Soybean protein-thymol nano-composite as well as preparation method and application thereof

Abstract

The invention discloses an application of a soybean protein-thymol nano-composite in the aspect of removing marine fouling algae. According to the invention, the excellent nano-delivery function of the SPI is utilized, insoluble thymol is loaded on SPI particles through hydrophobic interaction to form a nano-composite, the water solubility is greatly improved after the thymol is combined with the SPI, and the thymol can be more sufficiently and quickly dispersed in water, so that the stability of the nano-composite is improved, and the stability of the nano-composite is improved. Therefore, the thymol is in full contact with the target algae in the form of nano particles, so that the growth and spreading of marine fouling algae can be quickly and effectively controlled, and the effect of removing the algae is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of marine antifouling nanomaterials, and particularly relates to a soybean protein-thymol nanocomposite, a preparation method thereof, and an application thereof. Background Art

[0002] Marine fouling organisms, also known as marine attached organisms, mainly grow on the bottom of ships and the surfaces of artificial facilities in the sea, including complex communities mainly composed of sessile organisms, with a wide variety of species, including bacteria, attached diatoms, many large algae, and various phyla from protozoa to vertebrates.

[0003] Ships, marine engineering facilities, etc. come into frequent contact with seawater, resulting in the attachment of marine organisms such as seaweeds and shellfish, forming biological fouling. These biological foulings will not only increase the navigation resistance of ships, reduce the sailing speed, increase fuel consumption, accelerate the corrosion of metal materials such as ships and platforms, and shorten the service life; but also affect the optical or acoustic signal transmission of marine instruments, increasing the maintenance frequency and cost; in addition, the attachment and blockage of marine fouling organisms to the aquaculture netting will also cause the death of cultured organisms, causing great damage to the aquaculture industry. In short, the attachment of marine fouling organisms to artificial facilities is a major problem in human development and utilization of the ocean.

[0004] Natural active substances have outstanding performance in the field of marine antifouling and meet the requirements of sustainable development of the marine environment. Some active substances and their derivatives achieve antifouling effects by repelling and anesthetizing fouling organisms; others directly inhibit the activity of target organisms to achieve the purpose of control.

[0005] Thymol is the main active substance in essential oils such as thyme and oregano, and is a food additive recognized and actually applied in China. It is generally a colorless crystalline solid at room temperature and is relatively insoluble in water, with a strong musky smell. Thymol has good antioxidant activity and broad-spectrum antibacterial activity, and is widely used in the preservation and anti-damage during the transportation of agricultural products. As a commonly used antibacterial material in food packaging, Thymol has high safety and has no serious harm to the natural environment.

[0006] So far, no biological and chemical inhibition studies on Thymol as an antibacterial agent against the marine fouling diatom Phaeodactylum tricornutum have been found. Summary of the Invention

[0007] Therefore, the object of the present invention is to provide a soybean protein-thymol nanocomposite, a preparation method thereof, and an application thereof. The nanocomposite has good biological inhibitory activity against seaweeds at low addition amounts as an antifouling agent, and can quickly and effectively control the growth and spread of seaweeds.

[0008] The above object of the present invention is achieved by the following technical solutions: The first aspect of the present invention is to provide the application of soy protein-thymol nanocomposite in removing marine fouling algae, and the algae is Phaeodactylum tricornutum;

[0009] The preparation method of the soy protein-thymol nanocomposite includes the following steps: (1) Preparation of soy protein solution The soybean meal obtained by crushing soybeans is defatted; the obtained defatted soybean meal is enzymatically hydrolyzed; the obtained enzymatic hydrolysis product is dissolved in deionized water according to a solid-liquid ratio of 1:(10-14), the pH is adjusted to 10.0-11.0, after ultrasonic extraction and centrifugation, the supernatant obtained after centrifugation is adjusted to pH 4.0-5.0 and stirred, and after standing, centrifugation, collection, and drying, a crude soy protein extract is obtained; the crude soy protein extract is dissolved in deionized water and stirred evenly, and the obtained dispersion after centrifugation is the soy protein solution; (2) Preparation of soy protein-thymol nanocomposite The thymol solution dissolved in absolute ethanol is added dropwise to the soy protein solution, mixed and stirred thoroughly, centrifuged, and the obtained dispersion is freeze-dried and pulverized to obtain the soy protein-thymol nanocomposite powder.

[0010] In an optional embodiment, the crude soy protein extract is soy protein isolate SPI.

[0011] In an optional embodiment, in step (2), the mass ratio of thymol to soy protein is (1-5):12.

[0012] In an optional embodiment, in step (1), the steps for preparing defatted soybean meal are: mixing the soybean meal with petroleum ether according to a solid-liquid ratio of 1:5, extracting the oil at a constant temperature of 55-65 °C for 45-60 min to precipitate the oil, and obtaining defatted soybean meal after centrifugation, collection, and drying.

[0013] In an optional embodiment, the centrifugation conditions are: 5000-7500 rpm / min, and the time is 5-20 min.

[0014] In an optional embodiment, in step (1), the steps for preparing the enzymatic hydrolysis product are: dissolving the defatted soybean meal in deionized water according to a solid-liquid ratio of 1:(10-14), adjusting the pH to 4.0-6.0, adding cellulase, and enzymatically hydrolyzing at a constant temperature of 45-55 °C for 1-2 h, and collecting the precipitate after centrifugation to obtain the soybean enzymatic hydrolysis product.

[0015] In an optional embodiment, the centrifugation conditions are: 3000-7000 rpm / min, and the time is 5-20 min.

[0016] In an alternative embodiment, in step (1), the conditions for centrifugation are: 6500 - 1200 rpm / min, and the time is 10 - 25 min.

