Compound with food whitening effect as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a compound with a food whitening effect, which comprises the following steps: uniformly mixing an inulin solution and a titanium dioxide suspension, and mixing and incubating at room temperature for 20-24 hours; after the incubation is finished, centrifuging to remove supernate and precipitate, namely a titanium dioxide-inulin compound, washing the titanium dioxide-inulin compound with distilled water, removing uncombined inulin, and collecting the precipitate; and finally, carrying out vacuum freeze drying on the sample to obtain the titanium dioxide-inulin compound. The compound is stable in structure and good in biocompatibility, has the same whitening effect as titanium dioxide, has the potential of replacing titanium dioxide in food additives, and has important application prospects in the aspect of preparing low-toxicity food whitening agents.

Description

Technical Field

[0001] This invention relates to the field of materials, specifically to a compound with food whitening effect, its preparation method, and its application. Background Technology

[0002] Traditionally, titanium dioxide is a white pigment added to many foods and daily necessities, such as food additive E171, toothpaste, and pharmaceuticals. In the food industry, titanium dioxide is primarily used as a food additive. The cumulative exposure to titanium dioxide in the human diet can reach 0.06-2 mg per kilogram of body weight per day. In China, titanium dioxide can be used as a food coloring agent, with a maximum dosage of 10 g / kg, and can also be used in solid beverages according to production requirements (National Standard GB25577-2010). Titanium dioxide nanoparticles have many advantages in food applications, such as better taste, high refractive index, strong hiding power, and good whiteness. Furthermore, the photocatalytic activity of titanium dioxide nanoparticles gives them good antibacterial properties when used in food packaging. Due to these significant advantages, food-grade titanium dioxide nanoparticles are widely used as food additives to improve the optical properties of food. However, due to their small size, nanoparticles are more easily taken up by cells, participating in processes such as promoting inflammatory responses and altering cell signal transduction, leading to cytotoxicity, epigenetic changes, genotoxicity, and immunotoxicity. Studies have shown that orally administered titanium dioxide nanoparticles can trigger the secretion of pro-inflammatory cytokines through NLRP3 inflammasome activation, leading to their accumulation in human intestinal epithelial cells and macrophages, generating reactive oxygen species, and thereby increasing epithelial permeability and impairing the intestinal barrier. This suggests that titanium dioxide nanoparticles may pose a specific risk to patients with inflammatory bowel disease. Simultaneously, research has revealed that orally administered titanium dioxide nanoparticles induce colonic mucus layer disruption and microbiome dysbiosis, demonstrating their damage to the mucus layer through downregulation of the Muc2 gene.

[0003] Currently, numerous studies have shown that nano-titanium dioxide particles, used as a whitening agent in food, can cause various diseases, including bronchitis, lung cancer, and atherosclerosis. Nano-titanium dioxide particles in the gastrointestinal tract are linked to Crohn's disease and colon cancer. Therefore, it is crucial to address the safety of nano-titanium dioxide added to food, and there is an urgent need for a food additive that is biocompatible and possesses the whitening effect of nano-titanium dioxide.

[0004] Studies show that nanoparticles can adsorb biomolecules onto their surfaces to form complexes, which affect the interaction between nanoparticles and organisms, altering the bioabsorption, distribution, and toxicity of nanoparticles. Research has found that these complexes can, to some extent, mitigate the decline in cell viability caused by nanoparticles and prevent mitochondrial morphological abnormalities and functional disorders. Therefore, the damaging effects of titanium dioxide nanoparticles can be alleviated by addressing the issue from the perspective of complex formation. Summary of the Invention

[0005] Based on the aforementioned problems, there is an urgent need in this field to develop a food whitening agent with good whitening effect, high stability, and good biosafety to effectively replace titanium dioxide whitening agents. By preparing this novel composite, the biosafety issues of nano-titanium dioxide can be solved while ensuring its whitening effect, providing an innovative new solution for the field of food additives.

