Method for promoting Lithocarpus litseifolius leaf growth, dihydrochalcone synthesis and heavy metal content reduction

Abstract

The invention discloses a method for promoting Lithocarpus litseifolius leaf growth, dihydrochalcone synthesis and heavy metal content reduction. The selenium-enriched yeast is used as a leaf surface treating agent of lithocarpus litseifolius, so that leaf growth and dihydrochalcone synthesis can be synchronously promoted, and the content of heavy metal arsenic and lead in leaves can be reduced. Wherein the contents of trilobatin in young leaves and mature leaves can be increased by 190% and 83% respectively; the plant number proportion of newborn young leaves can be increased to 52%; and the contents of lead and arsenic can be respectively reduced by 92.2% and 347.7%. The method has remarkable quality improvement and yield increase effects, is simple and easy to implement, short in treatment time, environmentally friendly and free of pollution, has high economic benefits, is suitable for functional component improvement and heavy metal pollution prevention and control of lithocarpus litseifolius, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of improving plant functional components and resistant cultivation, and particularly relates to a method for promoting the growth of Lithocarpus litseifolius leaves, the synthesis of dihydrochalcone, and the decrease of heavy metal content. Background Art

[0002] Lithocarpus litseifolius (Hance) Chun is an evergreen broad-leaved tree of the genus Lithocarpus in the family Fagaceae, and is mostly found in Sichuan, Hunan, Yunnan and other places south of the Qinling Mountains in China. As a natural tea with both medicinal and edible properties, Lithocarpus litseifolius has the characteristics of lowering blood sugar, blood lipid and cholesterol, and thus has won the reputation of "cordyceps on the tree". Research shows that the main components of Lithocarpus litseifolius are dihydrochalcones, including compounds such as phloridzin and trifolin. However, this species is scattered in the wild, and the content of dihydrochalcone in field-cultivated seedlings is limited, and there is a serious risk of heavy metal (such as lead (Pb) and arsenic (As)) pollution, which cannot meet the market demand. Therefore, finding an effective measure to simultaneously promote the growth of Lithocarpus litseifolius and the synthesis of dihydrochalcone and reduce the heavy metal content has positive significance for the high-yield and high-quality utilization of its resources.

[0003] The application of selenium in plants is shown as an effective technique to regulate various biological functions. However, traditional inorganic selenium usually has high phytotoxicity to plants, thus increasing the risk of biosafety. Therefore, it is still necessary to find alternative supplementation methods. As an organic selenium with low toxicity, high bioavailability and cost-effectiveness, selenium-enriched yeast is widely used in the animal feed industry or health products, but it is rarely used in plants, and there is no report on its application in the cultivation of Lithocarpus litseifolius. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, the present invention provides a method for promoting the growth of Lithocarpus litseifolius leaves, the synthesis of dihydrochalcone, and the decrease of heavy metal content, which has significant quality improvement and yield increase effects, short treatment time, simple and easy operation, environmental protection and no pollution, and has high economic benefits, and has positive significance in the high-yield and high-quality cultivation of Lithocarpus litseifolius.

[0005] The first object of the present invention is to provide a method for promoting the growth of Lithocarpus litseifolius leaves, the synthesis of dihydrochalcone, and the decrease of heavy metal content, which includes the step of applying a selenium-enriched yeast solution on the surface of Lithocarpus litseifolius leaves.

[0006] The second object of the present invention is to provide a method for obtaining Lithocarpus litseifolius leaves with high dihydrochalcone content and low heavy metal content, including the following steps: spraying a selenium-enriched yeast solution on the surface of Lithocarpus litseifolius leaves 1-2 times, and then timely picking the Lithocarpus litseifolius leaves.

[0007] Preferably, the selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 5-25 g / L, and the selenium-enriched yeast solution is prepared through the following steps: adding selenium-enriched yeast into water according to the ratio of 5-25 g of selenium-enriched yeast to 1 L of water, and ultrasonically dissolving at 30°C-40°C to prepare the selenium-enriched yeast solution; the selenium-enriched yeast is Angel selenium-enriched yeast with 2000 ppm produced by Angel Yeast Co., Ltd.

[0008] Preferably, the selenium-enriched yeast solution is sprayed on the surface of the Lithocarpus litseifolius leaves twice. The second spraying is carried out 1-3 weeks after the first spraying, and the Lithocarpus litseifolius leaves are picked 1-3 weeks after the second spraying.

[0009] Preferably, the selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 25 g / L.

[0010] Preferably, the 1-3 weeks is 15 days.

[0011] Preferably, the picked Lithocarpus litseifolius leaves are young leaves or mature leaves of Lithocarpus litseifolius.

[0012] Preferably, the Lithocarpus litseifolius is 7-month-old seedlings of Lithocarpus litseifolius.

