Soy protein-thymol nanocomposites, methods of making and using the same

Abstract

The application discloses application of a soybean protein-thymol nanocomposite in removing marine fouling algae. The application utilizes the superior nanometer transportation function of soybean protein isolate SPI, so that the hardly soluble thymol is loaded on the soybean protein isolate SPI particles to form a nanocomposite through hydrophobic interaction. After the thymol is combined with the soybean protein isolate SPI, the water solubility of the thymol is greatly improved, and the thymol can be more fully and rapidly dispersed in water, so that the thymol fully contacts target algae in the form of nanometer particles, thereby the growth and spread of the 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 in particular to a soybean protein-thymol nanocomposite and a preparation method and application thereof. Background Art

[0002] Marine fouling organisms, also known as marine attached organisms, mainly grow on the bottom of ships and the surface of artificial facilities in the sea. They include complex communities with sessile organisms as the main body. There are many types of them, including bacteria, attached diatoms and many large algae, as well as various phyla from protozoa to vertebrates.

[0003] Frequent contact with seawater by ships and marine engineering facilities leads to the attachment of marine organisms such as algae and shellfish, forming biofouling. This biofouling not only increases the ship's navigational resistance, reduces speed, increases fuel consumption, accelerates corrosion of metal materials such as ships and platforms, and shortens their service life; it also affects the transmission of optical or acoustic signals from marine instruments, increasing maintenance frequency and costs. Furthermore, the attachment of marine fouling organisms to and clogging of aquaculture nets can cause the death of farmed organisms, causing significant damage to the aquaculture industry. In short, the attachment of marine fouling organisms to artificial facilities is a major challenge to human development and utilization of the ocean.

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

[0005] Thymol, the primary active ingredient in essential oils such as thyme and oregano, is a food additive recognized and used in my country. At room temperature, it is a colorless crystalline solid that is insoluble in water and has a strong musky aroma. Thymol exhibits strong antioxidant and broad-spectrum antibacterial activity and is widely used to preserve and prevent damage to agricultural products during transportation. As a commonly used antimicrobial material in food packaging, thymol is highly safe and poses no significant threat to the natural environment.

[0006] So far, no biological and chemical inhibition studies on Thymol as an antimicrobial 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 soy protein-thymol nanocomposite and its preparation method and application, which, as an antifouling agent, has good bioinhibitory activity against seaweed at a low addition amount and can quickly and effectively control the growth and spread of seaweed.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention is to provide the use of soy protein-thymol nanocomposite in removing marine fouling algae, wherein the algae is Phaeodactylum tricornutum;

[0010] The preparation method of the soy protein-thymol nanocomposite comprises the following steps:

[0011] (1) Preparation of soy protein solution

[0012] The soybean powder obtained by crushing soybeans is defatted; the defatted soybean powder is enzymatically hydrolyzed; the enzymatic hydrolysis product is dissolved in deionized water at a material-liquid ratio of 1:(10-14), the pH is adjusted to 10.0-11.0, ultrasonic extraction is performed, and then centrifugation is performed; the supernatant obtained after centrifugation is adjusted to pH 4.0-5.0 and stirred, and after standing, centrifugation, collection, and drying, a crude soybean protein extract is obtained; the crude soybean protein extract is dissolved in deionized water and stirred uniformly, and the dispersion obtained after centrifugation is a soybean protein solution;

[0013] (2) Preparation of soy protein-thymol nanocomplex

[0014] The thymol solution dissolved in anhydrous ethanol is added dropwise to the soy protein solution, fully mixed and stirred, and centrifuged. The obtained dispersion is freeze-dried and pulverized to obtain the soy protein-thymol nanocomposite powder.

[0015] In an optional embodiment, the soy protein crude extract is soy protein isolate SPI.

[0016] In an optional embodiment, in step (2), the mass ratio of thymol to soy protein is (1-5):12.

[0017] In an optional embodiment, in step (1), the defatted soy flour is prepared by mixing the soy flour with petroleum ether at a material-liquid ratio of 1:5, extracting the mixture at a constant temperature of 55 to 65° C. for 45 to 60 min to separate out the oil, and obtaining the defatted soy flour after centrifugation, collection, and drying.

[0018] In an optional embodiment, the centrifugal conditions are: 5000-7500 rpm / min, and the time is 5-20 min.

[0019] In an optional embodiment, in the step (1), the enzymatic hydrolysis product is prepared by dissolving defatted soy flour in deionized water at a material-liquid ratio of 1:(10-14), adjusting the pH to 4.0-6.0, adding cellulase, and performing enzymatic hydrolysis at a constant temperature of 45-55°C for 1-2 h, collecting the precipitate after centrifugation, and obtaining the soybean enzymatic hydrolysis product.

[0020] In an optional embodiment, the centrifugal conditions are: 3000-7000 rpm / min, and the time is 5-20 min.

[0021] In an optional embodiment, in step (1), the centrifugal conditions are: 6500-1200 rpm / min, and the time is 10-25 min.