[0006] The purpose of this invention is to provide a food whitening agent with good whitening effect, high stability and good biosafety to effectively replace titanium dioxide whitening agent.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a compound with a food whitening effect, the method comprising the following steps:

[0009] (1) Mix the inulin solution and titanium dioxide suspension evenly and incubate at room temperature for 20-24 hours;

[0010] (2) After the above incubation is completed, the supernatant is discarded by centrifugation and the precipitate is the titanium dioxide-inulin complex. It is washed with distilled water, the unbound inulin is discarded, and the precipitate is collected. Finally, the sample is freeze-dried under vacuum to obtain the titanium dioxide-inulin complex.

[0011] In the above preparation method, the mass-volume concentration of inulin solution in step (1) is 5~15 mg / mL, and the mass-volume concentration of titanium dioxide suspension is 5~15 mg / mL.

[0012] In the above preparation method, the mass-volume concentration of inulin solution in step (1) is 5~15 mg / mL, and the mass-volume concentration of titanium dioxide suspension is 5~15 mg / mL.

[0013] In the above preparation method, the volume ratio of inulin solution and titanium dioxide suspension in step (1) is 1~5:1~5.

[0014] In the above preparation method, the temperature of mixing and incubation in step (1) is 15~40℃, and the mixing and incubation is carried out under shaking conditions with a shaking speed of 180~220rpm.

[0015] In the above preparation method, the vacuum degree of vacuum freeze drying in step (2) is 0.08~0.133MPa and the temperature is -85~-80℃.

[0016] A compound with food whitening effect, which is prepared by the above method.

[0017] In the technical solution of this invention, the compound with food whitening effect is used as a food whitening agent.

[0018] Brightness measurement of nano-titanium dioxide-inulin composite:

[0019] The L, a, and b values ​​of titanium dioxide and titanium dioxide-inulin complex were determined using a colorimeter.

[0020] Structure and stability determination of nano-titanium dioxide-inulin complex: The structure and stability of uncoated and coated titanium dioxide nanoparticles in the gastrointestinal tract were investigated using an in vitro digestion model.

[0021] (1) Prepare simulated saliva, simulated gastric juice and simulated small intestinal juice respectively;

[0022] (2) The mixture of titanium dioxide-inulin complex and simulated saliva was placed in a constant temperature shaking box at 37°C for 15 min, and the sample was taken out and inactivated in a water bath at 100°C for 8 min.

[0023] (3) The mixture of the above solution and simulated gastric juice was placed in a constant temperature shaking box at 37°C for 2 hours, and the sample was taken out and inactivated in a water bath at 100°C for 8 minutes.

[0024] (4) Place the mixture of the above solution and simulated small intestinal fluid in a 37°C constant temperature shaking box for 3 hours, and then take out the sample and inactivate it in a 100°C water bath for 8 minutes.

[0025] In this technical solution, during the digestion process in step (2), the pH is maintained at 7.0; during the digestion process in step (3), the pH is maintained at 2.0; and during the digestion process in step (4), the pH is maintained at 7.5.

[0026] In this technical solution, the structural morphology of the nano-titanium dioxide-inulin composite was determined using transmission electron microscopy (TEM).

[0027] In this technical solution, the structural morphology of the nano-titanium dioxide-inulin composite was investigated by scanning electron microscopy (SEM).

[0028] In this technical solution, the potential changes of the titanium dioxide-inulin complex during in vitro simulated digestion were investigated by measuring the zeta potential of the above samples.

[0029] Biocompatibility determination of nano-titanium dioxide-inulin complex:

[0030] Three cell models—Raw264.7, Caco-2, and HT-29—were established to investigate the effects of nano-titanium dioxide-inulin complex on cell viability. The effects of lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) expression, and malondialdehyde (MDA) levels on cell membrane integrity and oxidative stress were investigated by detecting these parameters.

[0031] An acute DSS (sodium dextran sulfate) mouse model was established to investigate the effects of titanium dioxide and titanium dioxide-inulin complex on mouse body weight, disease activity index, colon length, colonic lipid peroxidation, colonic H&E staining, and gut microbiota.

[0032] In this technical solution, an acute DSS model is established, and mice are allowed free access to 2.5% DSS water.

[0033] This application discloses a method for preparing a food whitening agent (nano titanium dioxide-inulin complex) with good whitening effect, high stability and good biosafety, which can effectively maintain the whitening effect of nano titanium dioxide in food.