[0013] Preferably, the 7-month-old seedlings of Lithocarpus litseifolius are cultivated in an artificial culture medium, and the artificial culture medium is a mixture prepared by mixing rice husk, peat and loess according to the volume ratio of 6:3:1.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention uses selenium-enriched yeast as a foliar treatment agent for Lithocarpus litseifolius, which can simultaneously promote leaf growth, dihydrochalcone synthesis and reduce the content of heavy metals (arsenic and lead) in leaves. Among them, the trilobatin content in young leaves and mature leaves can be increased by 190% and 83% respectively; the proportion of the number of newly born young leaves can be increased to 52%; the lead and arsenic contents can be decreased by 92.2% and 347.7% respectively.

[0016] The present invention overcomes the problem that the conventional method has limited improvement in plant growth or leaf safety when promoting the accumulation of functional components, has significant quality improvement and yield increase effects, is simple and easy to operate, has a short treatment time, is environmentally friendly and pollution-free, and has high economic benefits, and has a wide application prospect in the efficient cultivation of Lithocarpus litseifolius and the improvement of functional components. Description of the Drawings

[0017] Figure 1 It is the high performance liquid chromatography diagrams of two dihydrochalcones (phloridzin and trilobatin); among them, the compounds represented by 1 and 2 are: 1: phloridzin; 2: trilobatin.

[0018] Figure 2 It shows the growth of Litsea cubeba seedlings after being treated with selenium-enriched yeast at different concentrations. Among them, SeY5, SeY15, and SeY25 correspond to the concentrations of selenium-enriched yeast solutions sprayed of 5 g / L (Example 1), 15 g / L (Example 2), and 25 g / L (Example 3) respectively; CK corresponds to the treatment conditions of Comparative Example 1. Detailed implementation manners

[0019] The following examples are further illustrations of the present invention rather than limitations thereof.

[0020] The selenium-enriched yeast used in the following examples or comparative examples is Angel selenium-enriched yeast 2000 ppm (Angel Yeast Co., Ltd.), purchased from Guansi Mycelium Department Store, Liuyang City, Hunan Province. Among them, moisture content ≤ 6.0%; ash content ≤ 10.0%; protein ≥ 40.0%; selenium (calculated as Se) 2000 mg / kg; lead (calculated as Pb) ≤ 1.0%; arsenic ≤ 1.0%; total mercury (calculated as Hg) ≤ 1.0%; proportion of organic selenium (accounting for total selenium) ≥ 97.0%.

[0021] Example 1

[0022] a. Pre-culture: Using 7-month-old Litsea cubeba seedlings as experimental materials, selecting seedlings with a height of 20 ± 5 cm, no pests and diseases, and at least 4 leaves, transferring them to non-woven bags filled with artificial culture medium, and culturing them in a culture room at a temperature of 25 °C, a relative humidity of 40%, and a light intensity of 2000 Lux; the artificial culture medium is a mixture prepared by mixing rice husk, peat, and loess in a volume ratio of 6:3:1.

[0023] b. Preparation of selenium-enriched yeast solution: Accurately weigh 5 g of selenium-enriched yeast granules, add them to 1 L of pure water, and dissolve them by ultrasonic wave at 30 °C - 40 °C to prepare a 5 g / L selenium-enriched yeast solution.

[0024] c. First treatment with selenium-enriched yeast: Spray the selenium-enriched yeast solution on both the front and back sides of the Litsea cubeba seedlings, and then continue to culture them in the culture room under the conditions of a temperature of 25 °C, a relative humidity of 40%, and a light intensity of 2000 Lux for 15 days.

[0025] d. Second treatment with selenium-enriched yeast: After the first treatment, repeat spraying the same concentration of selenium-enriched yeast solution, and then continue to culture them in the culture room under the conditions of a temperature of 25 °C, a relative humidity of 40%, and a light intensity of 2000 Lux for 15 days.

[0026] e. Sampling and detection: Young leaves and mature leaves of Lithocarpus litseifolius were collected respectively after 30 days of cultivation (counting from the first treatment). Among them, the young leaves were the first and second green leaves at the top of the Lithocarpus litseifolius seedlings; the mature leaves were the first and second green leaves at the bottom of the Lithocarpus litseifolius seedlings. After being washed clean with pure water and dried, they were placed in an oven at 55 °C for drying, ground, passed through a 65-mesh sieve, and stored in a drying oven for the detection of the content of dihydrochalcone (phyllodulcin, phloridzin) and heavy metal concentration. At the same time, the seedlings with newly grown young leaves (the purple-red leaves with a leaf length of more than 1 cm at the top of the Lithocarpus litseifolius seedlings) were counted, and the photosynthetic parameters of the young leaves and mature leaves were measured.