[0022] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0023] The present invention utilizes the superior nano-transport function of soy protein isolate (SPI) to load insoluble thymol onto soy protein isolate (SPI) particles through hydrophobic interaction to form a nanocomposite. After the thymol and soy protein isolate (SPI) are combined, their water solubility is greatly improved, allowing them to be more fully and quickly dispersed in water. This allows the thymol in the form of nanoparticles to fully contact the target algae, thereby quickly and effectively controlling the growth and spread of fouling algae and achieving the purpose of removing attached algae fouling. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The particle size distribution diagram of the nanocomposite obtained in Example 1, Example 2, and Example 3 of the present invention;

[0025] Figure 2 FTIR spectra of thymol, soy protein isolate SPI and the nanocomposite obtained in Example 2 of the present invention;

[0026] Figure 3 This is the XRD pattern of the nanocomposite obtained in Example 3 of the present invention;

[0027] Figure 4 The microstructure diagrams of the nanocomposites obtained in Examples 1, 2, and 3 of the present invention are shown;

[0028] Figure 5 This is a graph showing the relationship between the composite efficiency of thymol in the nanocomposites of Examples 1, 2, and 3 of the present invention;

[0029] Figure 6 1 is a graph showing the release of thymol from the nanocomposites of Examples 1, 2 and 3 of the present invention;

[0030] Figure 7 This is a graph showing the effects of the nanocomposites of Examples 1, 2, and 3 of the present invention on the growth of Phaeodactylum tricornutum cells at different concentrations;

[0031] Figure 8This is a graph showing the effects of the nanocomposites of Examples 1, 2, and 3 of the present invention on the chlorophyll a content in the cells of Phaeodactylum tricornutum;

[0032] Figure 9 This is a graph showing the effects of the nanocomplexes of Examples 1, 2, and 3 of the present invention on the carotenoid content in Phaeodactylum tricornutum cells;

[0033] Figure 10 This is a graph showing the effects of the nanocomplexes of Examples 1, 2, and 3 of the present invention on the growth of Phaeodactylum tricornutum cells at different action times;

[0034] Figure 11 This is a graph showing the effects of the nanocomposites of Examples 1, 2, and 3 of the present invention on the malondialdehyde content in Phaeodactylum tricornutum cells;

[0035] Figure 12 This is a graph showing the algae inhibition effect of the nanocomposite of Comparative Example 5 at different concentrations when it is not freeze-dried;

[0036] Figure 13 This is a cell micrograph of the nanocomplex of Example 2 of the present invention acting on Phaeodactylum tricornutum cells;

[0037] Figure 14 This is a SEM image of the nanocomposite of Example 3 of the present invention acting on Phaeodactylum tricornutum cells. DETAILED DESCRIPTION

[0038] In order to help those skilled in the art better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0039] In practical applications, the nanocomposite of the present invention can be added to coatings or directly added to seawater near a target object. When added to coatings, the coating is applied to marine engineering facilities. Due to the composite material's slow-release effect, the active ingredient slowly seeps out of the coating. When algae adhere to the coating surface, the composite material inhibits their normal growth, causing them to die, thereby achieving the purpose of removing marine algae and providing an anti-fouling effect. When added directly to seawater, the dispersed nanoparticles fully contact the algae with the action of the seawater, directly inhibiting their growth and causing them to die, thus achieving an anti-fouling effect.

[0040] Example 1

[0041] A method for preparing a soy protein-thymol nanocomposite comprises the following steps:

[0042] 1) Preprocessing

[0043] Commercially available soybeans with full grains and shiny appearance were selected as raw materials. 250 g were weighed and crushed with a small wall-breaking machine. The obtained powder was sieved with a 40-mesh stainless steel sieve and impurities were removed for later use.

[0044] 2) Low temperature leaching

[0045] Add 20 g of completely crushed soybean powder into a 250 mL beaker, and add 100 mL of petroleum ether. Cover the beaker mouth tightly with plastic wrap, and soak in a constant temperature water bath at 60°C for 50 min to precipitate the oil in the soybeans. After the oil precipitation is complete, centrifuge at 6000 rpm for 10 min, remove the supernatant, collect the precipitate, and dry it in a 55°C oven to obtain defatted soybean powder.

[0046] 3) Enzymatic hydrolysis

[0047] Accurately weigh 10 g of dried defatted soy flour and add it to deionized water at a material-liquid ratio of 1:12. Use 0.1 M HCl solution to adjust the pH of the system to 5.0. Then add cellulase at 0.5% enzyme mass, and enzymolysis is carried out in a constant temperature water bath at 50°C for 1 h. After the enzymolysis is completed, the precipitate is collected by centrifugation at 5000 rpm for 10 min.

[0048] 4) Alkali extraction

[0049] A certain mass of enzymatic hydrolysate was weighed and added to deionized water at a solid-liquid ratio of 1:12. The pH of the system was adjusted to 10 using 0.1 M NaOH. Ultrasonication was used for assisted extraction. After ultrasonication, the solution was allowed to stand for 20 minutes and centrifuged at 8000 rpm for 20 minutes. The supernatant obtained was a clear protein aqueous solution.

[0050] 0.1 M HCl was added dropwise to the supernatant while stirring to adjust the pH to approximately 4.5, allowing the protein to precipitate under isoelectric conditions. At this point, the system was observed to change from a pale yellow, clear liquid to a turbid white liquid. Stirring was stopped when the system reached the isoelectric point, and the solution was allowed to stand at room temperature for 30 minutes to allow the protein particles to settle. Centrifuge at 8000 rpm for 20 minutes, collect the precipitate, and dry it to obtain the crude soy protein isolate (SPI).

