A method for promoting growth of lindera leaf, synthesis of dihydrochalcone and reduction of heavy metal content

Abstract

The application discloses a method for promoting the growth of leaf of Lithocarpus polystachyus, synthesis of dihydrochalcone and reduction of heavy metal content. The method uses selenium-rich yeast as a leaf surface treatment agent of the Lithocarpus polystachyus, and can simultaneously promote the growth of leaf, synthesis of dihydrochalcone and reduction of the content of heavy metals arsenic and lead in the leaf. The content of trilobatin in the young leaf and mature leaf can be increased by 190% and 83% respectively, the proportion of the number of new young leaf plants can be increased to 52%, and the content of lead and arsenic can be reduced by 92.2% and 347.7% respectively. The method has remarkable quality improvement and yield increasing effects, is simple and easy to implement, has short treatment time, is environment-friendly and pollution-free, has high economic benefits, is suitable for functional component improvement and heavy metal pollution prevention and control of the Lithocarpus polystachyus, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant functional component enhancement and resistance cultivation, specifically involving a method for promoting the growth of Litsea cubeba leaves, dihydrochalcone synthesis, and reducing heavy metal content. Background Technology

[0002] *Lithocarpus litseifolius* (Hance) Chun is an evergreen broad-leaved tree belonging to the genus *Lithocarpus* in the family Fagaceae, commonly found in Sichuan, Hunan, and Yunnan provinces south of the Qinling Mountains in China. As a natural tea with medicinal and edible properties, *Lithocarpus litseifolius* possesses blood sugar, lipid, and cholesterol-lowering properties, earning it the nickname "Cordyceps of the Tree." Studies have shown that the main component of *Lithocarpus litseifolius* is dihydrochalcone, including compounds such as phlorizin and trifolin. However, this species is sparsely distributed in the wild, and the dihydrochalcone content in cultivated seedlings is limited, with a serious risk of heavy metal (such as lead (Pb) and arsenic (As)) contamination, failing to meet market demand. Therefore, finding an effective measure to simultaneously promote the growth of *Lithocarpus litseifolius* and dihydrochalcone synthesis while reducing heavy metal content is of positive significance for the high-yield and high-quality utilization of its resources.

[0003] Selenium is an effective technology for regulating various biological functions in plants. However, traditional inorganic selenium is often highly phytotoxic, increasing biosafety risks, thus necessitating the search for alternative supplementation methods. Selenium-enriched yeast, as an organic form of selenium with low toxicity, high bioavailability, and cost-effectiveness, is widely used in animal feed and health products, but its application in plants is rare, and its use in Litsea cubeba culture has not yet been reported. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, this invention provides a method for promoting leaf growth, dihydrochalcone synthesis, and reducing heavy metal content in Litsea cubeba. This method significantly improves quality and increases yield, has a short processing time, is simple and easy to implement, is environmentally friendly and pollution-free, and offers high economic benefits. It is of positive significance for the high-yield and high-quality cultivation of Litsea cubeba.

[0005] The first objective of this invention is to provide a method for promoting the growth of Litsea cubeba leaves, the synthesis of dihydrochalcone, and the reduction of heavy metal content, which includes the step of applying a selenium-enriched yeast solution to the surface of Litsea cubeba leaves.

[0006] The second objective of this invention is to provide a method for obtaining Litsea cubeba leaves with high dihydrochalcone content and low heavy metal content, comprising the following steps: spraying selenium-enriched yeast solution on the surface of Litsea cubeba leaves 1-2 times, and then harvesting Litsea cubeba leaves at an appropriate time.

[0007] Preferably, the selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 5-25 g / L, which is prepared by the following steps: adding selenium-enriched yeast to water at a ratio of 5-25 g selenium-enriched yeast to 1 L water, and dissolving it by ultrasonication at 30℃-40℃ to obtain the selenium-enriched yeast solution; the selenium-enriched yeast is 2000 ppm Angel selenium-enriched yeast produced by Angel Yeast Co., Ltd.

