Camel milk protein-polysaccharide composite nanoparticles and preparation method thereof

Abstract

The invention discloses camel milk protein-polysaccharide composite nanoparticles and a preparation method thereof, and belongs to the technical field of preparation of natural fermentation composite particles. The preparation method comprises the following steps: (1) inoculating phytobacterium plantarum into camel milk for fermentation, centrifuging, and collecting supernatant; and (2) carrying out ultrasonic treatment on the supernatant under an ice-water bath condition, adding absolute ethyl alcohol to precipitate after the ultrasonic treatment is finished, standing, centrifuging, and dialyzing to remove impurities, so as to finally obtain the camel milk protein-polysaccharide composite nanoparticles. According to the preparation method, camel milk is taken as a fermentation substrate, fermentation is carried out by utilizing phytobacterium plantarum LR-1, and the natural polysaccharide-protein nanoparticles (PPNPs) are successfully prepared in combination with ultrasonic treatment. In the fermentation process, the metabolic activity of lactic acid bacteria promotes the combination of protein and polysaccharide, and the particle size, the stability and the oxidation resistance are improved; the ultrasonic treatment further optimizes the particle size and structure, and enhances the oil-water interface adsorption capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of natural fermentation composite particle preparation, and particularly relates to camel milk protein-polysaccharide composite nanoparticles and a preparation method thereof. Background Art

[0002] Polysaccharide-protein nanoparticles (PPNPs) prepared through natural fermentation have garnered widespread attention in recent years due to their unique biocompatibility, functional diversity, and environmental friendliness. They have demonstrated significant application potential in a wide range of fields, including the food industry, biomedicine, environment and agriculture, and other emerging sectors. For example, PPNPs can serve as stabilizers for Pickering emulsions, replacing synthetic surfactants in low-fat foods and sauces, enhancing their stability and mouthfeel. They can also be used to encapsulate fat-soluble vitamins, polyphenols, or essential oils, protecting sensitive ingredients from photo-, thermal, or oxidative degradation through nanoparticle interfacial adsorption. They can also be used to manipulate food rheological properties, improving texture and water retention in applications such as low-fat ice cream and plant-based meats. Because naturally fermented particles align with the "clean label" trend and possess beneficial properties such as antioxidants, they are more aligned with the modern food industry's philosophy and cater to the needs of today's consumers. Furthermore, in the biomedical field, PPNPs can be used as targeted drug carriers, where the natural surface properties of fermentation products can reduce immunogenicity. They can also be used as matrices to prepare wound dressings or anti-infection coatings, potentially developing into antibacterial and anti-inflammatory materials. They can also be used to construct biomimetic 3D scaffolds, where the fermentation process can regulate particle porosity and mechanical properties to adapt to the needs of different tissues. Naturally fermented polysaccharide-protein nanoparticles are gradually becoming a model for the integration of green chemistry and biotechnology, and their application potential continues to expand with a deeper understanding of microbial metabolic mechanisms.

[0003] The preparation of naturally fermented polysaccharide-protein nanoparticles involves three key elements: the fermentation matrix, the fermentation strain, and nanoparticle preparation. Regarding the fermentation matrix, current research focuses primarily on cow's milk or its byproducts, while the exploration and utilization of other potential dairy resources or matrices is relatively limited. The preparation of natural polysaccharide-protein composite particles is simple and efficient, eliminating the need for separate extraction of proteins and polysaccharides followed by synthetic synthesis, thereby retaining the high bioactivity of the raw materials. Because the preparation process does not involve any chemical synthesis steps, these food-grade composite particles possess excellent biocompatibility. For example, Liu et al. extracted composite particles composed primarily of protein (55.6%) and carbohydrates (24.0%) from walnuts using media milling and demonstrated their ability to form a gel-like network structure in a pickering emulsion. Other studies have used ammonium sulfate precipitation to extract natural polysaccharide-protein composite particles with excellent emulsion stability and oil retention from an aqueous extract of red algae. Microscopic images revealed that fibrous polysaccharides were linked to the protein particles, forming stable cross-links. Fermentation can further promote the binding of polysaccharides and proteins. In fermentation systems, polysaccharides and proteins are linked through non-covalent interactions (such as electrostatic attraction, hydrogen bonding, and hydrophobic interactions) or covalent bonds to form nano- or micron-sized particle structures. These polysaccharide-protein composite particles exhibit excellent stability, interfacial activity, bioactivity, and controlled-release potential, making them suitable carriers for drugs, nutrients, or antioxidants. They also possess antioxidant, antibacterial, and immunomodulatory properties.