[0034] Based on the carbon dioxide-inulin complex prepared above, its structural stability was confirmed by establishing an in vitro simulated digestion model.

[0035] Based on the carbon dioxide-inulin complex prepared above, its biocompatibility was investigated by establishing cell and animal models.

[0036] Beneficial effects:

[0037] This invention discloses a titanium dioxide-inulin composite. The preparation method of this composite is simple, its brightness is close to that of titanium dioxide nanoparticles, and it exhibits good stability. Furthermore, it can improve the cell viability reduction, cell membrane damage, and oxidative stress caused by titanium dioxide, demonstrating good biocompatibility. Simultaneously, compared to titanium dioxide, administration of this composite to a mouse model of acute colitis induced by dextran sulfate sodium (DSS) resulted in improved mouse weight, increased colon length, and reduced inflammatory cell infiltration in the colon, thus improving the inflammatory phenotype of the colon. It also restored the homeostasis of the intestinal flora in mice and increased the abundance of beneficial bacteria. This composite has a stable structure, good biocompatibility, and the same whitening effect as titanium dioxide, showing potential to replace titanium dioxide in food additives and possessing significant application prospects in the preparation of low-toxicity food whitening agents. Attached Figure Description

[0038] Table 1. Brightness analysis of titanium dioxide and titanium dioxide-inulin composite.

[0039] Figure 1An in vitro simulated digestion model is shown in Figure a, which illustrates the establishment of the in vitro simulated digestion method. Figure b shows transmission electron microscopy (SEM) and scanning electron microscopy (SEM) images of simulated saliva (SSF), simulated gastric juice (SGF), and simulated small intestinal juice (SIF). Figure c shows the zeta point map at different digestion stages.

[0040] Figure 2 The effects of different concentrations of titanium dioxide, inulin, and titanium dioxide-inulin complex on cell viability are shown in Figure a, which presents the experimental results of Raw264.7 cells and Figure b, which presents the experimental results of Caco-2 cells.

[0041] Figure 3 The effects of different concentrations of titanium dioxide, inulin, and titanium dioxide-inulin complex on LDH release are shown in Figure a, where subfigure a represents the experimental results of Raw264.7 cells and subfigure b represents the experimental results of Caco-2 cells.

[0042] Figure 4 The effects of different concentrations of titanium dioxide, inulin, and titanium dioxide-inulin complex on MDA release were investigated.

[0043] Figure 5 Fluorescence images showing the effects of different concentrations of titanium dioxide, inulin, and titanium dioxide-inulin complex on ROS release are shown. Sub-image a shows the experimental results of Caco-2 cells, and sub-image b shows the experimental results of HT-29 cells.

[0044] Figure 6 This is a model of acute colitis induced by sodium dextran sulfate (DSS). Subfigure a shows the animal experimental design, subfigure b shows the body weight of mice in different groups: healthy group, DSS-normal group, DSS-TiO2 group, DSS-Inulin group, and DSS-TiO2-Inulin complex group, and subfigure c shows the disease activity index of mice.

[0045] Figure 7 The image shows the colon of an acute DSS mouse model. Subfigure a shows the colon length of mice in different groups, and subfigure b shows a photograph of the mouse colon.

[0046] Figure 8 The figures show the colonic lipid peroxidation level in an acute DSS mouse model. Subfigure a represents malondialdehyde (MDA), subfigure b represents glutathione peroxidase (GSH-PX), and subfigure c represents superoxide dismutase (SOD).

[0047] Figure 9 H&E staining of the colon in an acute DSS mouse model.

[0048] Figure 10 This is the gut microbiota of an acute DSS mouse model. Subplot a shows the Simpson index, subplot b shows the Shannon index, and subplot c shows the principal component analysis.