[0008] Preferably, the leaves of Litsea cubeba are sprayed with a selenium-enriched yeast solution twice, and the second spray is carried out 1-3 weeks after the first spray. The leaves of Litsea cubeba are harvested 1-3 weeks after the second spray.

[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 harvested leaves of Litsea cubeba are young or mature leaves of Litsea cubeba.

[0012] Preferably, the Litsea cubeba seedlings are 7-month-old Litsea cubeba seedlings.

[0013] Preferably, the 7-month-old seedlings of Litsea cubeba are cultivated in an artificial culture medium, which is a mixture of rice husks, peat, and loess in a volume ratio of 6:3:1.

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

[0015] This invention uses selenium-enriched yeast as a foliar treatment agent for Litsea cubeba, which can simultaneously promote leaf growth, dihydrochalcone synthesis, and reduce the content of heavy metals (arsenic and lead) in the leaves. Specifically, the content of trifolin in young leaves and mature leaves can be increased by 190% and 83%, respectively; the proportion of newly grown young leaves can be increased to 52%; and the content of lead and arsenic can be reduced by 92.2% and 347.7%, respectively.

[0016] This invention overcomes the problem that conventional methods have limited effect on improving plant growth or leaf safety when promoting the accumulation of functional components. It has significant effects on quality improvement and yield increase. The method is simple and easy to implement, has a short processing time, is environmentally friendly and pollution-free, and has high economic benefits. It has broad application prospects in the high-efficiency cultivation of Litsea cubeba and the enhancement of functional components. Attached Figure Description

[0017] Figure 1 These are high-performance liquid chromatograms of two dihydrochalcones (phlorizin and trifolin); where 1 and 2 represent the compounds respectively: 1: phlorizin; 2: trifolin.

[0018] Figure 2 The results show the growth of Litsea cubeba seedlings after treatment with different concentrations of selenium-enriched yeast; SeY5, SeY15, and SeY25 correspond to the concentrations of the sprayed selenium-enriched yeast solution 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

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] The selenium-enriched yeast used in the following examples or comparative examples was Angel Yeast Selenium-Enriched Yeast 2000ppm (Angel Yeast Co., Ltd.), purchased from Guankou Mycelium Department Store, Liuyang City, Hunan Province. The yeast contained the following: moisture ≤ 6.0%; ash ≤ 10.0%; protein ≥ 40.0%; selenium (as Se) 2000 mg / kg; lead (as Pb) ≤ 1.0%; arsenic ≤ 1.0%; total mercury (as Hg) ≤ 1.0%; and organic selenium content (as a percentage of total selenium) ≥ 97.0%.

[0021] Example 1

[0022] a. Pre-culture: Using 7-month-old seedlings of Litsea cubeba as experimental material, seedlings with a height of 20±5cm, free from diseases and pests, and with at least 4 leaves were selected and transferred to non-woven bags containing artificial culture medium. They were cultured in a culture room with a temperature of 25℃, a relative humidity of 40%, and a light intensity of 2000Lux. The artificial culture medium was a mixture of rice husks, peat, and loess in a volume ratio of 6:3:1.

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

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

[0025] d. Second treatment of selenium-enriched yeast: After the first treatment, spray the same concentration of selenium-enriched yeast solution once more, and then continue to culture in the culture room under the following conditions: temperature 25℃, relative humidity 40%, and light intensity 2000 Lux for 15 days.

[0026] e. Sampling and Detection: Young and mature leaves of *Litsea cubeba* seedlings (young leaves being the top 1 and 2 green leaves of the seedling; mature leaves being the bottom 1 and 2 green leaves of the seedling) were collected after 30 days of cultivation (starting from the first treatment). The leaves were cleaned with pure water, dried, and then dried in an oven at 55°C. After grinding, the leaves were passed through a 65-mesh sieve and stored in a drying oven for the detection of dihydrochalcone (trifolin, phlorizin) and heavy metal concentrations. Simultaneously, seedlings with newly sprouted young leaves (purplish-red leaves exceeding 1 cm in length at the top of the *Litsea cubeba* seedling) were counted, and the photosynthetic parameters of the young and mature leaves were measured.

