Application of olive pomace polyphenol extract-hydroxytyrosol as a biogenic nitrogen fertilizer synergist

Abstract

The present invention relates to the field of biogenic nitrogen fertilizer synergists, and in particular to an application of an olive pomace polyphenol extract-hydroxytyrosol as a biogenic nitrogen fertilizer synergist. The biogenic nitrogen fertilizer synergist component prepared by the present invention is an olive pomace polyphenol extract-hydroxytyrosol. After the olive pomace is deoiled, it is extracted under acid hydrolysis conditions, and an alkaline solution of sodium hydroxide is used to adjust the pH value to neutral to obtain the corresponding hydroxytyrosol. The abundance of soil nitrifying microorganisms (ammonia oxidizing bacteria and ammonia oxidizing archaea) and the conversion of mineral nitrogen in the soil can be effectively suppressed, the effectiveness of nitrogen in the soil can be improved, nitrogen can be more effectively absorbed and utilized by crops, the olive pomace is effectively recycled, and the problems of huge environmental pollution and waste of resources caused by the olive pomace can be effectively avoided.

Description

Technical Field

[0001] The invention relates to the field of biogenic nitrogen fertilizer synergists, and in particular to application of olive pomace polyphenol extract-hydroxytyrosol as a biogenic nitrogen fertilizer synergist. Background Art

[0002] Nitrogen is an essential nutrient for crop growth and a key factor influencing crop yields. Widespread application of nitrogen fertilizer has become essential for achieving high crop yields. According to statistics, over 55% of increased grain production comes from chemical fertilizers. Nitrogen fertilizer has played a key role in increasing grain production in my country. However, the biggest and most common problem facing agriculture today is low nitrogen fertilizer utilization. Statistics show that the utilization rate of seasonal nitrogen fertilizer is only 35% to 39%. The rational application of nitrogen fertilizer in the soil and improving its utilization in agriculture have attracted considerable attention. Precisely controlling nitrogen transformation in the soil is a key technology for reducing fertilizer application and increasing its efficiency.

[0003] The main reason for nitrogen loss is the loss of nitrogen fertilizer applied to the soil through nitrification and denitrification. The loss pathways of nitrogen include NO3 - By coupling the application of nitrification inhibitors with nitrogen fertilizers, the nitrogen source applied to the soil can be converted into NH4 + -N form remains in the soil for a long time for crops to absorb and utilize. Currently, most of the nitrification inhibitors available on the market are chemically synthesized, such as DMPP (3,4-dimethylpyrazole phosphate), NBPT (n-butylthiophosphoric acid triamine), HQ (hydroquinone), etc., which have the limitations of not being easy to decompose in the soil, affecting the biodiversity of farmland ecosystems and breaking the ecological balance. Therefore, resource-sustainable, green and safe biogenic nitrification inhibitors have broad application prospects, such as 1,9-decanediol, sorghumone, etc., which have good inhibitory activity against Nitrosomonas under pure culture conditions. Biogenic nitrification inhibitors are derived from plant root secretions and extracts, and have the characteristics of being green, sustainable, and recycling resources. They have little impact on the ecology, and the extraction of biogenic nitrogen fertilizer synergists with better effects in some wastes has practical significance for the development of green agriculture in my country.