[0017] Compared with the prior art, the technical solution of the present invention has the following advantages: The present invention utilizes the superior nano-delivery function of soy protein isolate (SPI), enabling the poorly soluble thymol to be loaded onto SPI particles through hydrophobic interaction to form a nano-complex. After thymol binds to SPI, its water solubility will be greatly improved, and it can be more fully and rapidly dispersed in water, so that thymol can fully contact the target algal cells in the form of nanoparticles, thereby rapidly and effectively controlling the growth and spread of fouling algae and achieving the purpose of removing attached algal fouling. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the particle size distribution diagram of the nano-complexes obtained in Example 1, Example 2, and Example 3 of the present invention; Figure 2 It is the FTIR spectra of thymol, soy protein isolate (SPI), and the nano-complex obtained in Example 2 of the present invention; Figure 3 It is the XRD pattern of the nano-complex obtained in Example 3 of the present invention; Figure 4 It is the microscopic structure diagram of the nano-complexes obtained in Example 1, Example 2, and Example 3 of the present invention; Figure 5 It is the relationship diagram of the complexation efficiency of thymol in the nano-complexes of Example 1, Example 2, and Example 3 of the present invention; Figure 6 It is the release curve diagram of thymol in the nano-complexes of Example 1, Example 2, and Example 3 of the present invention; Figure 7 It is the variation diagram of the growth effect of the nano-complexes of Example 1, Example 2, and Example 3 of the present invention on Phaeodactylum tricornutum cells at different concentrations; Figure 8 It is the variation diagram of the effect of the nano-complexes of Example 1, Example 2, and Example 3 of the present invention on the chlorophyll a content in Phaeodactylum tricornutum cells; Figure 9 It is the variation diagram of the effect of the nano-complexes of Example 1, Example 2, and Example 3 of the present invention on the carotenoid content in Phaeodactylum tricornutum cells; Figure 10 It is the variation diagram of the growth effect of the nano-complexes of Example 1, Example 2, and Example 3 of the present invention on Phaeodactylum tricornutum cells at different action times; Figure 11 Variation graph of the effect of the nano - composites of Example 1, Example 2, and Example 3 of the present invention on the malondialdehyde content in Phaeodactylum tricornutum cells; Figure 12 Algae - inhibition effect diagram of the nano - composite of Comparative Example 5 without freeze - drying treatment at different concentrations; Figure 13 Microscopic cell diagram of the effect of the nano - composite of Example 2 of the present invention on Phaeodactylum tricornutum cells; Figure 14 SEM diagram of the effect of the nano - composite of Example 3 of the present invention on Phaeodactylum tricornutum cells. Detailed implementation manners

[0019] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of this application.

[0020] The nano - composite of the present invention can be added to coatings for use or directly added to the seawater near the target object during actual application. When used in coatings, the coating is applied to marine engineering facilities. Due to the slow - release effect of the composite material, the active ingredients will slowly penetrate out of the coating film. When algae attach to the surface of the coating, it can inhibit the normal growth of algae, resulting in the death of algae, thereby achieving the purpose of removing marine fouling algae and playing an antifouling role. When directly added to seawater for use, the dispersed nano - particles will fully contact with algae under the action of seawater, directly inhibiting the growth of algae and resulting in the death of algae, thereby achieving the antifouling effect.

[0021] Example 1

[0022] A preparation method of a soybean protein - thymol nano - composite, comprising the following steps: 1) Pretreatment Select commercially available soybeans with plump grains and bright appearance as raw materials, weigh 250 g and crush them with a small - scale wall - breaker. The obtained powder is screened and purified with a 40 - mesh stainless - steel wire sieve and reserved after removing impurities.

[0023] 2) Low - temperature leaching Add 20 g of completely pulverized soybean powder into a 250 mL beaker, and add 100 mL of petroleum ether. Cover the beaker mouth tightly with plastic wrap, and extract at 60 °C in a constant temperature water bath for 50 min to precipitate the oil in soybeans. After the oil precipitation is complete, centrifuge at 6000 rpm for 10 min, then remove the supernatant and collect the precipitate, and dry it in an oven at 55 °C to obtain defatted soybean powder.

[0024] 3) Enzymatic hydrolysis Accurately weigh 10 g of dried defatted soybean powder, add it to deionized water according to a solid-liquid ratio of 1:12, adjust the pH of the system to 5.0 with 0.1 M HCl solution, then add cellulase according to 0.5% of the enzyme mass, and carry out enzymatic hydrolysis in a constant temperature water bath at 50 °C for 1 h. After the enzymatic hydrolysis is complete, centrifuge at 5000 rpm for 10 min to collect the precipitate.

[0025] 4) Alkaline extraction Weigh a certain mass of the enzymatic hydrolysis product, add it to deionized water according to a solid-liquid ratio of 1:12, adjust the pH of the system to 10 with 0.1 M NaOH, and use an ultrasonic instrument for assisted extraction. After ultrasonic extraction, let it stand for 20 min, and centrifuge at 8000 rpm for 20 min. The obtained supernatant is a clear protein aqueous solution.

[0026] Dropwise add 0.1 M HCl to the supernatant and stir to adjust the pH of the system to about 4.5, so that the protein forms a precipitate at the isoelectric point. At this time, it can be observed that the system changes from the original light yellow clear liquid to a white and relatively turbid liquid. When the system reaches the isoelectric point, stop stirring and let it stand at room temperature for 30 min to wait for the protein particles to precipitate. Centrifuge at 8000 rpm for 20 min and collect the precipitate and dry it to obtain the crude extract of soy protein isolate SPI.