[0027] The detection method is as follows:

[0028] Method for determining monomeric dihydrochalcones: Leaf samples of *Litsea cubeba* were vacuum-dried, ground into powder, and dissolved in 10 mL of methanol. The contents of representative dihydrochalcones of *Litsea cubeba*—trifolin and phlorizin—were determined by high-performance liquid chromatography (HPLC).

[0029] Photosynthetic parameter measurement method: The transpiration rate, photosynthetic rate and stomatal conductance of young and mature leaves of the plant were measured using a portable photosynthetic meter (Li-6800).

[0030] Method for determining heavy metal elements: Leaf samples of *Litsea cubeba* were placed in digestion tubes and soaked overnight in nitric acid. The content of heavy metal elements (As and Pb) was determined by inductively coupled plasma mass spectrometry.

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

[0032] Example 2

[0033] The method steps in this embodiment are the same as those in Embodiment 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 in this embodiment contains 15g of selenium-enriched yeast per liter.

[0034] Example 3

[0035] The method steps in this embodiment are the same as those in Embodiment 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 in this embodiment contains 25g of selenium-enriched yeast per liter.

[0036] Comparative Example 1

[0037] Compared with the method of Example 1, this comparative example has the same steps a and e, but no steps b, c, and d. That is, the Litsea cubeba 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 Litsea cubeba leaves in step e was 30 days after step a (that is, the same sampling time as in Example 1).

[0038] Parallel cultures (sample size 50 strains) were conducted according to the methods of Example 1, Example 2, Example 3, and Comparative Example 1 to identify the effects of different concentrations of selenium-enriched yeast on dihydrochalcone synthesis, leaf growth, and heavy metal content in young and mature leaves of Litsea cubeba. The results are shown in Tables 1-3.

[0039] Table 1. Effects of different concentrations of selenium-enriched yeast on the content of dihydrochalcone (trifolin, phlorizin) in Litsea cubeba leaves.

[0040]

[0041] (Note: Different letters after the numbers in the table indicate that the data are significantly different, P<0.05)

[0042] From Table 1 and Figure 1 As can be seen, the trifolin content in both young and mature leaves of *Litsea cubeba* was significantly higher in the selenium-enriched yeast treatments of Examples 1, 2, and 3 than in Comparative Example 1. Specifically, the trifolin content was highest in both young and mature leaves of Example 1. Meanwhile, there was no significant difference in phlorizin content in mature leaves between Examples 1-3 and Comparative Example 1. This indicates that the effect of applying selenium-enriched yeast is mainly reflected in the increased content of dihydrochalcone (trifolin, phlorizin) in young *Litsea cubeba* leaves.

[0043] Table 2. Effects of different concentrations of selenium-enriched yeast on the leaf growth of Litsea cubeba.

[0044]

[0045]

[0046] (Note: Different letters after the numbers in the table indicate that the data are significantly different, P<0.05)

[0047] From Table 2 and Figure 2 As can be seen, when using the selenium-enriched yeast treatment concentrations of Examples 1, 2, and 3, the number of newly sprouted leaves of Litsea cubeba seedlings was significantly higher than that of Comparative Example 1. Simultaneously, all photosynthetic parameters (transpiration rate, photosynthetic rate, and stomatal conductance) were also significantly higher than those of Comparative Example 1. Among them, Example 3 had the highest number of newly sprouted leaves.

[0048] Table 3. Effects of different concentrations of selenium-enriched yeast on heavy metal concentrations in Litsea cubeba leaves.

[0049]

[0050] (Note: Different letters after the numbers in the table indicate that the data are significantly different, P<0.05)

[0051] As shown in Table 3, the heavy metal contents (As and Pb) in the young and mature leaves of Litsea cubeba were significantly lower than those in Comparative Example 1 when the selenium-enriched yeast treatment concentrations of Examples 1, 2, and 3 were selected. Among them, Examples 2 and 3 achieved the lowest concentrations of As and Pb, respectively.

