide and selenium dual content).

[0035] Reference Figure 3 and Figure 4As shown, further, in a preferred embodiment of the present invention, the transplanting matrix containing selenium includes natural humus soil on the surface of the forest and a second selenium fertilizer, and the second selenium fertilizer is 1.10mg / L to 32.85mg / L of sodium selenite. Among them, when used for edible products, the application amount of the second selenium fertilizer is 1.10mg / L to 15.0mg / L; when used for selenium polysaccharide extraction, the application amount of the second selenium fertilizer is 15.0mg / L to 32.85.0mg / L. Specifically, dissolve sodium selenite with an appropriate amount of clean water, spray and mix it, and spread it on the surface of the ridge with a width of 0.9m and a ditch width of 0.4m after mixing evenly, with a thickness of about 4.5cm. About 120 plants are planted in about 4L to 5L of humus soil. After dividing the row spacing, the golden thread vine seedlings with a relatively uniform size (removing the seedlings) are cultivated and expanded for planting. Rat and bird prevention management and cultivation environment condition regulation and management are carried out to maintain the ambient light intensity of 4500Lx~70000Lx and the ambient temperature of ≤30℃. It can be harvested 4~6 months after transplanting.

[0036] The present invention produces raw material plants of Anoectochilus roxburghii rich in organic selenium polysaccharides by cultivating and expanding seedlings of Anoectochilus roxburghii combined with substrate transplanting and continuous propagation. Anoectochilus roxburghii plants rich in selenium polysaccharides are produced by adding selenium to the culture medium for cultivation and propagation, adding selenium fertilizer to the culture medium for cultivation and propagation of seedlings, and then spraying selenium on the leaves during the transplanting and planting process, adding selenium fertilizer to the transplanting and planting matrix combined with spraying selenium on the leaves, or planting with selenium fertilizer added to the humus soil under the forest, so that the plants can be used as raw materials for extracting selenium polysaccharides from Anoectochilus roxburghii. Give full play to the selenium (Se 4+ ) with a high bioconversion rate, and biosynthesizes and produces natural Anoectochilus roxburghii selenium polysaccharide. The invention uses sodium selenite as a selenium source, and artificially and continuously propagates and plants Anoectochilus roxburghii plant raw materials rich in organic selenium polysaccharide, so as to increase the edible and medicinal value of Anoectochilus roxburghii.

[0037] The present invention provides a roxburghii rich in organic selenium polysaccharide, which is cultivated according to the cultivation method. The present invention increases the content of selenium polysaccharide in roxburghii plants by increasing the timely spraying during the planting of roxburghii seedlings to extend the absorption and conversion period, and combines the application of small amounts of selenium elements on the leaves for multiple times, and strengthens the light to promote the lignification of plants, and compares and screens the improved extraction methods to increase the preparation rate of roxburghii selenium polysaccharide.

[0038] The present invention provides a method for preparing roxburghii selenium polysaccharide, comprising the following steps:

[0039] The organic selenium-rich polysaccharide roxburghii plant is harvested, washed and dried to obtain a dry product of the roxburghii plant. Preferably, after the organic selenium-rich polysaccharide roxburghii plant is harvested and washed, it is dried at 85°C±5°C for 10 hours to obtain a dry product of the roxburghii plant.

[0040] The dried product of the roxburghii plant is crushed and sieved to obtain a sieved powder, and then the sieved powder is subjected to selenium polysaccharide extraction, and the obtained crude selenium polysaccharide filtrate is purified to obtain a roxburghii selenium polysaccharide. Specifically, the crushing and sieving steps are: the dried product of the roxburghii plant harvested from planting is crushed and sieved through a 100-mesh sieve, and the coarse powder is repeatedly crushed and sieved through a 100-mesh sieve to obtain a sieved powder.