[0004] The selection of fermentation strains is also crucial for the preparation of naturally fermented polysaccharide-protein nanoparticles. Lactic acid bacteria are one of the best candidates due to their excellent fermentation-driving potential and prebiotic effects. When fermenting dairy matrices, lactic acid bacteria can partially hydrolyze milk proteins by secreting proteases, exposing hydrophobic groups and enhancing their interfacial binding ability with polysaccharides. They can also synthesize extracellular polysaccharides in situ, interacting with milk proteins to form "self-assembled" particles, and produce short-chain fatty acids, antibiotics, etc., giving the particles more functionality.

[0005] According to the definition of ISO, the International Organization for Standardization, nanoparticles are particles with at least one dimension between 1 and 100 nm. In application areas such as food, medicine, and materials science, the particle size range can be extended to 200-1000 nm, referred to as "generalized nanoparticles," which still possess significant nanoscale properties. Common nanoparticles include metal nanoparticles, inorganic oxide nanoparticles, and polymer nanoparticles. Research on naturally fermented nanoparticles is relatively limited, but due to their advantages such as good biocompatibility and biofunctional activity, they have received widespread attention in recent years. In the process of preparing naturally fermented polysaccharide-protein composite nanoparticles, uneven particle size, unstable bonding, and failure to meet nanoscale requirements may occur. Summary of the Invention

[0006] To address the aforementioned shortcomings of the existing technology, the present invention provides camel milk protein-polysaccharide composite nanoparticles and a method for their preparation. Using camel milk as the fermentation substrate, the present invention utilizes the plant-like strain Lactobacillus plantarum LR-1 for fermentation, combined with ultrasonic treatment, to successfully produce natural PPNPs. During the fermentation process, the metabolic activity of the lactic acid bacteria promotes the binding of the protein and polysaccharide, improving particle size, stability, and antioxidant capacity. Ultrasonic treatment further optimizes particle size and structure, enhancing adsorption capacity at the oil-water interface.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: The object of the present invention is to provide a method for preparing camel milk protein-polysaccharide composite nanoparticles, which comprises the following steps: (1) Inoculate Lactobacillus plantarum into camel milk, ferment at 26-30°C for 40-50 hours, centrifuge, and collect the supernatant; (2) The supernatant was ultrasonically treated in an ice-water bath. After the ultrasonic treatment, anhydrous ethanol was added for precipitation, and then the supernatant was centrifuged and dialyzed to remove impurities, and finally camel milk protein-polysaccharide composite nanoparticles were obtained.

[0008] Furthermore, the inoculation amount of Lactobacillus plantarum in step (1) is 1.5-3%.

[0009] Furthermore, the inoculation amount of Lactobacillus plantarum is 3%.

[0010] Furthermore, the plant-like Lactobacillus is plant-like Lactobacillus LR-1.

[0011] Furthermore, in step (1), the fermentation temperature is 26-30° C., and the fermentation time is 46-48 h.

[0012] Furthermore, the fermentation temperature is 28° C. and the fermentation time is 48 h.

[0013] Furthermore, the ultrasonic treatment conditions in step (2) are 20-25 kHz, 400-450 W.

[0014] Furthermore, the ultrasonic treatment conditions were 20 kHz, 400 W.

[0015] Furthermore, in step (2), the ultrasonic treatment uses a pulse mode of 2 s on and 2 s off, and the ultrasonic time is 11 to 15 minutes.

[0016] Furthermore, the temperature of the ice water bath in step (2) is 10±0.2°C.

[0017] Furthermore, dialysis removes impurities including small molecule monosaccharides and ethanol.

[0018] Another object of the present invention is to provide camel milk protein-polysaccharide composite nanoparticles, which are prepared by the above method.

[0019] Beneficial effects of the present invention: 1. Compared to cow's and goat's milk, camel milk contains higher concentrations of lactoferrin, lysozyme, and immunoglobulins, with a higher proportion of whey protein, making it more likely to form functional complexes. The protein content in camel milk is approximately 2.1%-4.9%, of which whey protein accounts for approximately 20%. Notably, camel milk does not contain β-lactoglobulin, a common allergenic protein, making it hypoallergenic. Furthermore, camel milk contains bioactive substances such as lactoperoxidase, lysozyme, and lactoferrin, which act as natural antioxidants and have immunomodulatory effects. Therefore, the hypoallergenicity and bioactivity of camel milk provide a natural advantage for functionalized particles.

[0020] 2. This invention uses the plant-like Lactobacillus plantarum LR-1 as the fermentation strain and camel milk, a unique biological resource, as the fermentation substrate. A fermentation-followed ultrasound method is employed to prepare natural polysaccharide-protein composite nanoparticles. These particles exhibit a uniform spherical structure, and their particle size meets the standard requirements for nanoparticles in the food industry, laying the foundation for their application in food-grade functional nanomaterials. PPNPs exhibit good stability in aqueous solutions, possessing certain osmotic pressure resistance, alkalinity resistance, and high temperature resistance, making them suitable for conventional dairy processing, storage, and transportation conditions, such as low-temperature, long-term pasteurization (LTLT).