[0051] 5) Preparation of soy protein-thymol nanocomplex

[0052] 2.4 g of crude soy protein isolate (SPI) was weighed and added to 50 mL of deionized water. The mixture was stirred on a magnetic stirrer for 1 h. The solution gradually became milky white and evenly dispersed. The mixture was centrifuged at 8000 rpm at room temperature for 15 min. The supernatant was transferred to a new beaker and magnetically stirred for later use. A thymol ethanol solution was then prepared. The thymol ethanol solution was then added dropwise to the supernatant of the SPI at a mass ratio of thymol: SPI = 1:12. After the solution was fully mixed, it was centrifuged again. The supernatant was freeze-dried to obtain a white flocculent soy protein-thymol nanocomposite (SPI-Thymol) powder, named ST1.

[0053] Example 2

[0054] The difference between this example and Example 1 is the mass ratio of thymol and soy protein isolate crude extract SPI when mixed. In this example, the mass ratio of thymol to soy protein isolate crude extract SPI is 2.5:12. The rest of the process is the same as in Example 1. The obtained white flocculent soy protein-thymol nanocomposite is named ST2.

[0055] Example 3

[0056] The difference between this example and Example 1 is the mass ratio of thymol and soy protein isolate crude extract SPI when mixed. In this example, the mass ratio of thymol to soy protein isolate crude extract SPI is 5:12. The rest of the process is the same as in Example 1. The obtained white flocculent soy protein-thymol nanocomposite is named ST3.

[0057] Example 4

[0058] The difference between this embodiment and embodiment 3 lies in the different material-liquid ratio when adding defatted soy flour to deionized water in step 3). In this embodiment, the ratio of defatted soy flour to deionized water is 1:10.

[0059] Example 5

[0060] The difference between this example and Example 3 lies in the different material-liquid ratio when adding defatted soy flour to deionized water in step 3). In this example, the ratio of defatted soy flour to deionized water is 1:14.

[0061] Comparative Example 1

[0062] The difference between this comparative example and Example 3 lies in the different material-liquid ratio when adding the defatted soy flour to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:6.

[0063] Comparative Example 2

[0064] The difference between this comparative example and Example 3 lies in the different material-liquid ratio when adding defatted soy flour to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:8.

[0065] Comparative Example 3

[0066] The difference between this comparative example and Example 3 lies in the different material-liquid ratio when adding defatted soy flour to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:16.

[0067] Comparative Example 4

[0068] The difference between this comparative example and Example 3 lies in the different material-liquid ratio when adding the defatted soy flour to deionized water in step 3). In this comparative example, the ratio of defatted soy flour to deionized water is 1:18.

[0069] Comparative Example 5

[0070] The difference between this comparative example and Example 3 lies in the freeze-drying treatment in step 5). In this comparative example, the freeze-drying treatment was not performed. The thymol ethanol solution was added to the supernatant of the soy protein isolate crude extract SPI and blended. The resulting soy protein-thymol nanocomposite solution was obtained, and the soy protein-thymol nanoparticles existed in the form of nanoparticles in the aqueous solution.

[0071] Test Example 1

[0072] 1. Effect of different feed-liquid ratios (defatted soy flour: deionized water) on the extraction rate of soy protein isolate

[0073] The protein content of the precipitates obtained after enzymatic hydrolysis in step 3) in Examples 3, 4, 5, Comparative Examples 1, 2, 3, and 4 was determined using the Folin-phenol method. The protein concentration of the samples was then calculated using a standard curve, and the extraction yield was then calculated. The soy protein extraction yield results are shown in Table 1.

[0074] Table 1 Extraction rate of soy protein isolate at different material-liquid ratios

[0075]

[0076] The contents in the above table show that the material-liquid ratio has a significant effect on the extraction rate of isolated protein after enzymatic hydrolysis. As the material-liquid ratio decreases, the extraction rate increases rapidly. More water can dissolve more protein. However, when the amount of water added exceeds a certain ratio, the extraction rate will decrease. Therefore, the material-liquid ratio can only improve the extraction rate of soy protein isolate within a certain range. When too little or too much water is added, the extraction rate of soy protein isolate will be reduced.

[0077] 2. Particle size analysis of thymol-soy protein isolate SPI nanocomplex

[0078] 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 were respectively weighed and dissolved in 10 mL of deionized water, and ultrasonicated at a power of 200 W at room temperature for 10 min to uniformly disperse the mixture solution in the system to obtain soy protein isolate SPI solution and three thymol-soy protein isolate SPI nanoparticle solutions. The particle size of the soy protein isolate SPI solution and the three composite nanoparticle solutions in different proportions were analyzed using a Zeta potential meter.

[0079] The results are as follows Figure 1 As shown, in Figure 1 In the curve, 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.

[0080] The particle size data of the Zeta potential instrument show that pure SPI exists in the form of nanoparticles with a particle size of several nanometers when dissolved in water. Because of this size effect, when SPI is mixed with Thymol (thymol), it can effectively complex with Thymol and form stable nanoparticles.

[0081] Depend on Figure 1 As shown in Figure A, when Thymol and SPI are compounded at a mass ratio of 1:12 (ST1), the particle size is mainly distributed around 10 nm, which is higher than that in the pure SPI dispersion solution. Figure 1 As shown in Figure B, when Thymol and SPI are compounded at a mass ratio of 2.5:12, the particle size increases significantly to about 17 nm, indicating that adding Thymol helps improve the compounding efficiency in the mixed solution and is conducive to the formation of composite nanoparticles. Figure 1 As shown in Figure C, when the mass ratio increases to 5:12, the complexation efficiency of Thymol no longer increases significantly, and the particle size only increases by 5% when twice the mass of Thymol is added, indicating that there is a certain upper limit for the complexation of Thymol with SPI.