[0041] Furthermore, in a preferred embodiment of the present invention, the step of extracting selenium polysaccharides from the sieved powder is: adding distilled water to the sieved powder and adjusting the pH to 6, then ultrasonically treating for 30 to 40 minutes, adding cellulase for water bath reaction, collecting the filtrate and repeating the above steps for extraction of the residue once, to obtain the crude selenium polysaccharide filtrate. Preferably, the mass ratio of sieved powder to distilled water is 1:18 to 22 (g / mL), and the mass ratio of cellulase to sieved powder is 0.03 to 0.06:1. The water bath reaction is first carried out at a water temperature of 52 to 58°C for 1.8 to 2.2 hours, and then at 92 to 97°C for 8 to 12 minutes.

[0042] It should be noted that in the present invention, the water extraction method can also be used to extract selenium polysaccharides from the sieved powder. The specific steps are: add distilled water (180-220 ml / 10 g powder) to the sieved powder, soak for 30 minutes, hot water bath at 85°C for 2 hours, and filter to obtain the filtrate; repeat the above steps with the filter residue to extract once to obtain the crude selenium polysaccharide filtrate.

[0043] Furthermore, in a preferred embodiment of the present invention, the purification step is: rotary evaporating the crude selenium polysaccharide filtrate to about 1 / 10 of the liquid volume, mixing the obtained concentrated solution with Sevag agent 5:1 and oscillating for 30 minutes, centrifuging and rotary evaporating again to obtain Anoectochilus roxburghii selenium polysaccharide.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1

[0046] This embodiment provides a polysaccharide rich in organic selenium, which is prepared according to the following steps:

[0047] (1) On the improved MS medium, axillary buds were germinated from the stem segments of the tissue culture seedlings of A. roxburghii and the roots were grown into seedlings. The new A. roxburghii plants were then regenerated into new plants. The plants were then moved into a seedling training greenhouse for about 55 days and the roots were washed with clean water to obtain A. roxburghii seedlings. The light intensity was about 5000 Lx and the number of days for the culture was about 125 days. The modified MS medium included (NH4)2SO4 190 g / L, NH4Cl 100 g / L, Ca(NO3)2·4H2O 620 g / L, KNO3 1900 g / L, KH2PO3 250 g / L, Mg(NO3)2·6H2O 380 g / L, FeSO4·7H2O 27.8 mg / L, Na2EDTA 37.3 mg / L, H3BO 36.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, naphthaleneacetic acid 1.2mg / L, banana puree 100g / L, potato 30g / L, peptone 0.8g / L, sucrose 30g / L, agar powder 5.6g / L, activated carbon 1.0g / L and sodium selenite 15mg / L, with a pH value of 5.8.

[0048] (2) Using peat: river sand = 3.0:1 as the matrix, add 1.2g / L of Huabao No. 2, 5g / L of biological bacteria agent and 25mg / L of sodium selenite. The order of mixing the transplanting matrix is ​​as follows: calculate the amount of Huabao No. 2 and sodium selenite to be added according to the volume of the matrix, first weigh the two fertilizer sources separately and dissolve them into aqueous solutions, and then spray them evenly on the surface of the river sand and then mix them with peat and biological bacteria fertilizer. After mixing, put the matrix into seedling trays (50cm×30cm×5cm), and each tray contains 5L of mixed matrix. Then take the golden thread lotus tissue culture seedlings from step (1), select seedlings with relatively uniform size (remove small seedlings), plant 120 golden thread lotus seedlings in each tray, and place the planting trays in the greenhouse, on the brick (or sand) cushion layer of the greenhouse or on the fence bed of the greenhouse. Carry out normal cultivation management. Use clean water to control the environmental humidity and matrix humidity. Control the environmental conditions, with temperature as the main control factor, that is, keep the environmental temperature ≤30℃. When the temperature drops, the light intensity is adjusted, and the light intensity is preferably 3500Lx-6000Lx. After transplanting and cultivating for 4 months, the plant material of golden thread lotus rich in organic selenium polysaccharide can be obtained.

[0049] This embodiment also provides a method for preparing selenium polysaccharide from Anoectochilus roxburghii, comprising the following steps:

[0050] The organic selenium polysaccharide-rich roxburghii was harvested, washed, dried at 85°C for 10 hours, and then stored at 5°C to obtain a dry product of the roxburghii plant.