[0021] In terms of antioxidant properties, PPNPs obtained after 11 minutes of ultrasonic treatment demonstrated superior antioxidant capacity compared to unfermented samples and other treatment groups, demonstrating promising potential for enhancing immune function and assisting in the treatment of chronic diseases. Lactic acid bacteria fermentation partially degrades camel milk proteins into small bioactive peptides, which not only enhances the stability of PPNPs but also their nutritional properties. The synergistic effect of fermentation and ultrasonication also improves the surface wettability of PPNPs, imparting them with a more hydrophilic-lipophilic neutral profile, providing a theoretical basis for their co-encapsulation with probiotics and prebiotics.

[0022] 3. The cavitation effect of ultrasound can break weak bonds (such as hydrogen bonds and hydrophobic interactions) in protein-polysaccharide complexes, promoting molecular rearrangement to form a denser nanostructure. Ultrasonic energy input can also regulate particle size, increase specific surface area, and enhance emulsion stability or drug loading efficiency. Ultrasound also exposes more active groups (such as hydroxyl and amino groups), enhancing the adsorption capacity of particles at the oil-water interface and making them suitable for the construction of high-internal-phase Pickering emulsions. During the preparation of nanoparticles, ultrasound technology achieves ion refinement, uniform dispersion, and structural remodeling through its unique acoustic cavitation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart for preparing the composite nanoparticles of the present invention; Figure 2 Camera images and optical microscope images of PPNPs in different groups; among them, ah is camera image, ad is ultrasound for 3-15 min; e, f are pH adjusted to 7, g is control after fermentation, h is not fermented; AH are optical microscope images, AD is ultrasound for 3-15 min, E, F are pH adjusted to 7, G is control after fermentation, H is not fermented; Figure 3 Scanning electron micrographs of PPNPs in different groups; among them, ah is magnified by 20,000 times, ad is sonicated for 3-15 minutes; e, f are adjusted to pH 7 and 9, g is a control after fermentation, and h is not fermented; AH is magnified by 5,000 times, AD is sonicated for 3-15 minutes; E, F are adjusted to pH 7 and 9, G is a control after fermentation, and H is not fermented; Figure 4 The particle size and distribution test diagram of PPNPs in different groups; A is the average particle size and dispersion index PDI; B is the particle size distribution; Figure 5 Detection diagram of structural changes of PPNPs in different groups; A is the Fourier transform infrared spectrum; B is the protein secondary structure distribution; Figure 6 UV spectra of PPNPs in different groups; Figure 7 The three-phase contact angle detection diagram of PPNPs in different groups; Figure 8 zeta potential detection diagram of PPNPs in different groups; Figure 9 This is a graph showing the molecular weight of PPNPs in different groups; Figure 10 The antioxidant activity test diagram of PPNPs in different groups; Figure 11 The stability test diagram of PPNPs treated under different conditions; A is the particle size and PDI test results; B is the PPNPs ζ potential test results. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0025] The strains and reagents used in the present invention are as follows: L. paraplantarum LR-1, deposited in the China Industrial Culture Collection Center (CICC, No. 24809).

[0026] Camel milk powder was purchased from Xinjiang Wangyuan Camel Milk Industry Co., Ltd., China. All other reagents used were of analytical grade.

[0027] Example 1 Camel milk protein-polysaccharide composite nanoparticles (PPNPs), the preparation method of which is as follows: Lactobacillus plantarum LR-1 was inoculated at a 3% inoculum into 12% (w / v) camel milk and fermented at 28°C for 48 h. The fermentation broth was centrifuged at 8000 g at 4°C for 10 min. The sediment was removed to remove the bacteria, and the suspended solids were removed to remove lipids. The supernatant was collected to obtain the PPNPs solution. The solution was sonicated by immersing the probe into the sample at a distance of 2 cm from the bottom. The sample was sonicated in an ice-water bath to maintain a temperature of 10 ± 0.2°C. Sonication conditions were set at 20 kHz, 400 W, and a 2 s on / 2 s off pulse mode.

[0028] The sample was sonicated for 11 minutes, and three volumes of anhydrous ethanol were added to the solution to precipitate the protein-polysaccharide composite particles. After standing at 4°C for 18 hours, the solution was centrifuged at 8000 g at 4°C for 10 minutes. The precipitate was collected and dissolved in deionized water. The precipitate was dialyzed at 4°C to remove impurities such as small monosaccharides and ethanol, and then freeze-dried after 3 days. The freeze-dried PPNPs were stored at -20°C.