[0082] 3. FTIR analysis of thymol-soy protein isolate SPI nanocomplex

[0083] The interaction between Thymol and SPI is key to maintaining the structural stability of composite nanoparticles. Thymol contains C=C, CO, and -CH3 groups. Commercially available Thymol (thymol), the soy protein isolate SPI prepared in Example 2, and the nanocomposite (ST1) prepared in Example 2 were placed in an agate mortar, and an appropriate amount of analytically pure anhydrous potassium bromide reagent was added. The mixture was then ground into a fine powder. After the powder was fully ground, a small amount of the mixed solid powder was evenly applied to the inside of a tablet press. Thin slices were then prepared in the tablet press for infrared testing. The results are shown in Figure 2. Figure 2 As shown, in Figure 2 In FIG, the SPI-Thymol curve represents the nanocomposite of Example 2.

[0084] from Figure 2 It can be seen that at 1620 cm -1 The antisymmetric deformation of -CH3 is at 1584 cm -1 The stretching vibration of C=C is 1285 cm -1 is the bending deformation of -CH3, 1156 cm -1 CO stretching vibration at 1655, 1542, and 1236 cm -1 There are three obvious characteristic peaks at 1657, 1547, and 1238 cm -1 The infrared absorption intensity at 855 and 805 cm-1 increases, indicating that some characteristics of thymol are superimposed on the characteristic peaks of SPI. -1 There are obvious characteristic peaks of thymol at 1600 to 1000 cm-1, while SPI has no characteristic peaks at these two locations. -1 In the range, after thymol was superimposed on SPI, the characteristic peaks of the mixture showed an enhanced absorption phenomenon, and the shift of some characteristic peaks may be due to the combined effects of hydrogen bonding or molecular conformational changes.

[0085] The spectra of SPI-Thymol and SPI are quite 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.

[0086] 4. X-ray diffraction (XRD) analysis of thymol-soy protein isolate SPI nanocomposite

[0087] 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, with a scanning rate of 0.02° / min in the 2θ region from 5° to 60°. The results are shown in Figure 2. Figure 3 shown.

[0088] 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 material prepared in Example 3 has a polycrystalline structure and good crystallinity.

[0089] 5. Microscopic morphological structure analysis of thymol-soy protein isolate SPI nanocomplex

[0090] The morphological characteristics of the nanocomposites prepared from thymol and soy protein isolate SPI at different mass ratios (1:12, 2.5:12, 5:12) were investigated as follows:

[0091] The nanocomposites 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 uniformly dissolved in deionized water to obtain mixed solutions containing nanocomposite particles. The mixed solutions were ultrasonically washed and centrifuged multiple times, and the supernatant was observed to be slightly light yellow and transparent. The supernatant was fully collected and solidified in a -80°C freezer overnight. The mixed solution was immediately placed in a freeze dryer in the solidified state for low-temperature freeze drying. After the water was fully evaporated, a small amount of powder was taken for field emission scanning electron microscopy (SEM, Mira3Tescan) morphology observation. The results are as follows: Figure 4 shown. Figure 4 From left to right in the middle are the microscopic structures under different magnifications (2 μm, 500 nm, 400 nm).

[0092] from Figure 4 It can be seen that under low magnification, the surface structure of the nanocomposites with different mass ratios is relatively compact and dense, and under high magnification, it can be clearly seen that the nanocomposites with different mass ratios have better morphology, indicating that the nanocomposites have better stability.

[0093] VI. Analysis of the composite efficiency of different concentrations of thymol and soy protein isolate SPI

[0094] A certain amount of the nanocomposites of Example 1, Example 2, and Example 3 were placed in a 10 mL glass bottle. The thymol in the complexes was extracted using the organic solvent n-hexane. The absorbance at 275 nm was measured using an ultraviolet-visible spectrophotometer (UV-1800). The specific steps are as follows:

[0095] 3 mL of n-hexane was added to 0.2 mL of the dispersion containing the nanocomplex and diluted with deionized water at a ratio of 1:10 (v / v) before extraction. The mixture was thoroughly mixed and allowed to stand at room temperature for 0.5 h. The separation of n-hexane and water was clearly observed. 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 n-hexane was measured at 275 nm. In addition, the specific content of thymol was calculated based on the standard curve of standard thymol in n-hexane (R2 = 0.995). The results are shown in Figure 2. Figure 5 shown. Figure 5 Figure A is a UV scan of thymol; Figure B is a graph showing the relationship between thymol concentration and OD quantity; Figure C is the loading rate of nanocomplexes with different mass ratios (thymol: soy protein isolate).

[0096] from Figure 5 As shown in Figure C, when thymol and soy protein isolate are compounded at a mass ratio of 1:12, the ultraviolet absorbance test shows that the compounding efficiency is 4.76%; when compounded at a mass ratio of 2.5:12, the compounding efficiency is 9.35%; when compounded at a mass ratio of 5:12, the compounding efficiency is 10.18%; as the proportion of thymol increases, the compounding efficiency continues to increase. It can be found that when thymol is compounded at a low mass ratio, its compounding efficiency increases approximately proportionally, from 4.76% to 9.35%. However, when the proportion of thymol in the mixture continues to increase exponentially, its compounding efficiency does not increase linearly. Therefore, the compounding efficiency of the crude extraction product soy protein isolate with thymol is limited.

