Pet treat formulation loaded with probiotics to modulate gut health and relieve inflammation

Abstract

The application discloses a pet snack formula loaded with probiotics for adjusting intestinal health and relieving inflammation, relates to the technical field of functional pet snacks, and uses casein and sodium alginate as nanoparticle raw materials, wraps Lactobacillus plantarum in the nanoparticles, loads the same using a gel matrix, uses 3D printing technology to prepare the functional pet snacks, studies the optimal preparation conditions of the casein and sodium alginate nanoparticles and the protection effect of the gel ink on the probiotics, and evaluates the effect of relieving pet colonitis and adjusting intestinal health through animal experiments. The application uses nanoparticles to wrap the probiotics, improves the preservation activity of the probiotics, uses a lotus root starch compound system gel as a blank pet snack gel matrix, loads the probiotic nanoparticles to prepare the pet snacks, further protects the activity of the probiotics, and obtains the functional pet snacks with the functions of improving immunity and resisting colonitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional pet snacks, in particular to a functional pet snack prepared by using casein and sodium alginate as raw materials, adding lactobacillus plantarum to prepare nanoparticles rich in probiotics, then loading the above-mentioned probiotic nanoparticles on lotus root starch hydrogel, and using 3D printing technology to prepare the functional pet snack, which can achieve the functions of relieving pet colitis and regulating intestinal health. BACKGROUND

[0002] With the improvement of people's living standards, pets are loved by more and more families, and their health has become a topic of increasing concern. At the same time, pet disease problems also emerge in an endless stream. First, antibiotic drugs are common in pet clinics. Correct use of antibiotic drugs can effectively cure sick pets and reduce the occurrence of drug side effects. However, due to the immaturity of pet disease diagnosis and treatment technology, improper use of drugs is very common, especially in the treatment of bacterial infectious diseases. The abuse of antibiotics can lead to bacterial resistance, and excessive drug residues can destroy the balance of intestinal flora, making pets prone to intestinal diseases such as constipation, diarrhea, and indigestion, which seriously endanger the health of pets. Second, due to changes in pet diet and living environment and other various internal and external factors, pet intestinal flora imbalance occurs from time to time, which in turn can lead to a series of intestinal diseases, such as diarrhea, abdominal pain, nausea and vomiting, and even other complications such as diabetes and liver damage. Under this background, probiotics, as a natural, healthy antibiotic substitute and effective intestinal regulator, have the characteristics of safety, non-toxicity, no accumulation, no pollution, and no drug resistance, and have a beneficial effect on pet health. Functional pet food containing probiotics such as lactobacillus plantarum and bifidobacterium can improve intestinal health, compete for adhesion to the intestinal epithelium in the intestine, strengthen the intestinal mucosal barrier, help improve intestinal microbiota, regulate intestinal health, promote nutrient absorption, help absorption and digestion of nutrients, and improve the nutritional utilization rate of pets, enhance the body's immunity, play an anti-inflammatory role by reducing the body's nuclear factor-B (NF-kB) response, and help restore the health of pets prone to gastrointestinal diseases. It can also improve diarrhea caused by environmental changes, dietary changes, or long-term use of antibiotics, and reduce gastrointestinal discomfort in pets. However, there are challenges in ensuring that probiotics have high activity and functionality in various aspects such as preparation, processing, and gastrointestinal digestion. One of the potential methods to protect and deliver probiotics is to encapsulate probiotics with nanoparticles. Nanotechnology has shown great potential in encapsulating and delivering bioactive substances in recent years. Nanoparticles wrapped around probiotics can potentially protect their activity, allowing probiotics to avoid the influence of the external environment as much as possible, and help extend the life of probiotics.

[0003] Food 3D printing technology offers advantages such as customization, minimal waste, and no need for equipment replacement, attracting widespread attention in recent years for the production of customized and functional foods. In the field of probiotics, 3D printing technology allows for the customization of probiotic dosages and strains for use in probiotic foods to meet specific individual needs. To more effectively protect probiotics, this invention uses sodium caseinate (Cas) and sodium alginate (SA) as raw materials to prepare nanoparticles, uses gel ink as a carrier to load *Lactobacillus plantarum* coated with nanoparticles, and then uses 3D printing technology to manufacture products capable of effectively carrying and delivering probiotics.