[0027] 5) Preparation of soy protein-thymol nanocomposite Weigh 2.4 g of the crude extract of soy protein isolate SPI, add it to 50 mL of deionized water and continuously stir on a magnetic stirrer for 1 h. It can be observed that the solution gradually becomes milky white and evenly dispersed. Centrifuge at 8000 rpm at room temperature for 15 min, transfer the centrifuged supernatant to a new beaker again and stir it magnetically for standby; then prepare a thymol ethanol solution, and then add the thymol ethanol solution dropwise to the supernatant of the crude extract of soy protein isolate SPI according to a mass ratio of thymol:crude extract of soy protein isolate SPI = 1:12 for co-mixing. After the solution is fully mixed, centrifuge again, take the supernatant and freeze-dry it to obtain a white flocculent soy protein-thymol nanocomposite (SPI-Thymol) powder, named ST1.

[0028] Example 2 The difference between this example and Example 1 lies in the mass ratio when thymol is mixed with the crude extract of soy protein isolate SPI. In this example, the mass ratio of thymol to the crude extract of soy protein isolate SPI is 2.5:12. The remaining procedures are the same as those in Example 1. The obtained white flocculent soy protein-thymol nanocomposite is named ST2.

[0029] Example 3 The difference between this example and Example 1 lies in the mass ratio when thymol is mixed with the crude extract of soy protein isolate SPI. In this example, the mass ratio of thymol to the crude extract of soy protein isolate SPI is 5:12. The remaining procedures are the same as those in Example 1. The obtained white flocculent soy protein-thymol nanocomposite is named ST3.

[0030] Example 4 The difference between this example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this example, the ratio of defatted soy flour to deionized water is 1:10.

[0031] Example 5 The difference between this example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this example, the ratio of defatted soy flour to deionized water is 1:14.

[0032] Comparative Example 1

[0033] The difference between this comparative example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:6.

[0034] Comparative Example 2

[0035] The difference between this comparative example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:8.

[0036] Comparative Example 3

[0037] The difference between this comparative example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:16.

[0038] Comparative Example 4

[0039] The difference between this comparative example and Example 3 lies in the material-liquid ratio when defatted soy flour is added to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:18.

[0040] Comparative Example 5

[0041] The difference between this comparative example and Example 3 lies in the lyophilization treatment in step 5). In this comparative example, without undergoing lyophilization treatment, thymol ethanol solution is added to the supernatant of the crude extract of soy protein isolate SPI for blending. What is obtained is a soy protein-thymol nanocomposite solution, and the soy protein-thymol nanoparticles exist in the form of nanoparticles in the aqueous solution.

[0042] Test Example 1 I. Influence of different material-liquid ratios (defatted soy flour: deionized water) on the extraction rate of soy protein isolate

[0043] Respectively take the precipitates obtained after enzymatic hydrolysis in step 3) in Example 3, Example 4, Example 5, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, use the Folin-phenol method to measure the protein content in the precipitates, then calculate the protein concentration of the samples through the standard curve, and further calculate the extraction rate. The extraction rate results of soy protein are shown in Table 1.

[0044] Table 1 Extraction rate of soy protein isolate with different material-liquid ratios

[0045] The content in the above table shows that the material-liquid ratio has a significant impact on the extraction rate of the separated protein after enzymatic hydrolysis. As the material-liquid ratio decreases, the extraction rate increases rapidly. More water can dissolve more proteins, but when the amount of added water exceeds a certain proportion, it will lead to a decrease in the extraction rate. Therefore, the material-liquid ratio can only improve the extraction rate of soy protein isolate within a certain range. When the amount of added water is too little or too much, the extraction rate of soy protein isolate will decrease.

[0046] II. Particle size analysis of thymol-soy protein isolate SPI nanocomposites Respectively weigh 1 g of soy protein isolate SPI prepared in Example 1, 1 g of the nanocomposite (ST1) prepared in Example 1, 1 g of the nanocomposite (ST2) prepared in Example 2, and 1 g of the nanocomposite (ST3) prepared in Example 3, dissolve them in 10 mL of deionized water, and ultrasonicate for 10 min at a power of 200 W at room temperature to make the mixture solution uniformly dispersed in the system, obtaining a soy protein isolate SPI solution and three thymol-soy protein isolate SPI nanoparticle solutions. Use a Zeta potential analyzer to perform particle size analysis on the soy protein isolate SPI solution and the three nanoparticle solutions with different compounding ratios.

[0047] The results are as Figure 1 shown, in Figure 1Among the curves, SPI represents the particle size of soy protein isolate SPI, ST1 represents the particle size of the nanocomposite prepared in Example 1, ST2 represents the particle size of the nanocomposite prepared in Example 2, and ST3 represents the particle size of the nanocomposite prepared in Example 3.

[0048] From the particle size data of the Zeta potentiometer, it can be seen that pure SPI exists in the form of nanoparticles when dissolved in water and the particle size is several nanometers. Because of this size effect of SPI, when it is mixed with Thymol, it can effectively complex with Thymol and form stable nanoparticles.

[0049] From Figure 1 Figure A in Figure 1 it can be seen that when Thymol and SPI are complexed at a mass ratio of 1:12 (ST1), the particle size is mainly distributed around 10 nm as shown in the figure, which is larger than the particle size in the pure SPI dispersion solution. From Figure 1 Figure B in

[0050] III. Fourier Transform Infrared Spectroscopy (FTIR) Analysis of Thymol-Soy Protein Isolate SPI Nanocomposites The interaction between Thymol and SPI is the key to maintaining the structural stability of the composite nanoparticles. Thymol contains C=C, C-O, and -CH3 groups. Commercially available Thymol, soy protein isolate SPI prepared in Example 2, and the nanocomposite (ST1) prepared in Example 2 were separately taken and placed in an agate mortar. An appropriate amount of analytical pure anhydrous potassium bromide reagent was added and mixed and ground finely. After the powder was sufficiently ground, a little of the mixed solid powder was evenly covered inside the tablet pressing device, and a thin slice was prepared in a tablet press for infrared testing. The results are as Figure 2 shown. In Figure 2 , the SPI-Thymol curve represents the nanocomposite of Example 2.