[0097] VII. Analysis of thymol release from thymol-soy protein isolate SPI nanocomplex

[0098] In order to test the release of thymol from the nanocomplex in artificial seawater, free thymol was used as a control, and the release of thymol from the free thymol and the nanocomplexes of Example 1, Example 2, and Example 3 was continuously measured within 24 hours. The specific steps are as follows:

[0099] The nanocomplexes prepared in Example 1, Example 2, and Example 3 were added to artificial seawater (f / 2 culture medium) at a concentration of 10 mg / L, shaken, and placed statically at room temperature. 3 mL of the solution was taken at 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, and the absorbance (275 nm) was measured using an ultraviolet spectrophotometer. The release process was calculated and simulated using the kinetic model of the following formula (1), where: Ct i for t i The cumulative amount of thymol released at any moment, a i for t i Thymol sampling concentration at time m 0 is the total mass.

[0100]

[0101] The results are as follows Figure 6 As shown, the release rate of free thymol was faster than that of the nanocomposite. Within the first four hours, the amount of free thymol released gradually increased and was significantly higher than that of the composite nanoparticles. After one hour, it was approximately 1.65 times that of the latter. After five hours, the free thymol release time began to stabilize and increase slightly, and remained at around 80% after 24 hours.

[0102] The release profiles of thymol from the three nanocomposites were similar to those of the free form, with rapid release during the first four hours and then reaching a steady state. ST1 released approximately 40% of its thymol content after 1 hour and maintained a stable level of approximately 80% after 24 hours. ST2 and ST3 released approximately 38% of its thymol content after 1 hour, followed by a slow, sustained release after 24 hours, ultimately reaching approximately 68%. This suggests that most thymol bound to the hydrophobic structures on the SPI surface through weak hydrophobic interactions, while some was tightly encapsulated in the core of the complex, preventing it from dissolving in n-hexane. The release rate after 24 hours decreased significantly with increasing thymol mass fraction (p < 0.05), indicating that at higher thymol fractions, thymol formed a more stable anodic complex with SPI, resulting in a more stable, compact, and dense nanoparticle structure and improved sustained-release performance.

[0103] Test Example 2: Algae Growth Inhibition

[0104] The experiment was conducted using algal cells in the logarithmic growth phase. The P. tricornutum solution was added to a glass conical flask and diluted with f / 2 culture medium to a final concentration of 3.5 × 107 pcs / mL, simulating the algae density when diatoms attach and grow to form a surface biofilm, and set aside.

[0105] 1. Inhibitory effects of different concentrations of thymol-soy protein isolate SPI nanocomplexes on P. tricornutum cells

[0106] The inhibitory effects of the thymol-soy protein isolate SPI nanocomplexes of Example 1, Example 2, and Example 3 on the cells of P. tricornutum at different concentrations were investigated, comprising the following steps: (1) the nanocomplex (ST1) prepared in Example 1 (thymol: soy protein isolate = 1:12) was added to the prepared algal solution, and algal solutions containing nanocomplexes at 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 solution containing different nanocomplexes was 25 mL. After preparation, the growth state of P. tricornutum was recorded by absorbance (OD = 680 nm), and then compared with the growth state of P. tricornutum in the algal solution without the addition of the nanocomplex. The results are as follows: Figure 7 As shown in Figure A and Figure A', Figure A shows the effect of ST1 on algal cell growth; Figure A' shows the inhibitory effect of different concentrations of ST1 on algal cell growth at 48 h.

[0107] Depend on Figure 7 As shown in Figures A and A', as the concentration of the nanocomposite increases in the system, 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, the growth of algal cells can be completely inhibited within 48 h. As the concentration of the nanocomposite continues to increase, a large number of P. tricornutum die.

[0108] (2) The nanocomposite (ST2) prepared in Example 2 (thymol: soy protein isolate = 2.5:12) was added to the prepared algae solution to prepare algae 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 algae solution containing different nanocomposites was 25 mL. After preparation, the growth status of the triangular spherical algae was recorded by absorbance (OD = 680 nm), and then compared with the growth status of the triangular spherical algae in the algae solution without the addition of the nanocomposite. The results are as follows: Figure 7 As shown in Figures B and B', Figure B shows the effect of ST2 on algal cell growth; Figure B' shows the inhibitory effect of different concentrations of ST2 on algal cell growth at 72 h.

[0109] Depend on Figure 7 As shown in Figures B and B', when the concentration of the nanocomposite reached 3 mg / L, the number of algal cells began to decrease after 72 h, and the composite material achieved 100% inhibition on algal cells.

[0110] (3) The nanocomposite (ST3) prepared in Example 3 (thymol: soy protein isolate = 5:12) was added to the prepared algae solution to prepare algae 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 algae solution containing different nanocomposites was 25 mL. After preparation, the growth status of the triangular algae was recorded by absorbance (OD = 680 nm), and then compared with the growth status of the triangular algae in the algae solution without the addition of the nanocomposite. The results are as follows: Figure 7 As shown in Figure C and Figure C', Figure C shows the effect of ST3 on algal cell growth; Figure C' shows the inhibitory effect of different concentrations of ST3 on algal cell growth at 72 h.