[0004] Olive pomace (OP) is the solid residue obtained from the olive oil production process (during pressing or centrifugation). Untreated OP has a high organic load and phenolic content. Burning or discarding it into the soil can be a potential source of soil, water, or air pollution. Consequently, OP raises widespread environmental concerns. Its value as a source of phenolic bioactive compounds is crucial for the sustainable growth of related industries. The phenolic compounds present in OP are primarily classified into simple phenols, such as hydroxytyrosol (HT) and oleuropein (OL). Numerous studies support the important antioxidant, anti-inflammatory, antimicrobial, and cytostatic properties of olive biophenols. HT is a simple phenol that can be obtained by acid or alkaline hydrolysis of olive pomace. HT has also been reported to exhibit antimicrobial, chemoprotective, skin-bleaching, antioxidant, anti-inflammatory, anti-atherosclerotic, and cardioprotective activities, with potent inhibitory effects against Gram-negative bacteria. HT derivatives are found not only in OP but also in olive mill wastewater, exhibiting significant antioxidant and anti-inflammatory activities. Hydroxytyrosol, a component of olives, acts as a highly active antioxidant in the human body. As bioactive molecules, antioxidants are found in many plants, but their activity varies. Hydroxytyrosol is considered one of the best antioxidants, and its commercial value is continuously increasing. Hydroxytyrosol is widely used in the food, medical, and cosmetic industries. However, to our knowledge, no studies have examined the antibacterial effects of hydroxytyrosol on soil nitrification. In agricultural production, chemically synthesized nitrification inhibitors are still the most widely used. Therefore, there is an urgent need to develop new, green and environmentally friendly biogenic nitrogen fertilizer synergists with inhibitory effects and optimize the compatibility of inhibitors—key technologies for synergistic enhancement. These can effectively extend the retention time of urea and ammonium nitrogen fertilizers in the soil, reduce the formation and accumulation of nitrate nitrogen in the soil, minimize nitrogen loss, and improve nitrogen fertilizer use efficiency. This will contribute to achieving my country's goal of reducing nitrogen and increasing fertilization efficiency, and have long-term practical significance for promoting the development of green agriculture, sustainable resource utilization, and the research and development of new, green and efficient nitrogen fertilizer products. Summary of the Invention

[0005] In order to solve the problems of the prior art and achieve the above-mentioned technical effects, the present invention provides an olive pomace polyphenol extract - hydroxytyrosol - for use as a biogenic nitrogen fertilizer synergist. Nitrogen fertilizer applied to the soil is rapidly converted into ammonium nitrogen and then converted into nitrate nitrogen through soil nitrification. Nitrate nitrogen is easily lost in the soil, and crops are unable to absorb nitrogen nutrients in a timely manner, resulting in low nitrogen utilization rate. Hydroxytyrosol can slow down the conversion of ammonium nitrogen, allowing it to be retained in the soil to a greater extent for crop absorption and utilization. In addition, the biogenic nitrification inhibitor is environmentally friendly and can avoid adverse effects on soil ecology, thereby solving the waste of resources and environmental problems caused by excessive application of nitrogen fertilizers, improving nitrogen utilization rate, and also broadening the industrial application field of hydroxytyrosol.

[0006] The technical solutions of the present invention are as follows:

[0007] First, the present invention claims protection for the use of hydroxytyrosol as a biogenic nitrogen fertilizer synergist.

[0008] The method for applying hydroxytyrosol as a nitrogen fertilizer synergist is as follows: hydroxytyrosol is applied simultaneously at 1 / 5-1 / 3 of the nitrogen application rate. Preferably, the amount of hydroxytyrosol used is 1 / 4 of the nitrogen application rate.

[0009] The present invention demonstrates that hydroxytyrosol has a good nitrification inhibition effect and nitrogen conversion can be effectively regulated by measuring the conversion of inorganic nitrogen and the abundance of typical ammonia oxidizing microorganisms AOA and AOB in a soil culture test.

[0010] Furthermore, the hydroxytyrosol is preferably derived from olive pomace.

[0011] A specific method for extracting hydroxytyrosol from olive pomace comprises the following steps:

[0012] (1) The olive pomace is deoiled by an organic solvent extraction method. The organic solvent is preferably n-hexane. Preferably, the olive pomace is sealed at -20°C before use.

[0013] The specific deoiling process is:

[0014] Select the crushed olive pomace, add n-hexane reagent at a solid-liquid ratio of 1:9, place it in a constant temperature shaking box at 55°C, with a rotation speed of 200r / min, and the extraction time is 3 hours.