[0004] This invention utilizes 3D printing technology to prepare functional pet food containing probiotics. Compared to traditional methods, it offers unique advantages: it allows for personalized customization based on each pet's specific needs, ensuring each pet receives the most suitable food; it protects the activity of probiotics, thereby increasing their survival rate; it precisely controls the volume and shape of the food, reducing waste caused by overproduction; and it can create pet foods with diverse shapes and rich flavors, enhancing pets' appetites; it can create food layers with different nutrient densities, ensuring pets gradually obtain the necessary nutrients during consumption, which helps them better absorb and utilize the nutrients in the food; furthermore, it is sustainable and environmentally friendly. In summary, 3D printing technology has significant advantages in preparing functional pet food containing probiotics. Summary of the Invention

[0005] Using casein and sodium alginate as nanoparticle raw materials, *Lactobacillus plantarum* was encapsulated in nanoparticles, loaded with a gel matrix, and prepared using 3D printing technology to produce functional pet snacks. The optimal preparation conditions of casein and sodium alginate nanoparticles and the protective effect of gel ink on probiotics were studied. Animal experiments were used to evaluate its effects in relieving pet colitis and regulating intestinal health.

[0006] The probiotic-loaded pet treat formula of this invention, which regulates intestinal health and relieves inflammation, is prepared from the following raw materials in parts by weight:

[0007] 1-20 parts lotus root starch

[0008] Xanthan gum 0.5-2 parts,

[0009] 0.5-2 parts whole egg powder,

[0010] 1-10 parts probiotic nanoparticles

[0011] Make up to 100 servings with purified water.

[0012] This preferred probiotic-loaded pet treat formula, designed to regulate gut health and alleviate inflammation, is prepared from the following ingredients in parts by weight:

[0013] 15 parts lotus root starch

[0014] 1.5 parts xanthan gum

[0015] 1 portion of whole egg powder

[0016] 10 portions of probiotic nanoparticles

[0017] Make up to 100 servings with purified water.

[0018] The protein-polysaccharide nanoparticle formulation is prepared from the following raw materials in parts by weight:

[0019] Casein 0.5-2 parts,

[0020] Sodium alginate 0.5-2 parts,

[0021] Add water to make up 100 servings.

[0022] The preferred protein-polysaccharide nanoparticle formulation is prepared from the following raw materials in parts by weight:

[0023] 1 part casein,

[0024] 1 part sodium alginate

[0025] Add water to make up 100 servings.

[0026] The preparation method of the protein-polysaccharide nanoparticles is carried out according to the following steps:

[0027] (1) Casein was dispersed in pure water at a concentration of 1 wt%;

[0028] (2) Sodium alginate was dispersed in pure water at a concentration of 1 wt%.

[0029] (3) Mix casein and sodium alginate in different proportions and stir thoroughly to make them evenly mixed;

[0030] (4) Adjust the pH of the mixed solution in step (3);

[0031] (5) The complex from step (4) is loaded into a dispensing bottle for testing.

[0032] The preferred ratio of casein and sodium alginate in step (3) is 1:1.

[0033] The preferred pH condition in step (4) is 4.0.

[0034] The preparation method of the probiotic nanoparticles is carried out according to the following steps:

[0035] (1) Prepare protein-polysaccharide nanoparticles according to the above method for later use;

[0036] (2) Centrifuge the Lactobacillus plantarum culture medium (8000 r / min) for 15 min to obtain bacterial sludge;

[0037] (3) Then add sterile distilled water to the obtained bacterial sludge, wherein the volume ratio of sterile distilled water to the above Lactobacillus plantarum culture medium is 1:1, and mix thoroughly.

[0038] (4) Centrifuge according to the centrifugation conditions in step (2) to obtain the washed bacterial sludge;

[0039] (5) Add an equal volume of the protein-polysaccharide nanoparticle solution from step (1) and mix thoroughly;

[0040] (6) Seal with sealing film to obtain probiotic-loaded nanoparticles.

[0041] The preparation method for probiotic-loaded pet treats that regulate gut health and alleviate inflammation is as follows:

[0042] (1) Lotus root starch was dispersed in purified water at a concentration of 15 wt%.

[0043] (2) Add xanthan gum and whole egg powder to the starch solution and stir thoroughly until completely dissolved;

[0044] (3) Place the mixed solution from step (2) into a water bath to gelatinize;

[0045] (4) Cool the gelatinized composite starch solution from step (3) to 40°C, add probiotic nanoparticles, and mix them evenly for later use.