[0051] The dried product of the plant of Anoectochilus roxburghii harvested from planting was crushed and passed through a 100-mesh sieve. The obtained coarse powder was repeatedly crushed and passed through a 100-mesh sieve and then sealed and stored at 1-5°C to obtain the sieved powder. The sieved powder was weighed, 20 times distilled water was added and the pH was adjusted to 6, and then ultrasonic treatment was performed for 35 minutes. Then, an appropriate amount of cellulase was added and the mixture was water bathed at 55°C for 2 hours, and then water bathed at 95°C for 10 minutes. After the filtrate was collected, the extraction was repeated once with the filter residue. The filtrate was collected and stored at 1-5°C to obtain the crude selenium polysaccharide filtrate.

[0052] Finally, the crude selenium polysaccharide filtrate was rotary evaporated to 1 / 10 of the liquid volume, and then the protein was removed by the Sevag method. After oscillation, centrifugation and re-rotary evaporation, the separated and purified material was collected, freeze-dried, and stored at -20°C to obtain Anoectochilus roxburghii selenium polysaccharide.

[0053] Example 2

[0054] This embodiment provides a method for preparing a roxburghii roxburghii polysaccharide rich in organic selenium and a roxburghii roxburghii selenium polysaccharide, which is mainly different from embodiment 1 in that:

[0055] (2) With peat: river sand = 3.0:1 as the matrix, add 1.2g / L of Huabao No. 2, 5g / L of biological bacteria agent and 5mg / L of sodium selenite to obtain the transplanting matrix. The matrix mixing order is as follows: Huabao No. 2 and selenium fertilizer are added according to the volume of the matrix, and the required amount is weighed and dissolved into aqueous solution respectively, and then sprayed evenly on the surface of the river sand and then mixed with peat and biological bacteria fertilizer. After the matrix is ​​mixed, it is placed in a seedling tray (50cm×30cm×5cm), and each tray is filled with 5L of mixed matrix. Then take the golden thread vine tissue culture seedlings of step (1), select the golden thread vine tissue culture seedlings of relatively uniform size (remove the small seedlings), plant 120 golden thread vine seedlings in each tray, and place the planting tray in the greenhouse, on the brick (or sand) cushion layer of the greenhouse or on the fence bed of the greenhouse. Spray selenium fertilizer on the leaves 25 to 30 days after planting. For foliar spraying of selenium fertilizer, use 100mg / L Na2SeO3 and spray 4 times, and the appropriate spray volume is 0.5mg / plant. Each spraying should be done 15 days apart. Regulate environmental climate conditions, with temperature as the main regulating factor, that is, keep the ambient temperature ≤30℃. In the hot summer season, the ambient humidity can be increased by spraying, and the ambient temperature around the transplanted seedlings can be reduced by cooling down with water evaporation. When cooling down, adjust the light intensity, and the light intensity should be 3500Lx~6000Lx. After 4 months of transplanting, you can get the plant material of golden thread vine rich in organic selenium polysaccharides.

[0056] Example 3

[0057] This embodiment provides a method for preparing a roxburghii roxburghii polysaccharide rich in organic selenium and a roxburghii roxburghii selenium polysaccharide, which is mainly different from embodiment 1 in that:

[0058] (2) The natural humus soil under the forest surface is added with selenium fertilizer as the cultivation medium, and the tissue culture seedlings of Anoectochilus roxburghii are transplanted under the forest. The application rate of selenium fertilizer under the forest is 22.0 mg / L. The specific steps are: dissolve sodium selenite in an appropriate amount of clean water, spray and mix it, and spread it on the surface of the ridge with a width of 0.9m and a ditch width of 0.4m after mixing evenly, with a thickness of about 4.5cm. About 120 plants are planted in about 5L of humus soil. After dividing the row spacing, the Anoectochilus roxburghii seedlings with relatively uniform size (remove the small seedlings) are planted. Anti-rat and anti-bird management is carried out, and the cultivation environment conditions are regulated and managed to maintain the ambient light intensity of 4000Lx~70000Lx, and the ambient temperature is maintained at ≤30℃. After 4 months of transplanting and planting, the Anoectochilus roxburghii plant material rich in organic selenium polysaccharides can be obtained.