[0015] The solvent and the solid residue were separated by filtration. After the residue was washed with n-hexane reagent, the de-oiled olive pomace was collected and vacuum dried to constant weight at 40°C for 12 h.

[0016] (2) The de-oiled olive pomace was crushed with an ultra-fine wall-breaking machine, passed through a 60-mesh sieve, and added with 0.14 mol / L hydrochloric acid solution at a material-liquid ratio of 1:50. The pomace was placed in a constant temperature oscillator at 60°C for 4 h, and then the pH was adjusted to neutral with 2 mol / L sodium hydroxide, and the pomace was filtered and purified.

[0017] The beneficial effects of the present invention are:

[0018] The present invention discovered that the polyphenol extract - hydroxytyrosol can not only inhibit the nitrification process of nitrogen, but also has a significant inhibitory effect on soil ammonia-oxidizing bacteria and ammonia-oxidizing archaea, and has no effect on the germination rate of dwarf tomatoes, which can be explained that it has no toxic effect on seeds. It provides new ideas and methods for new technologies to improve nitrogen utilization efficiency, further broadens the application field of hydroxytyrosol, and improves the industrial value of hydroxytyrosol. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the effects of hydroxytyrosol on the germination rate of dwarf tomatoes in Example 2.

[0020] Figure 2 This is a comparison chart of the effects of different treatments on soil ammonium nitrogen and nitrate nitrogen content in Example 3. DETAILED DESCRIPTION

[0021] The following is a detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned contents of the present invention fall within the scope of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies.

[0022] Example 1

[0023] In this embodiment, the olive pomace is the solid residue after oil is extracted from fresh olives, calculated on a dry weight basis with a moisture content of less than 2%, and the olive pits and other impurities are removed.

[0024] Olive pomace deoiling: An organic solvent extraction method (n-hexane is analytical grade) is used. The olive pomace is sealed at -20°C before use. 10 g of crushed olive pomace is placed in a stoppered Erlenmeyer flask. Hexane is added at a 1:9 ratio of solid to liquid. The flask is then placed in a constant temperature shaker at 55°C, with a rotation speed of 200 rpm. The extraction time is 3 hours.

[0025] The solvent and the solid residue were separated by filtration. After the residue was washed with a certain amount of n-hexane reagent, the de-oiled olive pomace was collected and vacuum dried to constant weight at 40°C for 12 h.

[0026] The olive pomace dried after de-oiling is selected to extract hydroxytyrosol.

[0027] The de-oiled olive pomace was crushed with an ultra-fine wall breaker, 4 g of solid olive pomace passing through 60 mesh was weighed and placed in a 250 ml stoppered conical flask, 0.14 mol / L hydrochloric acid solution was added according to the material-liquid ratio of 1:50, and the mixture was placed in a constant temperature oscillator at 60°C for 4 h.

[0028] The pH was adjusted to neutral with 2 mol / L sodium hydroxide, the volume was made up to 1000 ml, 1 ml was taken out, diluted 10 times, passed through a 0.45 μm filter membrane, and the hydroxytyrosol content was determined by high performance liquid chromatography. The concentration of hydroxytyrosol was 3.46 mg / g.

[0029] Example 2

[0030] Hydroxytyrosol 50 mg / kg was dissolved in deionized water to prepare a solution, and deionized water was used as a control. The solutions were used for germination of dwarf tomato seeds (other conditions were the same), and the germination rate of the dwarf tomato seeds was observed on the third day.

[0031] The results are as follows Figure 1 As shown, there is no significant difference in the germination rate of dwarf tomatoes under hydroxytyrosol aqueous solution and the germination rate of dwarf tomatoes under pure water conditions, and there is no inhibitory effect. It can be seen that hydroxytyrosol does not affect crop growth and has no toxic effect on tomato germination.