[0046] (5) After cooling the pet treat raw materials in the hopper of step (4) to room temperature, use a 3D printer to perform 3D printing behavior analysis;

[0047] (6) The printed product obtained in step (5) is dried and then processed.

[0048] In step (2), the preferred concentration of whole egg powder is 1 wt%; the concentration of xanthan gum is 1.5 wt%.

[0049] The gelatinization conditions in step (3) are 80℃ for 40 min;

[0050] The concentration of probiotic nanoparticles in step (4) is 10%;

[0051] The preferred 3D printer parameters mentioned in step (5) are: pressure 35PSI, syringe size 18G, and printing speed 30mm / s;

[0052] The preferred drying method in step (6) is freeze drying.

[0053] The beneficial effects of this invention are as follows:

[0054] (1) The present invention uses nanoparticles to encapsulate probiotics, thereby improving the preservation activity of probiotics.

[0055] (2) This invention uses lotus root starch compound system gel as blank pet snack gel matrix, loads probiotic nanoparticles to prepare pet snacks, and further protects the activity of probiotics.

[0056] (3) In this invention, Lactobacillus plantarum is selected as a functional nutrient and added to a blank gel matrix to obtain a functional pet snack that can improve immunity and fight colitis. Attached Figure Description

[0057] Figure 1 This invention illustrates the effect of different pH values ​​on the potentials of Cas, SA, and the complex.

[0058] Figure 2 The images show the appearance of the casein-sodium alginate complex under different pH conditions in this invention.

[0059] Figure 3 This invention illustrates the effect of different mass ratios of casein and sodium alginate on the turbidity of the complex.

[0060] Figure 4 The images show the appearance of the casein-sodium alginate complex at different mass ratios in this invention.

[0061] Figure 5 This is for particle size analysis of the complex in this invention.

[0062] Figure 6 This is an observation of the microscopic morphology of probiotics encapsulated in nanoparticles in this invention.

[0063] Figure 7 This is a 3D printing effect image of pet snacks loaded with probiotic nanoparticles in this invention.

[0064] Figure 8 The numbers represent the number of probiotics that survive after simulated in vitro digestion in this invention (A represents free probiotics; B represents probiotics encapsulated with nanoparticles; C represents probiotics loaded with gel ink).

[0065] Figure 9 This refers to the length of the mouse colon in this invention.

[0066] Figure 10 This is for the analysis of mouse serum components in this invention. Detailed Implementation

[0067] The terminology used in this invention, unless otherwise specified, will generally be understood by those skilled in the art. The invention is described in further detail below with reference to specific embodiments and data. It should be noted that these embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0068] (1) Preparation of casein-sodium alginate complex solution

[0069] 10g each of sodium caseinate and sodium alginate was weighed and dissolved in 1000mL of distilled water at 25℃. The polysaccharide and protein solutions were mixed in a certain ratio and stirred at 800rpm until homogeneous, yielding mixed solutions with mass ratios of 3:1, 3:2, 1:1, 2:3, and 1:3. The pH of the system was adjusted to 2.0, 3.0, 4.0, 5.0, and 6.0 with 0.5M HCl, respectively, to obtain casein-sodium alginate complex (Cas-SA) solutions.

[0070] (2) Preparation of probiotics

[0071] Lactobacillus plantarum 21804 (purchased from the China Industrial Microbial Culture Collection Center) was cryopreserved and streaked onto MRS plates using an aseptic inoculation loop. The plates were incubated at 37°C for 36 hours. Single colonies were picked and cultured in MRS liquid medium at 37°C and 140 rpm for 24 hours. The Lactobacillus plantarum culture was then centrifuged at 8000 rpm and 4°C for 15 minutes to collect the bacterial cells. The cells were resuspended in an equal volume of 0.85% sterile physiological saline. The bacterial suspension was then serially diluted (10⁻⁶ ppm) to different concentrations. -4 10 -5 10 -6 10 -7 ), take 100 μL of the diluted bacterial solution and spread it on MRS solid medium, incubate in a constant temperature incubator for 24 h and then count the viable bacteria, and record the number of viable bacteria as N (cfu / g).

[0072] (3) Preparation of probiotic-loaded nanoparticles

[0073] Prepare Cas / SA-NPS according to step (1). Centrifuge 9 mL of Lactobacillus plantarum culture medium (8000 r / min) for 15 min to obtain bacterial sludge. Then add sterile distilled water to the obtained bacterial sludge to 9 mL and mix thoroughly. Centrifuge under the same conditions to obtain washed bacterial sludge. Add an equal volume of 9 mL of nanoparticle solution and mix well. Seal with sealing film to obtain probiotic-loaded nanoparticles.