[0049] Figure 11 The differential bacterial genera in the acute DSS mouse model are shown in Figure a, where subfigure a is Coriobacteriaceae_UCG-002, subfigure b is Dubosiella, and subfigure c is Escherichia-Shigella. Detailed Implementation

[0050] The present invention will be further described below, but the scope of protection of the present invention is not limited thereto:

[0051] Example 1

[0052] This embodiment discloses a titanium dioxide-inulin composite, the specific preparation method of which is as follows:

[0053] Weigh 0.1 g of inulin powder and disperse it in 10 mL of distilled water to prepare a 10 mg / mL inulin solution. Similarly, prepare a 10 mg / mL titanium dioxide suspension and vortex to ensure uniform dispersion. Add the inulin solution and titanium dioxide suspension of the same concentration at a volume ratio of 1:1 and mix thoroughly. Incubate at room temperature for 24 h (incubation temperature: 20-30℃, incubation under shaking conditions, shaking speed: 200 rpm). After the incubation, centrifuge at 10000 g for 10 min, discard the supernatant, and the precipitate is the titanium dioxide-inulin complex. Wash the precipitate three times with distilled water, discarding unbound inulin, and collect the precipitate. Finally, freeze-dry the sample under vacuum (vacuum degree: 0.08-0.133 MPa, temperature: -80℃) to obtain the titanium dioxide-inulin complex.

[0054] Example 2

[0055] Brightness measurement of nano-titanium dioxide-inulin composite:

[0056] The brightness of titanium dioxide and titanium dioxide-inulin composite was measured using a colorimeter.

[0057] As shown in Table 1, the brightness of the composite is basically similar to that of titanium dioxide nanoparticles, and it has a good whitening effect.

[0058] Example 3

[0059] The structure and stability of titanium dioxide and titanium dioxide-inulin complex in the gastrointestinal tract were investigated using an in vitro digestion model.

[0060] 3.1 Establishing an in vitro simulated digestion model

[0061] Preparation of digestive juices: (1) Simulated saliva: Dissolve 0.34g NaCl, 0.08g CaCl2, and 0.75g KCl in 500mL distilled water, then adjust the pH to 7.0 with 0.1mol / L HCl and 0.1mol / L NaHCO3 to prepare a salivary electrolyte solution. The simulated saliva consists of 2.15g α-amylase and 500mL salivary electrolyte solution. (2) Simulated gastric juice: Dissolve 1.6g NaCl, 0.08g CaCl2, 0.55g KCl, and 0.30g NaHCO3 in 500mL distilled water to prepare a gastric juice electrolyte solution. Adjust the pH of the electrolyte solution to 2.0 with 1mol / L HCl. The simulated gastric juice consists of 0.18g pepsin and 500mL gastric electrolyte solution. (3) Simulated small intestinal fluid: 2.70 g NaCl, 0.18 g CaCl2, and 0.35 g KCl were dissolved in 500 mL of distilled water to prepare a small intestinal electrolyte solution. The pH was adjusted to 7.5 with 0.1 mol / L HCl and 0.1 mol / L NaHCO3. The simulated small intestinal fluid consisted of 0.08 g trypsin, 1.80 g porcine bile salts, and 500 mL of small intestinal electrolyte solution.

[0062] like Figure 1 As shown in Figure a, the mixture of titanium dioxide-inulin complex and simulated saliva was incubated in a 37°C constant temperature shaking incubator for 15 min, with the pH maintained at 7.0. The sample was then removed and inactivated in a 100°C water bath for 8 min. The mixture of the above solution and simulated gastric juice was incubated in a 37°C constant temperature shaking incubator for 2 h, with the pH maintained at 2.0. The sample was then removed and inactivated in a 100°C water bath for 8 min. The mixture of the above solution and simulated small intestinal juice was incubated in a 37°C constant temperature shaking incubator for 3 h, with the pH maintained at 7.5. The sample was then removed and inactivated in a 100°C water bath for 8 min.

[0063] 3.2 Transmission electron microscopy detection during simulated digestion

[0064] The morphology of the nano-titanium dioxide biocrest during simulated digestion was characterized using transmission electron microscopy. First, a titanium dioxide-inulin complex (10 mg / mL) was deposited on a carbon-coated copper mesh. Excess liquid was then removed with lint-free paper, and the sample was dried at room temperature before transmission electron microscopy images were acquired.

[0065] The morphology of the nano-titanium dioxide-inulin complex during simulated digestion was determined by transmission electron microscopy. Figure 1 b). Transmission electron microscopy images show that, under simulated digestion, digestive enzymes are adsorbed around the nano-titanium dioxide-inulin complex.