[0032] Example 3

[0033] Hydroxytyrosol and urea were dissolved in water to form a solution and applied to the soil together. The incubation time was 28 days. Destructive sampling was carried out at 3, 5, 7, 14, 21 and 28 days to determine the content of nitrate nitrogen and ammonium nitrogen in the soil. After the soil samples were taken at 7 and 28 days of incubation, the samples were stored at -20℃. Later, soil DNA was extracted to determine the changes in the abundance of ammonia-oxidizing bacteria and archaea under hydroxytyrosol conditions.

[0034] Test soil: The test soil type is fluvo-aquic soil with a texture type of sandy loam. It was collected from the 0-20 cm tillage layer of the Science and Technology Innovation Park of Shandong Agricultural University in Tai'an City, Shandong Province. After natural air drying, it was passed through a 2 mm sieve, mixed, and set aside. The basic physical and chemical properties are shown in Table 1.

[0035] Table 1 Basic physical and chemical properties of incubation test soil

[0036]

[0037]

[0038] Test reagents: Nitrification inhibitor-DCD (dicyandiamide, C2H4N4) is a commercially available nitrification inhibitor, and urea is regular urea with a nitrogen content of 46%.

[0039] Experimental treatments: No urea (CK), urea alone (U), the nitrification inhibitor DCD plus urea (U+DCD), and hydroxytyrosol plus urea (U+HT) were applied. Each treatment was replicated 18 times, and the nitrogen application rate was 0.2 g / kg. Soil moisture was adjusted to 60% of field capacity.

[0040] Experimental steps:

[0041] After weighing the total amount of experimental soil into a wide-mouth bottle, evenly add distilled water and adjust the soil moisture content to 60% of the maximum field water holding capacity. Then, seal the mouth of the wide-mouth bottle with a sealing film and evenly pierce holes to maintain air flow. Pre-cultivate in a 25°C incubator for three days.

[0042] a. The amount of soil 300g, and with a certain amount of deionized water to dissolve urea and DCD, urea dosage of each treatment 0.43g / kg (equivalent to a nitrogen application rate of 0.2g / kg), DCD each treatment 50mg / kg, hydroxytyrosol dosage of 50mg / kg.

[0043] b. Add a certain amount of deionized water to the soil in the previous step to keep the soil moisture content at 60% of the field water holding capacity.

[0044] c. Set up 18 replicates for each treatment and incubate the soil mixed in the previous step in a constant temperature incubator at 25°C for 28 days.

[0045] d. During the incubation period, destructive sampling was performed on triplicate at 3, 5, 7, 14, 21, and 28 days for each treatment to determine soil nitrate and ammonium nitrogen contents. Soil samples taken on days 7 and 28 of incubation were stored at -20°C. Soil DNA was later extracted, and real-time fluorescence quantitative PCR was used to determine the abundance changes of ammonia-oxidizing bacteria and archaea in different treatments.

[0046] Experimental results: Figure 2 As shown in the results, compared with the treatment with urea alone, the urea+hydroxytyrosol treatment could delay the transformation of inorganic nitrogen, prolong the retention time of ammonium nitrogen in the soil, and reduce the transformation of ammonium nitrogen to nitrate nitrogen.

[0047] Hydroxytyrosol exhibited the same inhibitory effect as DCD on typical soil ammonia-oxidizing microorganisms throughout the incubation period, significantly reducing the abundance of AOB. AOA abundance did not decrease significantly during the first seven days, but significantly decreased after 28 days of incubation.

Claims

1. The application of hydroxytyrosol as a biogenic nitrogen fertilizer synergist is characterized in that, Hydroxytyrosol is applied at the same time according to 1 / 5-1 / 3 of the nitrogen application rate, and the hydroxytyrosol is derived from olive pomace.

2. The application of hydroxytyrosol as a biogenic nitrogen fertilizer synergist according to claim 1, wherein The dosage of hydroxytyrosol is 1 / 4 of the nitrogen application amount.

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