[0051] It can be seen from Figure 2 that at 1620 cm -1 is the asymmetric deformation of -CH3, 1584 cm -1 is the stretching vibration of C=C, 1285 cm -1 is the bending deformation of -CH3, 1156 cm-1 C-O stretching vibration at -1 SPI has three relatively obvious characteristic peaks at -1 cm. The FTIR spectra of the SPI-Thymol mixture show increased infrared absorption intensity at -1 cm, indicating that some characteristics of thymol are superimposed on the characteristic peaks of SPI. The FTIR spectra of the SPI-Thymol mixture show relatively obvious characteristic peaks of thymol at -1 cm, where SPI has no characteristic peaks. In the range of 1600 to 1000 cm -1 Due to the superposition of thymol on SPI, the characteristic peaks of the mixture show enhanced absorption. The shift of some characteristic peaks may be the result of the combined effects of hydrogen bonding or molecular conformation changes.

[0052] The spectrograms of SPI-Thymol and SPI are relatively similar. After compounding with Thymol, the characteristic peaks of the former become more obvious than those of SPI, indicating that thymol is evenly dispersed in the structure of soy protein isolate and no new covalent bonds or chemical reactions are generated.

[0053] IV. X-ray diffraction (XRD) analysis of thymol-soy protein isolate SPI nanocomposites The XRD patterns of Example 3 (thymol:soy protein isolate = 5:12) were measured using an X-ray powder diffractometer (D8 Advance, Bruker, Germany) to analyze the structural differences between Thymol and SPI-Thymol (ST2). The test conditions were 45 kV and 40 mA, in the 2θ region from 5° to 60°, and the scanning rate was 0.02° / min. The results are as Figure 3 shown.

[0054] Figure 3 The data results from 5° to 60° were recorded. Thymol has a fixed characteristic peak at 20°, and the synthesized SPI-Thymol also has a peak at this position and contains diffraction peaks, indicating that the nanocomposite prepared in Example 3 is a polycrystalline structure with good crystallinity.

[0055] V. Microscopic morphological structure analysis of thymol-soy protein isolate SPI nanocomposites The morphological characteristics of the nanocomposites prepared from thymol and soy protein isolate SPI with different mass ratios (1:12, 2.5:12, 5:12) were investigated respectively, as follows: The nano - composites prepared in Example 1 (thymol: soy protein isolate SPI = 1:12), Example 2 (thymol: soy protein isolate SPI = 2.5:12), and Example 3 (thymol: soy protein isolate SPI = 5:12) were respectively and uniformly dissolved in deionized water to obtain a mixed solution containing nano - composite particles. After the mixed solution was ultrasonically washed and centrifuged multiple times, it was observed that the supernatant was slightly light yellow, transparent, and clear. The supernatant was fully collected and solidified overnight in a - 80 °C freezer. The solidified mixture was immediately placed in a freeze - dryer for low - temperature freeze - drying. After the water had fully evaporated, a small amount of the powder was taken for morphological observation using a field - emission scanning electron microscope (SEM, Mira3Tescan). The results are as Figure 4 shown. Figure 4 From left to right in the figure are the micrographs at different magnifications (2 μm, 500 nm, 400 nm).

[0056] From Figure 4 it can be seen that under low magnification, the surface structures of the nano - composite materials with different mass ratios are relatively compact and dense. Under high magnification, it can be clearly seen that the morphologies of the nano - composites with different mass ratios are good, indicating that the stability of the nano - composites is good.

[0057] VI. Analysis of the complexation efficiency of different concentrations of thymol and soy protein isolate SPI A certain amount of the nano - composites from Example 1, Example 2, and Example 3 was taken into a 10 - mL glass bottle. The thymol in the composite was extracted using the organic solvent n - hexane and determined using a UV - Vis spectrophotometer (UV - 1800). The absorbance value was measured at 275 nm. The specific steps are as follows: 3 mL of n - hexane was added to 0.2 mL of the dispersion containing the nano - composite and diluted with deionized water at a ratio of 1:10 (v / v) before extraction. After thorough mixing, it was left to stand at room temperature for 0.5 h. It could be clearly observed that the n - hexane and water were stratified. An appropriate amount of the mixture was taken from the n - hexane layer and centrifuged at 8000 rpm for 5 min. The supernatant was collected, and the absorbance of thymol in the n - hexane was measured at 275 nm. Additionally, the specific content of thymol was calculated according to the standard curve (R2 = 0.995) of standard thymol in n - hexane. The results are as Figure 5 shown. Figure 5 In Figure A of it is the UV - scanning diagram of thymol; Figure B is the relationship diagram between thymol concentration and OD value; Figure C is the loading rate of the nano - composites with different mass ratios (thymol: soy protein).

[0058] From Figure 5As can be seen from Figure C in , when thymol is combined with soy protein isolate at a mass ratio of 1:12, the combined efficiency detected by ultraviolet absorbance is 4.76%; when combined at a mass ratio of 2.5:12, the combined efficiency is 9.35%; when combined at a mass ratio of 5:12, the combined efficiency is 10.18%. As the proportion of thymol increases, its combined efficiency continues to rise. It can be found that when thymol is combined at a low mass ratio, its combined efficiency increases approximately proportionally, from 4.76% to 9.35%. However, when the proportion of thymol in the mixture continues to double, its combined efficiency does not increase in a linear trend. Therefore, the combined efficiency of soy protein isolate in the crude extract product for thymol is limited.