[0111] Depend on Figure 7 As shown in Figure C, the nanocomposite concentration of 2 mg / L achieved 100% inhibition on algal cells at 48 h, and the growth inhibition on the triangular algae cells was concentration-dependent. In the following period, the algal cells began to die in large numbers and white precipitates appeared. Figure 7 As shown in Figure C', when the concentration was 1.6 mg / L, the nanocomplex achieved 100% inhibition on the cells of Phaeodactylum tricornutum, with an IC50 of 0.83 mg / L.

[0112] 2. Effects of thymol-soy protein isolate SPI nanocomplex on the content of photosynthetic pigments (chlorophyll a and carotenoids) in P. tricornutum cells

[0113] The effects of the thymol-soy protein isolate SPI nanocomplexes prepared in Example 1, Example 2, and Example 3 on the content of photosynthetic pigments (chlorophyll a and carotenoids) in Phaeodactylum tricornutum cells were investigated respectively. The specific steps were as follows:

[0114] The nanocomposites prepared in Example 1 (ST1), Example 2 (ST2), and Example 3 (ST3) were respectively added to the algae solution. The concentrations of the nanocomposites of Example 1, Example 2, and Example 3 in the algae solution were half inhibitory concentrations, that is, the concentration of the nanocomposites of Example 1 in the algae solution was 2 mg / L, the concentration of the nanocomposites of Example 2 in the algae solution was 1.3 mg / L, and the concentration of the nanocomposites of Example 3 in the algae solution was 1.6 mg / L. After adding the nanocomposites of different examples to the algae solution, the volumes of the nanocomposites, f / 2 culture solution, and algae solution finally obtained were 25 mL.

[0115] After addition, 3 mL of algae solution was drawn into the corresponding conical flask at 0 h, 24 h, 48 h, 72 h, and 96 h respectively, and centrifuged at 7000 rpm and 4 ° C for 10 min. The precipitate was retained and 3 mL of acetone (90%) was added to it for re-suspending. After vortexing evenly, it was placed in a 4 ° C refrigerator for extraction under dark conditions for 24 h. After the dark extraction was completed, the supernatant was retained after centrifugation under the same conditions. The absorbance was then measured using an ultraviolet spectrophotometer at wavelengths of 440 nm, 644 nm, 630 nm, 647 nm, 664 nm, 662 nm and 750 nm. The blank control was to add 90% acetone solution to the algae solution, and the concentrations of chlorophyll a (Chla) and carotenoids (Car) were calculated using the following formulas (1) and (2) respectively, and the change trend graph was drawn. The results are shown in Figure 2. Figure 8 and Figure 9 shown. Figure 8 Figures A, B, and C show the effects of nanocomposite particles ST1, ST2, and ST3 on the chlorophyll a content in the cells of Phaeodactylum tricornutum. Figure 9 This is the effect of nanocomposite particles ST1, ST2, and ST3 on the carotenoid content in Phaeodactylum tricornutum cells.

[0116] Figure 8 Figure A shows the effect of Example 1 (ST1) on the chlorophyll a content of P. triangularis; Figure B shows the effect of Example 2 (ST2) on the chlorophyll a content of P. triangularis; and Figure C shows the effect of Example 3 (ST3) on the chlorophyll a content of P. triangularis.

[0117]

[0118]

[0119] from Figure 8It can be seen that the chlorophyll a content of the blank control group of P. tricornutum cells increased from 1.12 mg / L at the beginning to 1.89 mg / L, an increase of 68.75%. The chlorophyll a content of the control group began to increase significantly from the third day. This is because there are sufficient nutrients in the culture medium and suitable light conditions and temperature to accelerate the growth of algae cells. The chlorophyll a content in the ST1 experimental group decreased from 1.15 mg / L to 0.41 mg / L after 96 hours, a decrease of 64.35%, and continued to decrease; the chlorophyll a content in the ST2 experimental group decreased from 1.13 mg / L to 0.29 mg / L after 96 hours, a decrease of 74.34%, and showed a trend of continued decrease; the chlorophyll a content in the ST3 experimental group decreased from 1.14 mg / L to 0.09 mg / L after 96 hours, a decrease of 91.40%, and showed a trend of continued decrease; after the addition of the nanocomplex, the chlorophyll a content in the three experimental groups showed a significant decrease, and the addition of ST3 had the greatest effect 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).

[0120] Chlorophyll a is the main photosynthetic pigment in the cells of P. tricornutum. Due to the presence of long-chain alkyl groups and magnesium ions, chlorophyll a absorbs light energy and converts it into chemical energy. The addition of thymol-soy protein isolate SPI nanocomposite leads to a significant reduction in chlorophyll a. Therefore, the nanocomposite particles generated by loading thymol on soy protein isolate SPI have an inhibitory effect on the growth of algal cells.

[0121] from Figure 9 As shown in the data, the carotenoid content in the blank control group increased from 0.18 mg / L within 96 hours of natural growth to 0.82 mg / L after 96 hours. In a suitable growth environment, P. tricornutum cells rapidly grew and multiplied, resulting in a significant increase in carotenoid content within a short period of time. Conversely, after the addition of composite nanoparticles, the ST1 and ST2 experimental groups experienced a slight increase in carotenoid content in the early stages of the experiment. This may be due to the short-term stimulation of cell growth by the addition of a small amount of nanocomposite. However, starting on the second day, the carotenoid content in all three nanocomposite treatments began to decline sharply, reaching almost zero after 96 hours. This indicates that the vast majority of algal cells have lost their basic physiological functions or physiological activity.