[0066] 3.3 Scanning electron microscopy detection during simulated digestion

[0067] The stereomorphology of the nano-titanium dioxide-inulin composite during simulated digestion was obtained using scanning electron microscopy. The freeze-dried composite was mounted on an aluminum column, and a 10 nm gold layer was sputtered using a sputtering device. The sample was then examined at different magnifications (20000x, 50000x, and 100000x) at an accelerating voltage of 3 kV.

[0068] Scanning electron microscopy analysis further clarified the morphology of the nano-titanium dioxide-inulin complex during digestion in simulated saliva, gastric juice, and small intestinal juice. Figure 1 (b) Scanning electron microscopy (SEM) images show that the composite nanoparticles are approximately spherical in shape, but with some rough edges and exhibit overall aggregation, consistent with transmission electron microscopy (TEM) images. During simulated digestion in vitro, the complex continues to bind to digestive enzymes to varying degrees. SEM images at different digestion stages also show that the outer layer of the titanium dioxide nanoparticles is coated with inulin.

[0069] 3.4 Potential Measurement During Simulated Digestion

[0070] The zeta potential of titanium dioxide nanoparticles before and after incubation with inulin was measured using a dynamic light scattering instrument. Nano-titanium dioxide and the nano-titanium dioxide-inulin complex at a concentration of 500 μg / mL were ultrasonicated and vortexed to ensure uniform dispersion. 1 mL of the sample was then pipetteted into a measuring dish, and relevant parameters were set.

[0071] After incubation in digestive fluids, the surface potential of nano-titanium dioxide was measured to understand how digestive enzymes affect its electrical properties during simulated digestion. Under the influence of various ions in the digestive fluids, the surface potential of nanoparticles was negative during the simulated oral and small intestinal fluid stages and positive during the simulated gastric fluid stage. Compared to the control group, the experimental group's nanoparticles had more negative surface charge during the simulated intestinal fluid stage and less positive charge during the simulated gastric fluid stage, possibly due to the binding of nanoparticles to negatively charged digestive enzymes. During the simulated gastric fluid stage in the experimental group, there was no significant difference in surface potential between the EG-INU&TiO2 and EG-TiO2 groups, indicating that inulin has little effect on the potential of TiO2 nanoparticles during in vitro simulated digestion. Figure 1 c).

[0072] Example 4

[0073] Three cell models—Raw264.7, Caco-2, and HT-29—were established to investigate the effects of nano-titanium dioxide-inulin complex on cell viability. The effects of lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) expression, and malondialdehyde (MDA) levels on cell membrane integrity and oxidative stress were investigated by detecting these parameters.

[0074] 4.1 MTT assay for cell viability

[0075] Titanium dioxide suspensions, inulin solutions, and titanium dioxide-inulin complex solutions with concentrations of 12.5, 25, 50, 100, 200, 400, 800, and 1600 μg / mL were prepared using serum-free culture medium.

[0076] Raw264.7 and Caco-2 cells in logarithmic growth phase were digested with 0.25% trypsin, and cell counts were performed using a hemocytometer. The cell concentration was adjusted to 5000 cells / well, and the cell suspension was seeded into 96-well plates and cultured in a 5% CO2 cell culture incubator for 24 h. The original culture medium was discarded, and different concentrations of titanium dioxide suspension, inulin solution, and titanium dioxide-inulin complex solution were added respectively. The control group did not receive serum-free culture medium. Raw264.7 cells were cultured in a 5% CO2 cell culture incubator for another 24 h, and Caco-2 cells were cultured in a 5% CO2 cell culture incubator for another 48 h. 20 μL of 5 mg / mL LMT solution was added to each well, and the plates were incubated in a 5% CO2 cell culture incubator for 4 h. The original solution was discarded, and 100 μL of dimethyl sulfoxide solution was added to each well. The 96-well plates were shaken in a microplate reader for 1 min, and the absorbance was measured at a detection wavelength of 570 nm.