[0059] VII. Analysis of Thymol Release in Thymol-Soy Protein Isolate SPI Nanocomposites To test the release of thymol in the nanocomposite in artificial seawater, with free thymol as the control, the thymol release of free thymol and the nanocomposites of Example 1, Example 2, and Example 3 was continuously measured within 24 h. The specific steps are as follows: The nanocomposites prepared in Example 1, Example 2, and Example 3 were added to artificial seawater (f / 2 medium) at a concentration of 10 mg / L, shaken well, and statically placed at room temperature. At 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, 3 mL of the solution was taken to measure the absorbance (275 nm) using an ultraviolet spectrophotometer, and the release process was calculated and simulated using the kinetic model of the following formula (1), where Ct i is t i the cumulative amount of thymol released at time t, a i is t i the sampling concentration of thymol at time t, m 0 is the total mass.

[0060]

[0061] The results are as Figure 6 shown. The release rate of free thymol is faster than that of the nanocomposite. Within the first four hours, the release amount of free thymol gradually increases and is significantly higher than the release amount of thymol in the composite nanoparticles. After one hour, it is about 1.65 times that of the latter. The release time of free thymol starts to remain stable and slightly increases after reaching five hours, and remains at about 80% after 24 h.

[0062] The release profiles of thymol in the three kinds of nanocomposites were similar to that of the free form, showing a rapid release during the first four hours and then reaching a steady state. For ST1, the release amount reached about 40% after 1 h and remained stable at about 80% after 24 h. For ST2 and ST3, the release amount reached about 38% after 1 h, and then it was a slow and continuous release process, with the final release amount reaching about 68% after 24 h. This indicates that most of the thymol is combined with the hydrophobic structure on the surface of SPI through weak hydrophobic interactions, while some are tightly encapsulated in the core of the composite and cannot be dissolved from the loading structure under the dissolution of n-hexane. With the increase of the mass ratio of thymol, the release rate after 24 h decreased, showing a significant difference (p < 0.05), indicating that at a higher ratio, thymol can form a more stable anodic complex with SPI, thus having a more stable, compact and dense nanoparticle structure and better sustained-release performance.

[0063] Experimental Example 2 Growth Inhibitory Effect on Algae

[0064] Algal cells in the logarithmic growth phase were selected for the experiment. Fucus vesiculosus liquid was added to a glass conical flask and diluted with f / 2 culture medium to a final concentration of 3.5 × 10 7 cells / mL, mimicking the algal density when diatoms form surface biofilms during attachment growth, and reserved for use.

[0065] I. Inhibitory Effects of Thymol-Soybean Protein Isolate (SPI) Nanocomposites with Different Concentrations on Fucus vesiculosus Cells The inhibitory effects of the thymol-Soybean Protein Isolate (SPI) nanocomposites of Example 1, Example 2, and Example 3 at different concentrations on Fucus vesiculosus cells were investigated respectively, including the following steps: (1) The nanocomposite (ST1) prepared in Example 1 (thymol:soybean protein isolate = 1:12) was added to the prepared algal liquid, and algal liquids containing nanocomposite concentrations of 0.5 mg / L, 1 mg / L, 1.5 mg / L, 2.0 mg / L, 2.5 mg / L, 3.0 mg / L, 3.5 mg / L, 4.0 mg / L, and 8 mg / L were prepared respectively. The final volume of the algal liquid containing different nanocomposites was 25 mL. After preparation, the growth state of Fucus vesiculosus was recorded by absorbance value (OD = 680 nm), and then compared with the growth state of Fucus vesiculosus in the algal liquid without adding the nanocomposite. The results are as Figure 7 shown in Figure A and Figure A' of this text. Figure A shows the effect of ST1 on the growth of algal cells; Figure A' shows the inhibitory effect of different concentrations of ST1 on the growth of algal cells at 48 h.

[0066] From Figure 7As can be seen from Figure A and Figure A' in [reference], as the concentration of the nanocomposite in the system increases, its inhibitory effect on the growth of algal cells becomes more obvious, showing a concentration-dependent characteristic. When the material concentration reaches about 4 mg / L, it can completely inhibit the growth of algal cells at 48 h. As the concentration of the nanocomposite continues to increase, a large number of Phaeodactylum tricornutum cells die.

[0067] (2)Add the nanocomposite (ST2) prepared in Example 2 (thymol:soybean protein isolate = 2.5:12) to the prepared algal solution, and prepare algal solutions containing nanocomposite concentrations of 0.3 mg / L, 0.6 mg / L, 0.9 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 2 mg / L, 3 mg / L, and 5 mg / L respectively. The final volume of the algal solution containing different nanocomposites is 25 mL. After preparation, record the growth status of Phaeodactylum tricornutum by absorbance value (OD = 680 nm), and then compare it with the growth status of Phaeodactylum tricornutum in the algal solution without adding the nanocomposite. The results are as Figure 7 shown in Figure B and Figure B' of [reference]. Figure B shows the effect of ST2 on the growth of algal cells; Figure B' shows the growth inhibition effect of different concentrations of ST2 on algal cells at 72 h.

[0068] From Figure 7 Figure B and Figure B' in [reference], it can be seen that when the concentration of the nanocomposite reaches 3 mg / L, at 72 h, the number of algal cells begins to decrease, and at this time, the composite material reaches 100% inhibition of algal cells.