[0029] Example 2 Camel milk protein-polysaccharide composite nanoparticles (PPNPs), the preparation method of which is as follows: Lactobacillus plantarum LR-1 was inoculated at a 2% inoculum into 12% (w / v) camel milk and fermented at 29°C for 48 h. The fermentation broth was centrifuged at 8000 g at 4°C for 10 min. The sediment was removed to remove the bacteria, and the suspended solids were removed to remove lipids. The supernatant was collected to obtain the PPNPs solution. The solution was sonicated by immersing the probe 2 cm from the bottom of the sample and maintaining the sample temperature at 10 ± 0.2°C in an ice-water bath. Sonication conditions were set at 22 kHz, 420 W, and a 2 s on / 2 s off pulse mode.

[0030] The sample was sonicated for 15 minutes, and three volumes of anhydrous ethanol were added to the solution to precipitate the protein-polysaccharide composite particles. After standing at 4°C for 18 hours, the solution was centrifuged at 8000 g at 4°C for 10 minutes. The precipitate was collected and dissolved in deionized water. The precipitate was dialyzed at 4°C to remove impurities such as small monosaccharides and ethanol, and then freeze-dried after 3 days. The freeze-dried PPNPs were stored at -20°C.

[0031] Example 3 Camel milk protein-polysaccharide composite nanoparticles (PPNPs), the preparation method of which is as follows: Lactobacillus plantarum LR-1 was inoculated at a 2.5% inoculum into 12% (w / v) camel milk and fermented at 30°C for 46 h. The fermentation broth was centrifuged at 8000 g at 4°C for 10 min. The lower sediment was removed to remove the bacteria, and the upper suspended matter was removed to remove the lipids. The supernatant was collected to obtain the PPNPs solution. The solution was sonicated by immersing the probe into the sample at a distance of 2 cm from the bottom. The sample was sonicated in an ice-water bath to maintain a temperature of 10 ± 0.2°C. Sonication conditions were set at 25 kHz, 450 W, and a 2-s on / 2-s off pulse mode.

[0032] The sample was sonicated for 13 minutes, and three volumes of anhydrous ethanol were added to the solution to precipitate the protein-polysaccharide composite particles. After standing at 4°C for 18 hours, the solution was centrifuged at 8000 g at 4°C for 10 minutes. The precipitate was collected and dissolved in deionized water. The precipitate was dialyzed at 4°C to remove impurities such as small monosaccharides and ethanol, and then freeze-dried after 3 days. The freeze-dried PPNPs were stored at -20°C.

[0033] Example 4 1. Characterization of PPNPs (1) Morphological observation and scanning electron microscopy (SEM) The morphology and structure of PPNPs were observed using an industrial camera. The microscopic morphology of PPNPs was observed using a field emission scanning electron microscope (Zeiss Sigma 300). A small amount of powder sample was added to ultrapure water and ultrasonically dispersed uniformly. The sample was then dropped onto a silicon wafer and, after drying, sprayed with gold for approximately 60 seconds. The acceleration voltage was 3 kV, and the measurement mode was secondary electron mode.

[0034] (2) Fourier transform infrared spectroscopy (FT-IR) The infrared spectra of PPNPs were measured by the potassium bromide tablet method. 1 mg of sample was mixed with 100 mg of potassium bromide and ground into a uniform powder, which was then pressed into thin sheets using a tablet press. The infrared spectrum of 4000-500 cm was obtained on a Fourier transform infrared spectrometer. -1 Infrared spectra within the wavenumber range were plotted with potassium bromide as a blank control. Peak fitting software ThermoScientificTM OMNIC TM The spectra were smoothed and fitted using Peakfit v4.12 software. The amide I band (1600-1700 cm -1 ) Fourier transform and Gaussian deconvolution can reveal changes in the secondary structure of the protein.

[0035] (3) Three-phase contact angle The three-phase contact angle of PPNPs was measured using an SL 200 contact angle meter. Freeze-dried sample powder (50 mg) was pressed into tablets. 5 μL of deionized water was added to the pressed tablets using a microsyringe. The contact angle was determined by analyzing the drop shape using CAST 3.0 software and calculated according to Young's equation. The results are shown in Figure 4 .

[0036] (4) Zeta potential and particle size The zeta potential and particle size of a 1 mg / mL PPNP solution were measured using a zeta potential meter. DTS1070 and DTS0012 sample cells were used to measure the zeta potential and particle size, respectively. 2. Ultraviolet spectrum The UV spectrum of a 1 mg / mL PPNP solution was measured using a UV-visible spectrophotometer. The wavelength range was set to 200-400 nm, the speed was set to medium, and the scan interval was 1 nm. Three scans were performed. The sample temperature was always fixed at 25°C, and deionized water was used as a blank control.