[0059] Example 4

[0060] This embodiment provides a method for preparing selenium polysaccharide from Anoectochilus roxburghii, comprising the following steps:

[0061] The organic selenium polysaccharide-rich roxburghii plant material of Example 2 was harvested, washed, dried at 85° C. for 10 h, and then stored at 5° C. to obtain a roxburghii plant dry product.

[0062] The dried product of the plant of Anoectochilus roxburghii harvested from planting was crushed and passed through a 100-mesh sieve, and the obtained coarse powder was repeatedly crushed and passed through a 100-mesh sieve and sealed at 1-5°C to obtain a sieved powder. The sieved powder was weighed, and distilled water was added to the sieved powder to soak and ultrasonically treat for 35 minutes, and then CO2 supercritical liquid extraction was used at 31.1°C and 7.38MPa for 50 minutes, and the filtrate was collected and stored at 1-5°C to obtain a crude selenium polysaccharide filtrate.

[0063] Finally, the crude selenium polysaccharide filtrate was rotary evaporated to 1 / 10 of the liquid volume, and then the protein was removed by the Sevag method. After oscillation, centrifugation and re-rotary evaporation, the separated and purified material was collected, freeze-dried, and stored at -20°C to obtain Anoectochilus roxburghii selenium polysaccharide.

[0064] Test Example 1

[0065] This test example uses DBS42 / 002-2014 and GB 5009.93-2017 (first method) to detect the inorganic selenium and total selenium content in the golden thread vine plants of Example 1 and the golden thread vine plants of the control group. Among them, no selenium fertilizer was applied to the control group. The test was commissioned by the Fujian Product Quality Inspection Institute.

[0066] like Figure 5 The figure shows the test results of inorganic selenium and total selenium content in the roxburghii roxburghii plants grown by the organic selenium-rich polysaccharide seedling culture and propagation in Example 1 and the roxburghii roxburghii plants in the control group. Figure 5It can be seen that the total selenium content of the organic selenium-rich Anoectochilus roxburghii plant, which was cultured for 125 days and trained for 55 days, reached 51.1 mg / kg·dw, while the total selenium content of the control Anoectochilus roxburghii plant (cultured without adding selenium) was 0.058 mg / kg·dw, a difference of 881 times. The organic selenium content of the Anoectochilus roxburghii plant cultured with selenium-added seedlings accounted for 71.0%, and the organic selenium content could be increased to more than 80% after subsequent transplanting and planting, that is, the conversion rate of selenium and polysaccharide binding was further improved.

[0067] Test Example 2

[0068] This test example uses SN / T 4260-2015 to study the effects of different extraction methods on the preparation rate of Anoectochilus roxburghii selenium polysaccharide in Example 2 and Example 4. The test was specifically commissioned to Jike Testing Technology (Fujian) Co., Ltd. As shown in Table 1, the Anoectochilus roxburghii used in the experiment and the selenium content of the Anoectochilus roxburghii selenium polysaccharide after extraction and selenium content are shown.

[0069] Table 1 Selenium polysaccharides and selenium content of Anoectochilus roxburghii

[0070]

[0071] As can be seen from Table 1, the polysaccharide extraction effect of enzyme + ultrasonic extraction is 17.1% higher than that of water + ultrasonic extraction, and the selenium content is increased by 10.7% accordingly.

[0072] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for cultivating aroxithium rich in organic selenium polysaccharide, characterized in that: The following steps are involved: S1. Inoculating a single bud stem segment of a tissue culture seedling of Anoectochilus roxburghii on a modified MS medium, and performing seedling propagation and cultivation in a manner that axillary buds germinate from the stem segment and grow roots into seedlings, and obtaining Anoectochilus roxburghii seedlings after training the seedlings, washing and drying, wherein the modified MS medium includes selenium, and uses (NH4)2SO4, NH4Cl and Ca(NO3)2 instead of ammonium nitrate, and no inositol; S2. Transplanting the roxburghii seedlings into a transplanting medium containing selenium to obtain roxburghii rich in organic selenium polysaccharides.