[0069] (3)Add the nanocomposite (ST3) prepared in Example 3 (thymol:soybean protein isolate = 5:12) to the prepared algal solution, and prepare algal solutions containing nanocomposite concentrations of 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, 1.2 mg / L, 1.4 mg / L, 1.6 mg / L, and 2 mg / L respectively. The final volume of the algal solution containing different nanocomposites is 25 mL. After preparation, record the growth status of Phaeodactylum tricornutum by absorbance value (OD = 680 nm), and then compare it with the growth status of Phaeodactylum tricornutum in the algal solution without adding the nanocomposite. The results are as Figure 7 shown in Figure C and Figure C' of [reference]. Figure C shows the effect of ST3 on the growth of algal cells; Figure C' shows the growth inhibition effect of different concentrations of ST3 on algal cells at 72 h.

[0070] From Figure 7As can be seen from Figure C in [reference], the nano - composite concentration of 2 mg / L achieved 100% inhibition of algal cells at 48 h. Meanwhile, the growth inhibition of Phaeodactylum tricornutum cells was concentration - dependent. In the following time, a large number of algal cells began to die and white precipitates appeared. From Figure 7 As can be seen from Figure C' in [reference], when the concentration was 1.6 mg / L, the nano - composite achieved 100% inhibition of Phaeodactylum tricornutum cells, and the IC50 was 0.83 mg / L.

[0071] II. Effects of thymol - soy protein isolate (SPI) nano - composites on the contents of photosynthetic pigments (chlorophyll a and carotenoids) in Phaeodactylum tricornutum cells The effects of thymol - soy protein isolate (SPI) nano - composites prepared in Example 1, Example 2, and Example 3 on the contents of photosynthetic pigments (chlorophyll a and carotenoids) in Phaeodactylum tricornutum cells were investigated respectively. The specific steps are as follows: The nano - composites prepared in Example 1 (ST1), Example 2 (ST2), and Example 3 (ST3) were respectively added to the algal solution. The concentrations of the nano - composites of Example 1, Example 2, and Example 3 in the algal solution were the half - inhibitory concentrations, that is, the concentration of the nano - composite of Example 1 in the algal solution was 2 mg / L, the concentration of the nano - composite of Example 2 in the algal solution was 1.3 mg / L, and the concentration of the nano - composite of Example 3 in the algal solution was 1.6 mg / L. The final volumes of the nano - composites, f / 2 culture medium, and algal solution obtained by adding the nano - composites in different examples were 25 mL.

[0072] After adding, 3 mL of algal solution was taken from the corresponding conical flasks at 0 h, 24 h, 48 h, 72 h, and 96 h respectively, centrifuged at 7000 rpm and 4°C for 10 min, the precipitate was retained and 3 mL of acetone (90%) was added to resuspend it. After vortex - shaking evenly, it was placed in a 4°C refrigerator for dark extraction for 24 h. After the dark extraction was completed, it was centrifuged under the same conditions and the supernatant was retained. Subsequently, the absorbance was measured at wavelengths of 440 nm, 644 nm, 630 nm, 647 nm, 664 nm, 662 nm, and 750 nm using a UV - visible spectrophotometer. The blank control was adding 90% acetone solution to the algal solution, and the concentrations of chlorophyll a (Chla) and carotenoids (Car) were calculated respectively using the following formulas (1) and (2), and the change trend graph was drawn. The results are as Figure 8 and Figure 9 shown. Figure 8 Figures A, B, and C in [reference] respectively show the effects of nano - composite particles ST1, ST2, and ST3 on the content of chlorophyll a in Phaeodactylum tricornutum cells. Figure 9Effects of nano - composite particles ST1, ST2, and ST3 on the carotenoid content in Phaeodactylum tricornutum cells.

[0073] Figure 8 In Figure A of [reference], the effect of Example 1 (ST1) on the chlorophyll a content of Phaeodactylum tricornutum; Figure B shows the effect of Example 2 (ST2) on the chlorophyll a content of Phaeodactylum tricornutum; Figure C shows the effect of Example 3 (ST3) on the chlorophyll a content of Phaeodactylum tricornutum.

[0074]

[0075]

[0076] From Figure 8 it can be seen that the chlorophyll a content in the blank control group of Phaeodactylum tricornutum cells increased from the initial 1.12 mg / L to 1.89 mg / L, an increase of 68.75%. The chlorophyll a content in the control group began to increase significantly from the third day. This is because there are sufficient nutrient elements, suitable light conditions, and temperature in the culture medium, which promote the accelerated growth of algal cells. The chlorophyll a content in the ST1 experimental group decreased from 1.15 mg / L to 0.41 mg / L after 96 h, a decrease of 64.35%, and continued to decline; the chlorophyll a content in the ST2 experimental group decreased from 1.13 mg / L to 0.29 mg / L after 96 h, a decrease of 74.34%, and there was a continuous downward trend; the chlorophyll a content in the ST3 experimental group decreased from 1.14 mg / L to 0.09 mg / L after 96 h, with a decrease of 91.40%, and there was a continuous downward trend; after adding the nano - composite, the chlorophyll a content in the three experimental groups all decreased significantly, and the addition of ST3 had the greatest impact on the chlorophyll content of algal cells, followed by ST2. The addition of ST1 also significantly reduced the chlorophyll a content compared with the control group (P < 0.05).

[0077] Chlorophyll a is the most important photosynthetic pigment in Phaeodactylum tricornutum cells. Due to the presence of long - chain alkyl and magnesium ions, chlorophyll a absorbs light energy and converts it into chemical energy. The addition of thymol - soy protein isolate SPI nano - composite particles leads to a significant reduction in chlorophyll a. Therefore, the nano - composite particles formed by loading thymol on soy protein isolate SPI have an inhibitory effect on the growth of algal cells.