[0037] 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The protein molecular weight distribution of PPNPs was determined by SDS-PAGE. A 1 mg / mL PPNP solution was mixed with SDS-PAGE Loading Buffer at a volume ratio of 4:1, heated at 100°C for 30 minutes, and then cooled. SDS-PAGE analysis was performed on a 12% separating gel containing a 5% stacking gel at a voltage of 120 V. The gel was stained with 0.25% Coomassie Brilliant Blue R250, and molecular weights were estimated by comparison with prestained protein markers.

[0038] 4. Antioxidant activity To determine the DPPH radical scavenging rate of PPNPs, 150 μL of sample solution (1 mg / mL) and an equal volume of DPPH-ethanol solution (0.1 mM) were taken, and the mixture was shaken thoroughly and reacted in the dark at 37°C for 30 min. The absorbance at a wavelength of 517 nm was measured (A1). At the same time, the absorbance value after mixing 150 μL of sample and 150 μL of ethanol was measured (A2), and the absorbance value after mixing 150 μL of anhydrous ethanol and 150 μL of DPPH solution was measured (A3).

[0039] DPPH free radical scavenging rate (%) =

[0040] Determination of ABTS of PPNPs + Free radical scavenging rate, ABTS was prepared by mixing equal volumes of 7 mmol / L ABTS aqueous solution and 2.45 mmol / L potassium persulfate aqueous solution. + Free radical working solution was prepared and the absorbance of the working solution at 734 nm was adjusted to 0.7 ± 0.02 with distilled water for later use. 15 μL of sample was mixed with 285 μL of working solution and reacted in the dark for 5 min. The absorbance value A1 was measured at 734 nm. At the same time, the absorbance value (A2) of 15 μL sample and 285 μL of anhydrous ethanol was measured. + The absorbance value (A3) of the working solution after it is evenly mixed.

[0041] ABTS + Clearance rate (%) =

[0042] 5. Storage stability The particle size and zeta potential of PPNPs were measured at different pH values ​​(3.0-11.0). The pH of the samples was adjusted using 0.1 and 1.0 M hydrochloric acid or sodium hydroxide solutions. The particle size and zeta potential of PPNPs were also measured at different temperatures (-20, 4, 25, 37, and 60°C). The particle size and zeta potential of PPNPs were also measured at different ionic strengths (0-1000 mM NaCl). All PPNPs were tested at a concentration of 1 mg / mL and at 25°C. All groups were equilibrated under the corresponding conditions for 2 hours before testing.

[0043] 6. Results (1) Morphological observation The morphology of camel milk particles was observed using a camera. The fermented particles showed a fluffy but dispersed sponge-like structure ( Figure 2ag). As the ultrasonic time increases, the color of the fermented particles gradually changes from off-white to pure white. When the pH of the fermented particles is adjusted to neutral or alkaline, the color of the particles becomes whiter and the structure becomes a uniform and complete fluffy sponge. Figure 2 h shows that unfermented camel milk is a dense pure white powder. The microscopic morphology of the particles was further observed using a microscope. The microscopic morphology of the fermented particles is flocculent, and sharp spike structures can be observed in the sparser areas at the edge of the particles ( Figure 2 AG). Figure 2 It can be seen from H that the microscopic morphology of unfermented camel milk is granular crystals.

[0044] (2) SEM The microstructure and particle size of camel milk particles were observed using a scanning electron microscope at 20,000 and 5,000 magnifications, respectively. All the results are presented in Figure 3 The fermented particles showed uniform spherical shapes of different sizes, with no signs of aggregation. The diameter of the spheres varied from 1 to 3 μm, and there were some porous structures near the spheres. As the ultrasonic time increased, the diameter of the fermented camel milk particles gradually decreased ( Figure 3 AD). High-intensity ultrasonic treatment can destroy the structure of proteins and obtain smaller particles. Compared with the untreated control group after fermentation, the particle diameter of the fermented products adjusted to neutral or alkaline pH is reduced ( Figure 3 EG). When the pH is adjusted to alkaline, the particle surface shows more obvious wrinkles ( Figure 3 F, f). Unfermented camel showed a fuzzy layered structure with a dense, brittle texture scattered with distinct sharp edges ( Figure 3 H, h). It can be seen that the camel milk powder freeze-dried after ultrasound is more uniform and stable, while the camel milk powder directly freeze-dried is more aggregated.