[0122] Carotenoids, a class of unsaturated fatty acids with conjugated double bonds, play a crucial role in cell growth and reproduction. They participate in all stages of photosynthesis, acting as photosensitizers or signaling molecules. A significant reduction in their content will inevitably severely impact the normal physiological activities of algal cells. Therefore, the addition of nanocomposite particles can lead to a sharp decrease in carotenoid content, inhibiting algal cell growth and potentially causing cell death.

[0123] 3. Effect of thymol-soy protein isolate SPI nanocomplex on algae inhibition at different action times

[0124] The growth inhibition effects of the thymol-soy protein isolate SPI nanocomplexes prepared in Example 1, Example 2, and Example 3 on the Phaeodactylum tricornutum cells were investigated over time. The specific steps are as follows:

[0125] The nanocomplexes prepared in Examples 1, 2, and 3 were added to the algae solution, and experiments were conducted at a concentration that reached a half-maximal inhibitory effect on the cells within 72 hours. These groups were ST1, ST2, and ST3, respectively. The algae solution without the addition of the nanocomplexes and normal growth was used as a blank control group. The results are shown in Figure 2. Figure 10 shown.

[0126] Depend on Figure 10 It can be seen that the blank control group, with the increase of time, the triangular algae cells continue to grow, after 5 days, the absorbance value of the algae cells exceeds 1, compared with the blank control group, the treatment group began to show an upward trend from the second day, and began to decline on the fourth day. Figure 10 It can be seen that in different treatment groups, the higher the proportion of thymol, the more obvious the inhibitory effect of the nanocomplex on the growth of algal cells. After the treatment time reaches 4 days, due to the inhibitory effect of the nanocomplex on the growth of algal cells, the treatment group ST3 reaches 100% inhibition on the triangular brown algae, and begins to have a destructive effect on the cells in the following period, and a large number of algal cells die.

[0127] Effect of thymol-soy protein isolate SPI nanocomplex on malondialdehyde (MDA) content in algal cells

[0128] The effects of the nanocomposites of Examples 1, 2 and 3 on the malondialdehyde (MDA) content of algal cells were investigated. The nanocomposites prepared in Examples 1, 2 and 3 were added to algal liquid. The changes in the MDA content of algal cells were continuously measured within 96 hours, and the activity change trends were plotted. The results were compared with those of the algal liquid control group without the addition of the nanocomposites. Figure 11 As shown. Figure 11, Figure A represents the effect of Example 1 (ST1) on the change of MDA content in algae cells; Figure B represents the effect of Example 2 (ST2) on the change of MDA content in algae cells; and Figure C represents the effect of Example 3 (ST3) on the change of MDA content in algae cells.

[0129] Depend on Figure 11 As shown in the data, MDA content in the control group remained essentially unchanged at around 0.01 mol / g. However, after adding the nanocomposite, MDA content increased significantly within 24 hours (p < 0.05). The addition of thymol caused the algal cells to produce a large amount of reactive oxygen species, leading to an excess of reactive oxygen species, which ultimately caused severe damage and inhibited normal algal growth. MDA content in the ST2 and ST3 groups gradually decreased after 24 hours, reaching around 0.02 mol / g after 96 hours. MDA content in the ST1 group began to decline after 48 hours. Ultimately, MDA content in all three treatment groups converged, but remained higher than that in the control group.

[0130] V. Effect of Freeze-Drying and Non-Freeze-Drying on the Anti-Algae Effect of Thymol-Soy Protein Isolate SPI Nanocomplex The soy protein-thymol nanocomplex solution prepared in Comparative Example 5 was directly added to algal cells to study its growth inhibitory effect on P. tricornutum cells. The details are as follows:

[0131] 1, 3, 5, 8, and 10 mL of the nanocomposite solution prepared in Comparative Example 5 were added to the triangular brown algae solution to finally form a 30 mL algae solution system, thereby forming a series of nanoparticle solution gradients.

[0132] Its effect on the growth of algal cells is as follows Figure 12 As shown in the figure, with the increase of the amount of nanoparticle solution added, its inhibitory effect on algal cells increased significantly. When more than 8 mL was added, the inhibitory effect on algal cells reached 100% after 72 h.

[0133] Compared to the direct addition of the lyophilized nanocomposite (SPI & Thymol) to the algal cell system, a total volume of 8 mL of nanoparticle solution was required to achieve complete inhibition of algal cell growth after 72 hours. Since the original thymol and soy protein isolate were combined at a mass ratio of 5:12 and 40 mL of deionized solution were added, the mass of thymol in the 8 mL mixed nanoparticle solution was 3.33 mg / L. As shown above, in the preparation of the ST3 nanocomposite, only a concentration of 1.6 mg / L of the lyophilized nanocomposite material was required to achieve complete inhibition of P. tricornutum cells after 72 hours. Therefore, compared to the direct addition of the nanoparticle mixture to the algal cells, the lyophilized nanoparticle powder increased the inhibitory efficiency of algal cells by 52%.