[0078] From Figure 9As can be seen, the blank control group grew naturally within 96 h, and the carotenoid content increased from 0.18 mg / L to 0.82 mg / L after 96 h. In a suitable growth environment, the cells of *Phaeodactylum tricornutum* grew and reproduced rapidly, and the carotenoid content increased significantly within a short time. On the contrary, after adding the composite nanoparticles, the carotenoid content in the ST1 and ST2 experimental groups increased slightly in the early stage of the experiment due to the influence on the carotenoid content. This may be because the addition of a small amount of the nanocomposite stimulated cell growth in a short time. However, starting from the second day, the carotenoid content under the treatment of the three nanocomposites began to decrease sharply and was almost zero after 96 h, indicating that the vast majority of algal cells lost their basic physiological functions or physiological activities.

[0079] As a class of unsaturated fatty acids, carotenoids have conjugated double bonds and play a very important role in the growth and reproduction of cells. They participate in all stages of photosynthesis and act as photosensitizers or signal molecules. A significant decrease in their content will surely seriously affect the normal physiological activities of algal cells. Therefore, the addition of the nanocomposite particles will lead to a sharp decrease in the carotenoid content, thereby inhibiting the growth activity of algal cells and causing the death of algal cells.

[0080] III. Effects of thymol-soybean protein isolate (SPI) nanocomposites on algal inhibition effect at different action times The inhibitory effects of the thymol-soybean protein isolate (SPI) nanocomposites prepared in Example 1, Example 2, and Example 3 on the growth of *Phaeodactylum tricornutum* cells over time were investigated respectively. The specific steps are as follows: The nanocomposites prepared in Example 1, Example 2, and Example 3 were added to the algal solution, and experiments were conducted on the concentration at which the cells reached the half-inhibition effect at 72 h, namely treatment group ST1, treatment group ST2, and treatment group ST3. The algal solution that grew normally without adding the nanocomposite was used as the blank control group. The results are as Figure 10 shown.

[0081] As can be seen from Figure 10 , as time increased, the cells of *Phaeodactylum tricornutum* in the blank control group grew continuously. After 5 days, the absorbance value of the algal cells exceeded 1. Compared with the blank control group, the treatment groups showed an upward trend starting from the second day and a downward trend starting from the fourth day. As can be seen from Figure 10 , among the different treatment groups, the higher the proportion of thymol in the nanocomposite, the more obvious the inhibitory effect of the nanocomposite on the growth of algal cells. When the treatment time reached 4 days, due to the inhibitory effect of the nanocomposite on the growth of algal cells, treatment group ST3 achieved 100% inhibition on *Phaeodactylum tricornutum* and began to damage the cells in the following time, resulting in a large number of algal cells dying.

[0082] IV. Effect of thymol-soybean protein isolate (SPI) nanocomposite on the content of malondialdehyde (MDA) in algal cells The effects of the nanocomposites of Example 1, Example 2, and Example 3 on the content of malondialdehyde (MDA) in algal cells were investigated. The nanocomposites prepared in Example 1, Example 2, and Example 3 were added to the algal solution, and the changes in the MDA content of the algal cells were continuously measured within 96 h, and the activity change trend was plotted and compared with the algal solution control group without adding the nanocomposite. The results are as Figure 11 shown. In Figure 11 , Figure A represents the effect diagram of Example 1 (ST1) on the change in the MDA content of algal cells; Figure B represents the effect diagram of Example 2 (ST2) on the change in the MDA content of algal cells; Figure C represents the effect diagram of Example 3 (ST3) on the change in the MDA content of algal cells.

[0083] As can be seen from Figure 11 , the MDA content in the control group remained basically unchanged, at about 0.01 mol / g. After adding the nanocomposite, the content of malondialdehyde increased significantly within 24 h (p < 0.05). Adding thymol caused a large amount of reactive oxygen species to be produced in algal cells and resulted in an excess of reactive oxygen species content, finally leading to serious damage to algal cells, thereby inhibiting the normal growth of algal cells. The ST2 and ST3 groups began to gradually decline after 24 h and became about 0.02 mol / g after 96 h. The ST1 group began to decline after 48 h. The MDA contents of these three treatment groups finally tended to be the same, but were all higher than the control group.

[0084] V. Effect of freeze-drying and non-freeze-drying on the algicidal effect of thymol-soybean protein isolate (SPI) nanocomposite. The soybean protein-thymol nanocomposite solution prepared in Comparative Example 5 was directly added to algal cells to study its growth inhibitory effect on Phaeodactylum tricornutum cells. The specific steps are as follows: 1, 3, 5, 8, and 10 mL of the nanocomposite solution prepared in Comparative Example 5 were respectively added to the Phaeodactylum tricornutum solution and finally formed a 30 mL algal solution system, thus forming a series of nanoparticle solution gradients.

[0085] Its growth effect on algal cells is as Figure 12 shown. As the addition amount of the nanoparticle solution increases, its inhibitory effect on algal cells increases significantly. When the addition amount exceeds 8 mL, the inhibitory effect on algal cells reaches 100% after 72 h.

[0086] Compared with adding the nanocomposite (SPI&Thymol) directly to the algal cell system after freeze-drying, the amount of the nanoparticle solution directly added needs to reach a total volume of 8 mL to achieve a complete inhibitory effect on algal cells after 72 h. Since the original thymol and soy protein isolate are compounded at a mass ratio of 5:12 and 40 mL of deionized solution is added, the mass of thymol in the 8 mL mixed nanoparticle solution is 3.33 mg / L. As known from the above, when preparing the ST3 nanocomposite, for the freeze-dried nanocomposite material, only a concentration of 1.6 mg / L can achieve a complete inhibitory effect on Phaeodactylum tricornutum cells after 72 h. Therefore, compared with directly adding the nanoparticle mixed solution to the algal cells, the inhibitory efficiency of the freeze-dried nanoparticle powder on algal cells is increased by 52%.