[0077] like Figure 2 As shown in a and b, the MTT results indicated that titanium dioxide treatment reduced the viability of Raw264.7 and Caco-2 cells in a dose-dependent manner. Treatment with the titanium dioxide-inulin complex significantly restored cell viability, and compared with the titanium dioxide group, the titanium dioxide-inulin complex significantly improved this cell damage effect.

[0078] 4.2 Release of cellular lactate dehydrogenase (LDH)

[0079] Titanium dioxide suspensions, inulin solutions, and titanium dioxide-inulin complex solutions of 50 and 100 μg / mL were prepared using serum-free culture medium.

[0080] Raw264.7 and Caco-2 cells in logarithmic growth phase were digested with 0.25% trypsin, and cell counts were performed using a hemocytometer. The cell concentration was adjusted to 50,000 cells / well, and the cell suspension was seeded into 96-well plates and cultured in a 5% CO2 cell culture incubator for 24 hours. The original culture medium was discarded, and different concentrations of titanium dioxide suspension, inulin solution, and titanium dioxide-inulin complex solution were added respectively. The control group did not receive serum-free culture medium. Raw264.7 cells were cultured in a 5% CO2 cell culture incubator for another 24 hours, and Caco-2 cells were cultured in a 5% CO2 cell culture incubator for another 48 hours. One hour before the assay, 20 μL of LDH release reagent was added to the positive control wells and incubated for 1 hour. Collect the supernatant from each group into centrifuge tubes, centrifuge at 400g for 5 min, add 120 μL of the supernatant to each well into a new 96-well plate, add 60 μL of LDH detection working solution, place in an ELISA reader and shake for 1 min to mix thoroughly, incubate at room temperature in the dark for 30 min, and measure the absorbance at a detection wavelength of 490 nm.

[0081] like Figure 3 Figures a and 3b show that LDH release from cells increased after titanium dioxide treatment, indicating that titanium dioxide damages the cell membrane and releases more LDH; administration of the titanium dioxide-inulin complex significantly reduced LDH release and restored the cell membrane damage.

[0082] 4.3 Determination of Malondialdehyde (MDA) Content in Cells

[0083] Titanium dioxide suspensions, inulin solutions, and titanium dioxide-inulin complex solutions of 100 and 400 μg / mL were prepared using serum-free culture medium.

[0084] Caco-2 cells in the logarithmic growth phase were digested with 0.25% trypsin, and cell counts were performed using a hemocytometer to adjust the cell concentration to 1×10⁻⁶. 6Cells were seeded per well into 6-well plates and incubated in a 5% CO2 cell culture incubator for 24 h. The original culture medium was discarded, and different concentrations of titanium dioxide suspension, inulin solution, and titanium dioxide-inulin complex solution were added. The control group received serum-free culture medium without sample addition. Cells were incubated in a 5% CO2 cell culture incubator for another 48 h. The supernatant was discarded, and the cells were washed once with sterile phosphate buffer. 500 μL of extraction reagent was added and shaken to mix. Cells were scraped off and collected into centrifuge tubes. Cells were disrupted in a cell disruptor for 3 min at 250 W for 5 seconds with a 15-second interval. 100 μL of the suspension was then transferred to a centrifuge tube, and different reagents were added according to the instructions for the Nanjing Jiancheng Cell Malondialdehyde Detection Kit. The mixture was vortexed and incubated in a 95°C metal bath for 1 h. After cooling to room temperature under running water, the absorbance was measured at 530 nm using a microplate reader.

[0085] like Figure 4 As shown, the MDA content in cells increased after titanium dioxide treatment, indicating that titanium dioxide treatment stimulated increased lipid peroxidation in cells, resulting in oxidative stress. Compared with titanium dioxide, the complex treatment inhibited lipid peroxidation in cells.

[0086] 4.4 Release of Reactive Oxygen Species (ROS) from Cells

[0087] Titanium dioxide suspensions, inulin solutions, and titanium dioxide-inulin complex solutions of 200 and 800 μg / mL were prepared using serum-free culture medium.

[0088] As described above, perform cell seeding and adjust the cell concentration to 2 × 10⁻⁶. 5 Cells / well. After incubation with the added sample, discard the original culture medium, wash once with phosphate buffer, add 600 μL DCFH-DA solution to each well, incubate the cell culture plate in a cell culture incubator for 20 min, wash three times with serum-free culture medium on ice, and take pictures using an inverted fluorescence microscope.