[0045] (3) Particle size and distribution The size of camel milk in aqueous solution was determined by dynamic light scattering (DLS) technique. Figure 4 A shows that the particle size of the fermented particles is 100-150 nm, which is significantly smaller than the 470 nm of the unfermented ones. Ultrasound and adjusting the pH of the particles to neutral or alkaline can reduce the particle size. As the ultrasonic time increases, the particle size decreases from 150 nm to 100 nm. The lower dispersion index (PDI) can reflect the stability of the solution. The PDI of the unfermented camel milk is 0.76, which is significantly higher than that of the fermented group (about 0.3). As the ultrasonic time increases, the PDI of the solution decreases from 0.4 to 0.2. Figure 4B found that unfermented camel milk showed a multi-peak distribution, while fermented camel milk particles showed a more uniform peak. All groups had a low-intensity peak at 5000 nm, which was more obvious under alkaline conditions, which may be related to particle aggregation or component characteristics.

[0046] (4) Structural changes The chemical structure of camel milk was analyzed by FT-IR spectroscopy. Figure 5 A shows that there are four main absorption peaks of unfermented camel milk. They appear at 3417 cm -1 The peak at 2924 cm is attributed to the stretching vibration of hydroxyl (-OH) and amino (-NH) groups; -1 and 2854 cm -1 The peaks at 1745 cm-1 are attributed to the asymmetric and symmetric stretching vibrations of CH in the fatty acid chain; -1 The peak at 1080 cm-1 is attributed to the stretching vibration of the ester carbonyl group (C=O); -1 Compared with the unfermented camel milk, the fermented group has two new characteristic peaks, one at 1410 cm -1 The peaks nearby are attributed to carboxylates (COO - ) symmetrical stretching vibration; the other is 545 cm -1 The absorption peak near 3400 cm is attributed to the CH bending vibration. Compared with the unfermented camel milk, the fermented camel milk has a -1 The peak near 2921cm is blue-shifted. -1 and 2854 cm -1 The intensity of the two characteristic peaks at 1255 cm decreased. No new peaks or peak shifts appeared after ultrasound. The pH was adjusted to alkaline so that the peak at 1255 cm -1 The absorption peak at 147° disappears, attributable to the vibration of the P=O / COC bond in phospholipids. These results suggest that unfermented camel milk contains fat, while the fat content decreases after fermentation. This is likely due to centrifugation to remove suspended fat and fermentation during processing. Active lactic acid bacteria metabolism during fermentation, accompanied by lactose conversion and protein degradation, leads to the accumulation of carboxylic acid compounds. Under alkaline conditions, saponification or hydrolysis of milk fat, lactose phospholipids, and other structures occurs.

[0047] The peakfit software was used to analyze the amide I band (1600-1700 cm) of the camel milk Fourier transform infrared spectrum. -1 ) Fourier transform and Gaussian deconvolution can roughly determine the changes in the secondary structure of the protein. The results are as follows Figure 5As shown in Figure B. The β-turn content of the protein secondary structure in all samples remained stable at 18%-28%. Unfermented camel milk contained no β-sheets, and the random coil content was 35%, higher than that in the fermented group. In the fermented group, the β-sheet content increased with increasing ultrasound time, while the random coil content decreased. Within protein secondary structure, β-sheets correspond to rigid, structurally supported conformations. These results indicate that the spatial conformation of proteins in unfermented camel milk is relatively loose, lacking a highly ordered, stable structure. Fermentation enhances the spatial stability of camel milk. Ultrasound promotes the transition from disordered to ordered protein conformations.

[0048] (5) Ultraviolet spectrum The molecular structure, composition and properties of camel milk were studied by ultraviolet spectroscopy. Figure 6 All sample groups exhibited a high absorption peak near 275 nm and a shoulder peak at 295 nm. Unfermented camel milk exhibited higher absorbance in the UV spectrum, with a sharper peak. The fermented group exhibited a small absorption peak at 415 nm, while unfermented camel milk lacked this peak. Adjusting the pH to neutral or alkaline decreased the absorbance of camel milk, with this decrease being more pronounced under alkaline conditions. The absorbance decreased after ultrasound compared to the control group, but the decrease was not significant with prolonged ultrasound time.

[0049] (6) Particle surface wettability and zeta potential The surface wettability of camel milk is reflected by the three-phase contact angle. Figure 7 As shown in the figure. Unfermented camel milk had the highest contact angle, approximately 109°, while the fermented, untreated group had the lowest contact angle, approximately 62°. The contact angle of ultrasound-treated camel milk ranged from 71 to 87°, showing an upward trend over time. Adjusting the pH to neutral or alkaline levels increased the contact angle compared to the fermented, untreated control group. It is generally believed that a three-phase contact angle greater than 90° reflects the hydrophobicity of the material. Unfermented camel milk contains more fat, resulting in a stronger hydrophobicity than the fermented group with a lower fat content. Ultrasound may induce conformational changes in protein molecules, exposing hydrophobic groups, increasing interactions between proteins, and forming a more compact structure. This corresponds to the previous discussion of protein secondary structure.