[0087] VI. Microscopic Observation of the Inhibition of Algal Cells by Thymol-Soy Protein Isolate SPI Nanocomposite

[0088] Prepare three conical flasks each containing 3×10 7 cells / mL (OD = 1.7). Add the nanocomposite prepared in Example 1 to the algal solutions in two of the conical flasks so that their concentrations are 1 μg / mL and 4 μg / mL respectively. Then, take 10 μL of the algal solution and slowly drip it along the cover glass. After standing for 1 min, observe the morphology of the algal cells under a 50× objective lens of an inverted electron microscope. The results are as Figure 13 shown. In Figure 13 , Figure A represents the microscopic morphology diagram of algal cells without adding the nanocomposite; Figure B represents the microscopic morphology diagram of algal cells after adding the nanocomposite with a concentration of 1 μg / mL; Figure C represents the microscopic morphology diagram of algal cells after adding the nanocomposite with a concentration of 4 μg / mL.

[0089] From Figure 13 Figure A in

[0090] From Figure 13 Figure B and Figure C in

[0091] VII. SEM Observation of the Inhibition of Thymol-Soybean Protein Isolate (SPI) Nanocomposites on Algal Cells

[0092] To observe the effect of the nanocomposites on the microscopic structure of Phaeodactylum tricornutum cells, the nanocomposites prepared in Example 3 were added to the algal solution. The concentration of the nanocomposites in the algal solution was 1.6 mg / L. After 72 h, 5 mL of the algal solution was evenly pipetted from the conical flask into a centrifuge tube and centrifuged at 6000 rpm and 4 °C for 10 min to collect the precipitate. 3 mL of 2.5% glutaraldehyde solution was added to the precipitate. After vortexing and shaking evenly, it was placed in a refrigerator at 4 °C for dark treatment for 24 h. The samples were washed and purified with 0.1 M phosphate buffer solution with pH = 7.0. Finally, the samples were dehydrated with ethanol solutions with concentrations of 30%, 50%, 80%, 90% and absolute ethanol for 10 min respectively. After completing the above steps, the processed samples were frozen in a refrigerator at -80 °C, and the microscopic structure of the cells was observed under a scanning electron microscope and compared with the algal solution control group without adding the nanocomposites. The results are as Figure 14 shown.

[0093] From Figure 14 Figure A of Figure 14 it can be seen that the algal cell structure in the control group was complete and plump, showing a spindle shape, and the structure of individual algal cells could be clearly observed; from Figure 14 Figure B of

[0094] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the protection scope of the present invention. Any person skilled in the art, without departing from the spirit and scope of the present invention, making various changes and modifications to the above embodiments still belongs to the scope protected by the present invention.

Claims

1. Application of soybean protein-thymol nanocomposite in removing marine fouling algae, wherein the algae is Phaeodactylum tricornutum; It is characterized in that The preparation method of the soybean protein-thymol nanocomposite comprises the following steps: (1) Preparation of soybean protein solution Defat the soybean powder obtained by pulverizing soybeans; enzymatically hydrolyze the obtained defatted soybean powder; dissolve the obtained enzymatic hydrolysis product in deionized water at a solid-liquid ratio of 1:(10-14), adjust the pH to 9.0-10.0, perform ultrasonic extraction and then centrifugation, adjust the pH of the supernatant obtained after centrifugation to 4.0-5.0 and stir, and obtain the crude soybean protein extract after standing, centrifugation, collection and drying; dissolve the crude soybean protein extract in deionized water and stir evenly, and the obtained dispersion after centrifugation is the soybean protein solution; (2) Preparation of soybean protein-thymol nanocomposite Dropwise add the thymol solution dissolved in absolute ethanol to the soybean protein solution, fully mix and stir, centrifuge, and perform freeze-drying and pulverization on the obtained dispersion to obtain the soybean protein-thymol nanocomposite powder.

2. The application according to claim 1, wherein The crude soybean protein extract is soy protein isolate SPI.

3. The application according to claim 1, wherein In the step (2), the mass ratio of thymol to soybean protein is (1-5):

12.

4. The application according to claim 1, wherein In the step (1), the steps for preparing defatted soybean powder are: mix the soybean powder with petroleum ether at a solid-liquid ratio of 1:5, extract the oil at a constant temperature of 55-65 °C for 45-60 min to precipitate the oil, and obtain the defatted soybean powder after centrifugation, collection and drying.

5. The application according to claim 4, characterized in that The centrifugation conditions are: 5000-7500 rpm / min, and the time is 5-20 min.

6. The application according to claim 1, wherein In the step (1), the steps for preparing the enzymatic hydrolysis product are: dissolve the defatted soybean powder in deionized water at a solid-liquid ratio of 1:(10-14), adjust the pH to 4.0-6.0, add cellulase, and perform enzymatic hydrolysis at a constant temperature of 45-55 °C for 1-2 h, and collect the precipitate after centrifugation to obtain the soybean enzymatic hydrolysis product.

7. The application according to claim 6, characterized in that, The centrifugation conditions are: 3000-7000 rpm / min, and the time is 5-20 min.

8. The application according to claim 1, characterized in that, In the step (1), the centrifugation conditions are: 6500-1200 rpm / min, and the time is 10-25 min.

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