[0052] In summary, the concentrations of selenium-enriched yeast selected in Examples 1, 2, and 3 all promoted the synthesis of dihydrochalcone (trifolin, phlorizin), especially in young leaves. Furthermore, Examples 1, 2, and 3 all promoted the growth and photosynthesis of both young and mature leaves, and reduced the heavy metal content in the leaves.

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

[0054] Comparative Example 2

[0055] The methods and steps of this comparative example are the same as those of comparative example 1, except for the following differences; the difference is that in step e, samples are collected and cultured for 15 days after step a for testing.

[0056] Comparative Example 3

[0057] The methods and steps of this comparative example and Example 3 are the same except for the following differences: step d is omitted, that is, the sampling and testing of Litsea cubeba leaves are carried out on the 15th day after the first treatment with selenium-enriched yeast.

[0058] Parallel cultures (50 strains each) were performed according to the methods in Example 3 and Comparative Examples 1-3 to identify the effect of different treatment times on the content of dihydrochalcone in Litsea cubeba leaves. The results are shown in Table 4.

[0059] Table 4. Effects of selenium-enriched yeast treatment times on the content of dihydrochalcone (trifolin, phlorizin) in Litsea cubeba leaves.

[0060]

[0061] (Note: The units for trifolin and phlorizin content are mg / gDW. Different letters after the numbers in the table indicate that the data are significantly different. Lowercase letters represent the differences when the number of treatments (selenium-enriched yeast) is 2, and uppercase letters represent the differences when the number of treatments (selenium-enriched yeast) is 1. P < 0.05. The numbers in parentheses below the numbers represent the multiples of the corresponding control (CK) data. When the number of treatments (selenium-enriched yeast) is 2, CK is control example 1, and when the number of treatments (selenium-enriched yeast) is 1, CK is control example 2.)

[0062] As shown in Table 4, when the number of treatments with selenium-enriched yeast was 1, the content of trifolin in the young leaves of Comparative Example 3 was significantly increased, while the content of phlorizin in the young and mature leaves did not change significantly. Furthermore, under the same concentration of selenium-enriched yeast, the fold change in trifolin content was 1.31 when the number of treatments was 1 (Comparative Example 3 vs. Comparative Example 2), which was lower than the fold change of 2.81 when the number of treatments was 2 (Example 3 vs. Comparative Example 1). In conclusion, when the number of treatments was 2, the promoting effect of selenium-enriched yeast on the dihydrochalcone content in Litsea cubeba leaves was better than when the number of treatments was 1.

Claims

1. A method for obtaining Litsea cubeba leaves with high trifoliol content and low lead and arsenic content, characterized in that, Includes the following steps: Spray the surface of Litsea cubeba leaves with a selenium-enriched yeast solution twice. The second spraying is carried out 15 days after the first spraying. The young leaves of Litsea cubeba are harvested 15 days after the second spraying. The selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 5-25 g / L.

2. The method according to claim 1, characterized in that, The selenium-enriched yeast solution was prepared by the following steps: selenium-enriched yeast was added to water at a ratio of 5-25 g selenium-enriched yeast to 1 L water, and dissolved by ultrasonication at 30℃-40℃ to obtain the selenium-enriched yeast solution; the selenium-enriched yeast was 2000 ppm Angel Yeast Co., Ltd.

3. The method according to claim 1, characterized in that, The selenium-enriched yeast solution is a selenium-enriched yeast solution with a concentration of 25 g / L.

4. The method according to claim 1, characterized in that, The Litsea cubeba mentioned is a 7-month-old seedling of Litsea cubeba.

5. The method according to claim 4, characterized in that, The 7-month-old seedlings of Litsea cubeba were cultivated in an artificial culture medium, which was a mixture of rice husks, peat, and loess in a volume ratio of 6:3:1.

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