[0050] The stability of the dispersed system was measured by zeta potential. Figure 8As shown in the figure. The zeta potential of all samples was negative, and the absolute value of the zeta potential of unfermented camel milk was significantly lower than that of the other groups, at only 5.63 mV. Among the fermented groups, the PPNPs adjusted to an alkaline pH had the highest absolute value of 19.3 mV. In the ultrasound group, the absolute value of the zeta potential showed an increasing trend with increasing ultrasound time. The larger the absolute value of the zeta potential, the stronger the electrostatic repulsion between the particles and the more stable the system. These results indicate that fermentation can significantly enhance the stability of PPNPs.

[0051] (7) Protein molecular weight The molecular weight of camel milk protein was characterized by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Figure 9 As shown in Figure 2, markers with molecular weights ranging from 15 to 130 kDa were used for comparison. The molecular weight of the unfermented PPNPs was concentrated around 15 kDa, with varying degrees of tailing. Unfermented camel milk, on the other hand, exhibited two additional major bands around 25 and 35 kDa. These results indicate that the proteins in the fermented NPs are primarily small proteins and peptides.

[0052] 7. Physicochemical properties of PPNPs (1) Protein polysaccharide content The total protein and total polysaccharide concentrations of camel milk were determined, and the protein and polysaccharide contents were calculated. The results are shown in Table 1. The protein content of unfermented camel milk was 4.91%, significantly lower than that of fermented PPNPs. The protein content of PPNPs under alkaline conditions increased by 7% compared to the unadjusted pH group. As the ultrasonic treatment time increased, the protein content showed a decreasing trend. The protein content of NPs sonicated for 15 minutes increased by 5% compared to the control group. The polysaccharide content of unfermented camel milk was 13.29%, significantly higher than that of the fermented group. In the fermented group, adjusting the pH to neutral or alkaline decreased the polysaccharide content. As the ultrasonic treatment time increased, the polysaccharide content of PPNPs increased from 9.48% to 10.05%. Ultrasonic treatment can disrupt non-covalent bonds between protein molecules, such as hydrogen bonds and hydrophobic interactions, thereby affecting protein structure.

[0053] Table 1 Protein and polysaccharide concentration and content of PPNPs in different groups

[0054] (2) Antioxidant properties By measuring DPPH and ABTS + The free radical scavenging rate reflects its in vitro antioxidant capacity. The measured scavenging rate is converted to 100 mg / L ascorbic acid equivalent. The results are shown in Figure 10 The free clearance rate of the two substances in unfermented camel milk is the lowest.+ The free radical scavenging rate increased by 0.013 mL, and the DPPH free radical scavenging rate increased by 0.027 mL. In the ultrasound group, within a certain range (0-11 min), the scavenging rate of the two free radicals of PPNPs increased with the increase of ultrasound time. The scavenging rate of the two free radicals was the best after ultrasound for 11 min, among which ABTS + The equivalent weight of PPNPs was 0.174 mL, and the equivalent weight of DPPH was 0.168 mL. Among the groups with different pH values, the scavenging rate of both free radicals was highest when the pH was adjusted to alkaline, followed by neutral, and then acidic. The antioxidant capacity of camel milk PPNPs increased with increasing pH. Smaller particles have a larger specific surface area, exposing more functional groups (such as amino, carboxyl, and thiol groups). These groups have more sites for interaction with free radicals or metal ions, enhancing their free radical scavenging ability.

[0055] (3) Stability of PPNPs The particle size, PDI and ζ potential of PPNPs balanced under different conditions were measured to reflect their stability. PPNPs fermented with lactic acid bacteria and ultrasonicated for 11 minutes were selected for relevant research. Since the samples were stored in a refrigerator at -20°C, the particle size of the samples increased compared to when they were first prepared, and the absolute value of the ζ potential decreased. The measured results are as follows: Figure 11 shown.

[0056] The effects of osmotic pressure on PPNPs were simulated by equilibrating the PPNPs in saline solutions of varying concentrations (0-1000 mM NaCl). Compared to the control group, the particle size of the PPNPs decreased in saline solutions, but the particle size did not change significantly between the different saline concentration groups. The PDI of the PPNPs in saline solutions of varying concentrations was also relatively low, remaining around 0.3. As the salt concentration increased, the absolute value of the zeta potential of the PPNPs gradually decreased, from 7.76 mV to 4.59 mV.