[0089] Figure 5 Figures a and b show that titanium dioxide treatment stimulates cells to produce more ROS, leading to excessive oxidative stress in cells. Complex treatment reduces ROS release and alleviates cell damage.

[0090] Example 5

[0091] A mouse model of acute colitis induced by dextran sulfate sodium (DSS) was established to investigate the effects of titanium dioxide and titanium dioxide-inulin complex on mouse body weight, disease activity index, colon length, colonic lipid peroxidation, colonic H&E staining, and gut microbiota.

[0092] 5.1 Establishment of a mouse model of acute colitis induced by sodium dextran sulfate (DSS)

[0093] like Figure 6 As shown in Figure a, 6-week-old (20±1g) male C57BL / 6J mice were randomly divided into 5 groups of 10 mice each according to their body weight: Healthy, DSS-Normal, DSS-TiO2, DSS-Inulin, and DSS-TiO2-Inulin complex. After three days of acclimatization, the mice were given free access to 2.5% DSS for seven days. Then, they were administered samples via gavage at a dose of 100 mg / kg body weight for seven days. Mouse body weight, water intake, and disease activity index were recorded daily. After gavage, the mice were sacrificed, and their feces were collected for microbial analysis. Their colons were also collected for subsequent experiments.

[0094] 5.2 Mouse body weight, disease activity index, and colon length

[0095] After three days of adaptive culture, mouse body weight and disease activity index were recorded daily. Results showed that after seven days of free access to DSS, mouse body weight continued to decrease. Figure 6 b), the disease activity index increased daily ( Figure 6 c), and at the same time, the mouse colon was significantly shortened ( Figure 7 (a and 7b) indicates that the acute DSS mouse model was successfully established. After stopping DSS consumption, the mice began to recover their body weight. Compared with the DSS-normal group, mice given titanium dioxide by gavage had lower body weight, higher disease activity index, and the shortest colon length. Gavage of titanium dioxide-inulin complex resulted in partial recovery of mouse body weight, a decrease in disease activity index, and relief of colon shortening caused by DSS consumption. Figure 6 ,7).

[0096] 5.3 Colonic lipid peroxidation

[0097] After the mice were euthanized, a colonic segment was harvested for subsequent experiments. First, the colon was longitudinally dissected, feces were removed, and the tissue was washed once with phosphate-buffered saline. A 30mg colonic fragment was weighed, and the tissue weight was accurately measured. Nine times the volume of physiological saline was added at a weight (g):volume (mL) ratio of 1:9. The tissue was minced and homogenized in a tissue homogenizer. Centrifugation was performed at 4°C, 2500–4000 rpm for 10 minutes. The supernatant (10% homogenate supernatant) was collected for analysis. MDA, SOD, and GSH-PX levels in the mouse colon were measured sequentially according to the Nanjing Jiancheng reagent kit instructions.

[0098] The results showed that, compared with the healthy control group, the colonic MDA level was increased in DSS-induced colitis mice ( Figure 8 a), while inhibiting the activity of GSH-PX and SOD ( Figure 8b, c) Increased lipid peroxidation; in mice with DSS-induced colitis, gavage treatment with titanium dioxide increased the level of lipid peroxidation in the colon; while feeding with titanium dioxide-inulin complex resulted in lower MDA content and significantly increased GSH-PX and SOD activity in colonic tissue. Figure 8 It effectively inhibits lipid peroxidation in the colon of mice with DSS-induced colitis.

[0099] 5.4 Colon H&E staining

[0100] After the mice were euthanized, the colonic segment was harvested for subsequent experiments. First, the colon was longitudinally dissected, feces were removed, and the colonic segment was rolled into a "Swiss roll" and fixed in Carnoy's fixative for 48 hours. The fixed colonic segment was then dehydrated, cleared, paraffin-embedded, and sectioned for hematoxylin-eosin staining. The sections were then scanned using a digital pathology slide scanner (NanoZoomer S60) and analyzed using NDP software.