[0027] The detection methods are as follows:

[0028] Determination method of monomeric dihydrochalcone: The Lithocarpus litseifolius leaf samples were vacuum-dried and ground into powder, and then dissolved in 10 mL of methanol. The contents of the representative monomeric dihydrochalcones of Lithocarpus litseifolius, phyllodulcin and phloridzin, were determined by high performance liquid chromatography (HPLC).

[0029] Determination method of photosynthetic parameters: The transpiration rate, photosynthetic rate and stomatal conductance of the young leaves and mature leaves on the plants were measured using a portable photosynthesis measuring instrument (Li-6800).

[0030] Determination method of heavy metal elements: The Lithocarpus litseifolius leaf samples were put into digestion tubes and soaked in nitric acid overnight. The contents of heavy metal elements (As and Pb) were determined by inductively coupled plasma mass spectrometry.

[0031] Regarding the concentration of selenium-enriched yeast:

[0032] Example 2

[0033] The method steps of this example are the same as those of Example 1 except for the following differences; the difference is that the selenium-enriched yeast solution used in step b preparation and steps c and d treatment in this example contains 15 g of selenium-enriched yeast per liter.

[0034] Example 3

[0035] The method steps of this example are the same as those of Example 1 except for the following differences; the difference is that the selenium-enriched yeast solution used in step b preparation and steps c and d treatment in this example contains 25 g of selenium-enriched yeast per liter.

[0036] Comparative Example 1

[0037] Compared with the method of Example 1, steps a and e of this comparative example are the same, but there are no steps b, c, and d, that is, the Lithocarpus litseifolius seedlings were not treated with selenium-enriched yeast (pure water was sprayed at the corresponding time of the treatment in Example 1), and the sampling and identification time of the Lithocarpus litseifolius leaves in step e was 30 days after cultivation through step a (that is, the same sampling time as in Example 1).

[0038] Parallel cultures were carried out according to the methods of Example 1, Example 2, Example 3, and Comparative Example 1 respectively (sample size: 50 plants) to identify the effects of different treatment concentrations of selenium-enriched yeast on the synthesis of dihydrochalcone, leaf growth, and heavy metal content in the young leaves and mature leaves of Lithocarpus litseifolius. After statistics, the results are shown in Tables 1-3.

[0039] Table 1 Effects of different concentrations of selenium-enriched yeast on the content of dihydrochalcone (phloridzin, phloretin) in the leaves of Lithocarpus litseifolius

[0040]

[0041] (Note: Different letters after the numbers in the table indicate significant differences in the data, P<0.05)

[0042] As can be seen from Table 1 and Figure 1 It can be seen that when the treatment concentrations of selenium-enriched yeast in Example 1, Example 2, and Example 3 were selected, the phloridzin content in the young leaves and mature leaves of Lithocarpus litseifolius was significantly higher than that in Comparative Example 1. Among them, the phloridzin content in the young leaves and mature leaves in Example 1 was the highest. At the same time, there was no significant difference in the phloretin content in the mature leaves among Example 1-3 and Comparative Example 1. This shows that the effect of applying selenium-enriched yeast is mainly reflected in the increase in the content of dihydrochalcone (phloridzin, phloretin) in the young leaves of Lithocarpus litseifolius.

[0043] Table 2 Effects of different concentrations of selenium-enriched yeast on the leaf growth of Lithocarpus litseifolius

[0044]

[0045]

[0046] (Note: Different letters after the numbers in the table indicate significant differences in the data, P<0.05)

[0047] As can be seen from Table 2 and Figure 2 It can be seen that when the treatment concentrations of selenium-enriched yeast in Example 1, Example 2, and Example 3 were selected, the number of newly emerged young leaves of Lithocarpus litseifolius seedlings was significantly higher than that in Comparative Example 1, and at the same time, each photosynthetic parameter (transpiration rate, photosynthetic rate, stomatal conductance) was also significantly higher than that in Comparative Example 1. Among them, the number of newly emerged young leaves in Example 3 was the largest.

[0048] Table 3 Effects of different concentrations of selenium-enriched yeast on the heavy metal concentration in the leaves of Lithocarpus litseifolius

[0049]

[0050] (Note: Different letters after the numbers in the table indicate significant differences in the data, P<0.05)

[0051] As can be seen from Table 3, for the selenium-enriched yeast treatment concentrations selected in Example 1, Example 2, and Example 3, the heavy metal contents (As and Pb) in the young leaves and mature leaves of Lithocarpus litseifolius were significantly lower than those in Comparative Example 1. Among them, Example 2 and Example 3 obtained the lowest concentrations in As and Pb, respectively.