[0057] The effect of pH on PPNPs was simulated by balancing PPNPs at different pH levels (3, 5, 7, 9, and 11). When PPNPs were stable at pH 3 and pH 5, the particle sizes were 2000 nm and 1150 nm, respectively. Compared with the control group, the particle size of PPNPs increased under acidic conditions. When PPNPs were stable at pH 9 and pH 11, the particle sizes were 174 nm and 166 nm, respectively. Compared with the control group, the particle size of PPNPs decreased under alkaline conditions. The particle size of PPNPs did not change significantly in a pH 7 environment. As pH increased, the absolute value of the zeta potential of PPNPs increased, from 5.55 mV at pH 3 to 23.9 mV at pH 11. Unlike others, the zeta potential was positive at pH 3.

[0058] The effects of temperature on PPNPs were simulated by equilibrating the PPNPs at different temperatures (-20, 4, 25, 27, and 60°C). At freezing temperature (-20°C), the particle size of the PPNPs increased by 15 nm, and the absolute value of the zeta potential decreased by 0.63 mV. Refrigeration temperature (-4°C), simulated room temperature (25°C), simulated human body temperature (37°C), and simulated high temperature (60°C) had no effect on the zeta potential, particle size, or PDI of the PPNPs.

[0059] The concentration and type of ions in a solution affect the thickness of the double layer. High ionic strength can compress the double layer, reducing the absolute value of the zeta potential, affecting electrostatic interactions between particles, and thus their stability. The isoelectric point of camel milk proteins (such as casein) is approximately pH 4.6. At pH 3–5, proteins are near or at their isoelectric point, with net surface charge approaching zero. This weakens electrostatic repulsion, leading to aggregation of particles through hydrophobic interactions or hydrogen bonding, resulting in larger aggregates. During freezing, water freezes to form ice crystals, which squeeze PPNPs closer together and may lead to aggregation. The denaturation temperature of milk proteins is typically ≥70°C; temperatures at 60°C may only induce partial conformational changes, insufficient to trigger large-scale aggregation.

[0060] In summary, this study successfully prepared camel milk protein-polysaccharide composite nanoparticles (PPNPs) through lactic acid bacteria fermentation and ultrasonic treatment. Scanning electron microscopy revealed that the PPNPs exhibited a uniform spherical structure, with a particle size that met the standard requirements for nanoparticles in the food industry, paving the way for their application in food-grade functional nanomaterials. PPNPs exhibited good stability in aqueous solution, possessing certain osmotic pressure resistance, alkaline resistance, and high temperature resistance, making them suitable for conventional dairy processing, storage, and transportation conditions, such as low-temperature, long-term pasteurization (LTLT).

[0061] In terms of antioxidant properties, PPNPs obtained after 11 minutes of ultrasonic treatment demonstrated superior antioxidant capacity compared to unfermented samples and other treatment groups, demonstrating promising potential for enhancing immune function and assisting in the treatment of chronic diseases. Lactic acid bacteria fermentation partially degrades camel milk proteins into small bioactive peptides, which not only enhances the stability of PPNPs but also their nutritional properties. The synergistic effect of fermentation and ultrasonication also improves the surface wettability of PPNPs, imparting them with a more hydrophilic-lipophilic neutral profile, providing a theoretical basis for their co-encapsulation with probiotics and prebiotics.

[0062] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing camel milk protein-polysaccharide composite nanoparticles, characterized in that: The following steps are involved: (1) Inoculate Lactobacillus plantarum into camel milk, ferment at 26-30°C for 40-50 hours, centrifuge, and collect the supernatant; (2) The supernatant was ultrasonically treated in an ice-water bath. After the ultrasonic treatment, anhydrous ethanol was added for precipitation, and then the supernatant was centrifuged and dialyzed to remove impurities, and finally camel milk protein-polysaccharide composite nanoparticles were obtained.

2. The preparation method according to claim 1, characterized in that The inoculation amount of Lactobacillus plantarum in step (1) is 1.5-3%.

3. The preparation method according to claim 1 or 2, characterized in that The Lactobacillus plantarum-like strain is Lactobacillus plantarum-like strain LR-1.

4. The preparation method according to claim 1, characterized in that In step (1), the fermentation temperature is 31-33°C and the fermentation time is 46-48 hours.

5. The preparation method according to claim 1, characterized in that The ultrasonic treatment conditions in step (2) are 20~25kHz, 400~450W.

6. The preparation method according to claim 1, characterized in that In step (2), the ultrasonic treatment uses a pulse mode of 2 s on and 2 s off, and the ultrasonic time is 3 to 15 minutes.

7. The preparation method according to claim 1, characterized in that The temperature of the ice water bath in step (2) is 10±0.2°C.

8. The preparation method according to claim 1, characterized in that Dialysis removes impurities including small molecule monosaccharides and ethanol.

9. A camel milk protein-polysaccharide composite nanoparticle, characterized in that: It is prepared by the method according to any one of claims 1 to 8.

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