[0101] Figure 9 The results showed that, compared with the healthy control group, DSS-induced acute colitis in mice resulted in an abnormal increase in colonic inflammatory cell infiltration (yellow circle), crypt cysts (yellow arrow), and crypt abnormalities (torsion and branching). Titanium dioxide treatment exacerbated colonic inflammatory cell infiltration. Compared with titanium dioxide treatment, inulin and titanium dioxide-inulin complex treatment reduced colonic inflammatory infiltration and crypt structure tended to be normal.

[0102] 5.5 Microbial Community Analysis

[0103] Mouse feces were collected in a sterile environment and processed by the CAS New Life Bioinformatics Cloud Platform. The V3-V4 variable regions were amplified and sequenced using the Illumina Novaseq 6000 sequencing platform. To ensure accurate and reliable data analysis, the raw data underwent quality control analysis using Qiime2 default parameters, including quality filtering, noise reduction, assembly, and chimera removal. Sequences with an abundance less than 10 (summed across all samples) were filtered out to obtain amplicon sequence variants. Based on the flattened amplicon sequence variants, various diversity indices and sequencing depth analyses were performed. Statistical analysis of community structure at various taxonomic levels was conducted based on taxonomic information.

[0104] 5.5.1 Analysis of α- and β-diversity of microbial communities

[0105] Compared to the DSS-normal group, the Simpson index (α-diversity) and Shannon index (community evenness) of the gut microbiota in mice administered titanium dioxide by gavage were both statistically significantly reduced. Figure 10(a and 10b) indicates that titanium dioxide can cause an imbalance in the gut microbiota of mice. However, after administration of the complex, both the Simpson and Shannon indices significantly increased, confirming that the titanium dioxide-inulin complex can effectively alleviate the gut microbiota imbalance caused by DSS-titanium dioxide treatment. Figure 10 c. Principal component analysis shows that DSS treatment can significantly affect the bacterial community structure.

[0106] 5.5.2 Differential bacterial genus analysis

[0107] Compared to the DSS-titanium dioxide group and the titanium dioxide-inulin complex group, the abundance of Coriobacteriaceae_UCG-002 (Rhodotorulaceae-UCG-002) was significantly increased. Figure 11 a) This genus of bacteria can regulate the host's immune response and neurological function by producing short-chain fatty acids (SCFAs), bile acids, and other metabolites. Simultaneously, it may affect the host's metabolic state and immune response by altering the levels of these metabolites; the abundance of *Dubosiella* was also significantly increased. Figure 11 (b) This genus of bacteria can alleviate colitis by improving colonic mucosal barrier function (such as increasing the expression of tight junction proteins and promoting mucus secretion), regulating T cell subsets in the intestinal immune microenvironment, and inhibiting inflammatory responses.

[0108] Table 1

[0109] .

Claims

1. A method for preparing a complex with a food whitening effect, characterized in that, The method includes the following steps: (1) Mix the inulin solution and titanium dioxide suspension evenly and incubate at room temperature for 20-24 hours; (2) After the above incubation is completed, the supernatant is discarded by centrifugation and the precipitate is the titanium dioxide-inulin complex. It is washed with distilled water, the unbound inulin is discarded, and the precipitate is collected. Finally, the sample is freeze-dried under vacuum to obtain the titanium dioxide-inulin complex.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-volume concentration of the inulin solution is 5~15 mg / mL, and the mass-volume concentration of the titanium dioxide suspension is 5~15 mg / mL.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass-volume concentration of the inulin solution is 5~15 mg / mL, and the mass-volume concentration of the titanium dioxide suspension is 5~15 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (1), the volume ratio of inulin solution to titanium dioxide suspension is 1~5:1~5.

5. The preparation method according to claim 1, characterized in that, In step (1), the temperature for mixing and incubation is 15~40℃, and the mixing and incubation is carried out under shaking conditions with a shaking speed of 180~220rpm.

6. The preparation method according to claim 1, characterized in that, In step (2), the vacuum degree of vacuum freeze drying is 0.08~0.133MPa and the temperature is -85~-80℃.

7. A complex having a food whitening effect, characterized in that, The complex was prepared by the method described in claim 1.

8. The use of the complex with food whitening effect as described in claim 1 as a food whitening agent.

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