[0052] In summary, the selenium-enriched yeast concentrations selected in Example 1, Example 2, and Example 3 can all promote the synthesis of dihydrochalcone (phloridzin, phloretin), especially significantly in young leaves. At the same time, Example 1, Example 2, and Example 3 can all promote the growth and photosynthesis of young leaves and mature leaves, and reduce the heavy metal content in the leaves.

[0053] Regarding the number of times of selenium-enriched yeast treatment:

[0054] Comparative Example 2

[0055] This comparative example is the same as Comparative Example 1 in all method steps except for the following differences; the difference lies in that in step e, samples cultured for 15 d after step a are collected for detection.

[0056] Comparative Example 3

[0057] This comparative example is the same as Example 3 in all method steps except for the following differences; the difference lies in that there is no step d, that is, sampling and detection of Lithocarpus litseifolius leaves are carried out on the 15th d after the first treatment with selenium-enriched yeast.

[0058] Parallel cultures were carried out according to the methods of Example 3 and Comparative Examples 1 - 3 respectively (the sample size was 50 plants) to identify the effects of different numbers of times of selenium-enriched yeast treatment on the dihydrochalcone content of Lithocarpus litseifolius. After statistics, the results are shown in Table 4.

[0059] Table 4 Effects of the number of times of selenium-enriched yeast treatment on the dihydrochalcone (phloridzin, phloretin) content in Lithocarpus litseifolius leaves

[0060]

[0061] (Note: The units of phloridzin and phloretin contents are mg / gDW. Different letters between the numbers in the table represent significant differences in the data. Among them, lowercase letters represent the differences in each data when the number of times of (selenium-enriched yeast) treatment is 2, and uppercase letters represent the differences in each data when the number of times of (selenium-enriched yeast) treatment is 1, P < 0.05. The numbers in parentheses below represent the multiples of the corresponding CK data. Among them, CK is Comparative Example 1 when the number of times of selenium-enriched yeast treatment is 2, and CK is Comparative Example 2 when the number of times of selenium-enriched yeast treatment is 1)

[0062] As can be seen from Table 4, when the number of times of treating with selenium-enriched yeast is 1, the trilobatin content in the young leaves of Comparative Example 3 increases significantly, and the phloridzin content in the young leaves and mature leaves does not change significantly. At the same time, under the condition of the same concentration of selenium-enriched yeast, the change multiple of trilobatin is 1.31 (Comparative Example 3 relative to Comparative Example 2) when the number of treatment times is 1, which is lower than the multiple of 2.81 (Example 3 relative to Comparative Example 1) when the number of treatment times is 2. In summary, when the number of treatment times is 2, the promoting effect of selenium-enriched yeast on the content of dihydrochalcone in the leaves of Litsea cubeba Pers. is better than that when the number of treatment times is 1.

Claims

1. A method for promoting the growth of leaves of Zingiber officinale, the synthesis of dihydrochalcone, and the reduction of heavy metal content, characterized in that: The method comprises the steps of applying selenium-enriched yeast solution on the surface of leaves of the Artemisia selengensis.

2. A method for obtaining leaves of Ligusticum chuanxiong with high dihydrochalcone content and low heavy metal content, characterized in that: The following steps are involved: Spray the selenium-rich yeast solution on the surface of the leaves of the Alpinia zeylanicum 1-2 times, and then pick the leaves of the Alpinia zeylanicum at the right time.

3. The method according to claim 2, characterized in that The selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 5-25g / L, and the selenium-enriched yeast solution is prepared by the following steps: adding selenium-enriched yeast to water at a ratio of 5-25g selenium-enriched yeast: 1L water, and ultrasonically dissolving at 30°C-40°C to prepare a selenium-enriched yeast solution; the selenium-enriched yeast is 2000ppm Angel selenium-enriched yeast produced by Angel Yeast Co., Ltd.

4. The method according to claim 2, characterized in that: Spray the selenium-rich yeast solution on the surface of the leaves of the Artemisia selengensis twice, spray the second time 1-3 weeks after the first spraying, and pick the Artemisia selengensis leaves 1-3 weeks after the second spraying.

5. The method according to claim 3, characterized in that: The selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 25 g / L.

6. The method according to claim 4, characterized in that The 1-3 weeks is 15 days.

7. The method according to claim 2, characterized in that The picking of leaves of the Alpinia zeylanicum var. truncatum is picking young leaves or mature leaves of the Alpinia zeylanicum var. truncatum var.

8. The method according to claim 2, characterized in that: The described Ligusticum chuanxiong is a 7-month-old seedling of Ligusticum chuanxiong.

9. The method according to claim 8, characterized in that The 7-month-old seedlings of Artemisia selengensis are cultivated in an artificial culture matrix, which is a mixture of rice husk, peat and loess in a volume ratio of 6:3:1.

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