2. The cultivation method according to claim 1, characterized in that In step S1, the improved MS medium includes (NH4)2SO4 190-220g / L, NH4Cl 100-120g / L, Ca(NO3)2·4H2O 620g / L, KNO3 1900-2200g / L, KH2PO3 170-250g / L, Mg(NO3)2·6H2O 380-420g / L, FeSO4·7H2O 27.8mg / L, Na2EDTA37.3mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, KI 0.83mg / L, Na2MoO4·2H2O 0.25 mg / L, glycine 2 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.5 mg / L, naphthaleneacetic acid 0.5-1.2 mg / L, banana puree 80-100 g / L, potato 30-40 g / L, peptone 1.0-1.5 g / L, sucrose 30 g / L, agar powder 5.2-5.6 g / L, activated carbon 0.6-1.0 g / L and sodium selenite 0.2-15 mg / L, wherein the pH value of the improved MS medium is 5.

8.

3. The cultivation method according to claim 1, characterized in that In step S1, the light intensity of the culture is 3500Lx-5000Lx, the culture time is 120-128 days, and the time for training the seedlings is 52-60 days.

4. The cultivation method according to claim 1, characterized in that In step S2, the planting is carried out by bag-type matrix transplanting or seedling tray-type matrix transplanting, and the planting time is 4 to 6 months.

5. The cultivation method according to claim 1, characterized in that: In step S2, the transplanting matrix containing selenium includes peat, river sand, Huabao No. 2, biological bacteria and sodium selenite, wherein the concentration of Huabao No. 2 is 0.8-1.2 g / L, the concentration of the biological bacteria is 4.8-5.2 g / L, and the concentration of the sodium selenite is 0.5 mg / L-25 mg / L.

6. The cultivation method according to claim 1, characterized in that: In step S2, transplanting the golden thread vine seedlings into a transplanting matrix containing selenium for planting also includes spraying a first selenium fertilizer on the leaves of the golden thread vine seedlings, and the transplanting matrix containing selenium includes peat, river sand, Huabao No. 2, biological bacteria and sodium selenite, wherein the volume ratio of the peat to the river sand is 3.0-4.2:1, the concentration of Huabao No. 2 is 0.8-1.2 g / L, the concentration of the biological bacteria is 4.8-5.2 g / L, the concentration of the sodium selenite is 0.5 mg / L-25 mg / L, and the first selenium fertilizer is a 100-200 mg / L Na2SeO3 solution.

7. The cultivation method according to claim 1, characterized in that: In step S2, the transplanting matrix containing selenium element includes natural humus soil on the surface of the forest and a second selenium fertilizer, and the second selenium fertilizer is 1.10 mg / L to 32.85 mg / L of sodium selenite.

8. A kind of golden thread vine rich in organic selenium polysaccharide, characterized in that: The method according to any one of claims 1 to 7 is used for cultivation.

9. A method for preparing selenium polysaccharide from Anoectochilus roxburghii, characterized in that: The following steps are involved: The organic selenium-rich polysaccharide roxburghii as claimed in claim 8 is harvested, washed, and dried to obtain a dry product of the roxburghii plant; The dried product of the roxburghii plant is crushed and sieved to obtain sieved powder, and then the sieved powder is subjected to selenium polysaccharide extraction. The obtained crude selenium polysaccharide filtrate is purified to obtain roxburghii selenium polysaccharide.

10. The preparation method according to claim 9, characterized in that: The steps of extracting selenium polysaccharides from the sieved powder are as follows: adding distilled water to the sieved powder and adjusting the pH to 6, then performing ultrasonic treatment, adding cellulase for water bath reaction, collecting the filtrate and repeating the above steps to extract the residue once to obtain the crude selenium polysaccharide filtrate.

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