[0134] VI. Microscopic observation of the inhibition of algal cells by thymol-soy protein isolate SPI nanocomplex

[0135] Prepare three samples containing 3 × 10 algal cells. 7 The algae solution in two conical flasks with a concentration of 1 μg / mL (OD = 1.7) was added with the nanocomplex prepared in Example 1 to make the concentrations of 1 μg / mL and 4 μg / mL, respectively. Then, 10 μL of algae solution was taken from each flask and slowly dripped onto the coverslip. After standing for 1 minute, the morphology of the algae cells was observed under a 50x objective lens using an inverted electron microscope. The results are shown in Figure 2. Figure 13 As shown. Figure 13 In the figure, Panel A represents the microscopic morphology of algal cells without the addition of nanocomplexes; Panel B represents the microscopic morphology of algal cells after the addition of nanocomplexes at a concentration of 1 μg / mL; Panel C represents the microscopic morphology of algal cells after the addition of nanocomplexes at a concentration of 4 μg / mL.

[0136] from Figure 13 As shown in Figure A, the algae cells without the addition of nanocomplexes were not damaged, the particles were full and had a radial triangular shape, and were relatively uniform, and single algae cells could be observed dispersed in the culture medium.

[0137] from Figure 13 As shown in Figures B and C, after the addition of the nanocomplex, algal cells began to aggregate in large numbers, and the aggregation trend became more pronounced as the nanocomplex concentration increased. This is because the addition of the nanocomplex causes growth stress to the algal cells in the system, and the nanocomplex drives them away, leading to bioaggregation. This suggests that the addition of the nanocomplex can severely impact the normal growth of algal cells, preventing them from fully absorbing nutrients and receiving sufficient light, ultimately inhibiting their growth.

[0138] VII. SEM Observation of Thymol-Soybean Protein Isolate SPI Nanocomplex Inhibiting Algal Cells

[0139] In order to observe the effect of the nanocomplex on the microstructure of the cells of P. tricornutum, the nanocomplex prepared in Example 3 was added to the algae solution. The concentration of the nanocomplex in the algae solution was 1.6 mg / L. After 72 h, 5 mL of algae solution was evenly aspirated 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, vortexed and placed in a 4°C refrigerator for dark treatment for 24 h. The sample was washed with 0.1 M, pH = 7.0 phosphate buffer for purification. Finally, the sample was dehydrated for 10 min using ethanol solutions of 30%, 50%, 80%, and 90% and anhydrous ethanol, respectively. After completing the above steps, the treated samples were placed in a -80°C refrigerator to freeze the samples, and the cell microstructure was observed under a scanning electron microscope. The results were compared with those of the algae solution control group without the addition of the nanocomplex. Figure 14 shown.

[0140] from Figure 14 As shown in Figure A, the algal cell structure of the control group is complete and full, and presents a spindle-shaped morphology, and the structure of a single algal cell can be clearly observed; Figure 14 As shown in Figure B, after adding the nanocomplex, the algae cells collapsed and broke, and it was difficult to find intact algae cells under a high-power microscope. Figure 14 As shown in Figure C, the addition of the nanocomposite caused the algal cell walls to rupture, releasing intracellular substances that mixed with the broken algal cells. This indicates that the addition of the nanocomposite caused irreversible rupture of the algal cells and that the nanocomposite had a strong biological and chemical inhibitory effect on P. tricornutum cells.

[0141] Although the present invention has been described using the above preferred embodiments, they are not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. Application of soy protein-thymol nanocomposite in removing marine fouling algae, wherein the algae is Phaeodactylum tricornutum; The preparation method of the soy protein-thymol nanocomposite comprises the following steps: (1) Preparation of soy protein solution The soybean powder obtained by crushing soybeans is defatted; the defatted soybean powder is enzymatically hydrolyzed; the enzymatic hydrolysis product is dissolved in deionized water at a material-liquid ratio of 1:(10-14), the pH is adjusted to 9.0-10.0, ultrasonic extraction is performed, and then centrifugation is performed; the supernatant obtained after centrifugation is adjusted to pH 4.0-5.0 and stirred, and after standing, centrifugation, collection, and drying, a crude soybean protein extract is obtained; the crude soybean protein extract is dissolved in deionized water and stirred uniformly, and the dispersion obtained after centrifugation is a soybean protein solution; in, The enzymatic hydrolysis product is prepared by dissolving defatted soybean flour in deionized water at a material-liquid ratio of 1:(10-14), adjusting the pH to 4.0-6.0, adding cellulase, performing enzymatic hydrolysis at a constant temperature of 45-55°C for 1-2 hours, collecting the precipitate after centrifugation, and obtaining the soybean enzymatic hydrolysis product. (2) Preparation of soy protein-thymol nanocomplex A thymol solution dissolved in anhydrous ethanol was added dropwise to a soy protein solution, thoroughly mixed and stirred, and centrifuged. The obtained dispersion was freeze-dried and pulverized to obtain a soy protein-thymol nanocomposite powder, wherein the mass ratio of thymol to soy protein was (1-5):

12.

2. The use according to claim 1, characterized in that In the step (1), the defatted soy flour is prepared by mixing the soy flour with petroleum ether at a material-liquid ratio of 1:5, extracting the soy flour for 45-60 minutes at a constant temperature of 55-65°C to separate the oil, and obtaining the defatted soy flour after centrifugation, collection and drying.

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