[0074] (4) Freeze-drying of probiotic nanoparticles

[0075] The prepared probiotic-loaded nanoparticle solution was dispensed into petri dishes, wrapped with plastic wrap, and pre-frozen at -80°C for 6 hours. The pre-frozen solution was then placed in a vacuum freeze dryer, maintaining a vacuum of -100 MPa, and freeze-dried for 36 hours.

[0076] (5) Determination of probiotic encapsulation rate

[0077] Dissolve 1g of probiotic-loaded nanoparticles in PBS solution and place the solution in a sterile tube. Place the solution on a shaker and mix thoroughly. Shake conditions: temperature 37℃, shaker speed 100 rpm, shake time 1 hour. Then, take 0.5mL of the solution and perform serial dilutions in 4.5mL of physiological saline. Take 10g of each solution... 4 10 5 10 6 100 μL of bacterial suspensions at multiple concentrations were spread and counted on solid MRS culture dishes, and the viable cell count was recorded as N1 (cfu / g).

[0078]

[0079] Note: N (cfu / g) is the original viable count.

[0080] (6) Observation by laser confocal microscopy (CLSM)

[0081] The loading of probiotics on nanoparticles was observed using a Leica TCS SP5 CLSM system. Using Syto9 dye, 3 μL of the dye premix was added to 1 mL of the probiotic-loaded nanoparticle solution, mixed thoroughly, and incubated at 25°C in the dark for 15 min. 10 μL of the stained sample was then dropped onto a glass slide, covered with a coverslip, and observed using a laser confocal microscopy system at a wavelength of 488 nm.

[0082] (7) Preparation of ink loaded with probiotic nanoparticles

[0083] Lotus root starch (LRS) at a concentration of 15% (w / v) was weighed and dissolved in distilled water at 25°C. Xanthan gum (XG) at a concentration of 1.5% (w / v) was also weighed and dissolved in the LRS solution at 25°C. The mixture was stirred at 800 rpm until homogeneous. Different concentrations of whole egg powder (0%, 0.5%, 1.0%, 1.5%, and 2.0%) were added to the mixed solution, and stirring continued until homogeneous, resulting in mixed solutions of different concentrations of whole egg powder. These mixed solutions were placed in an 80°C magnetically stirred water bath and gelatinized for 40 minutes to obtain ink. After gelatinization, the mixture was cooled to 45°C, and probiotic-loaded nanoparticles were added to the hydrogel (the amount of probiotic nanoparticles added was set at 10%). After stirring until homogeneous, the mixture was loaded into the printer cartridge and refluxed at 4°C for 12 hours.

[0084] (8) 3D Printing Analysis

[0085] After the gelatinized sample in (5) was naturally cooled to room temperature, it was placed into the printer cartridge, sealed with plastic wrap, and then placed in a 4°C refrigerator for recrystallization for 12 hours to obtain 3D printing ink. The printing parameters were set as follows: the print head size was 18G, the printing pressure was 35PSI, and the printing speed was 30mm / s.

[0086] (9) In vitro simulated gastrointestinal digestion test

[0087] Stomach stage: Prepare gastric digestion stock solution according to Table 1. Heat the stock solution to 37℃ and add pepsin to achieve a protease activity of 2000 U / mL. Then adjust the pH to 2.0 to obtain simulated gastric juice (SGF). Take 0.1 mL of probiotic suspension, 0.1 g of lyophilized nanoparticles, and 0.1 g of 3D printed sample and add them to 9 mL of the prepared gastric juice. Shake thoroughly to mix and place on a shaker. Set the shaker temperature to 37℃, the rotation speed to 100 r / min, and the time to 2 h.

[0088] Intestinal stage: Prepare intestinal digestion stock solution according to Table 1. Heat the stock solution to 37℃, then add pancreatic enzymes, pancreatic lipase, and porcine bile salts to achieve a protease activity of 100 U / mL, a lipase activity of 2000 U / mL, and a bile salt concentration of 10 mM. After thorough mixing, adjust the pH to 8.0 to obtain simulated intestinal fluid (SIF). Add 5 mL of the gastric digestion solution to 5 mL of the prepared intestinal fluid, mix well, and place on a shaker at 37℃ and 100 r / min for 2, 4, and 6 hours. Dilute 0.5 mL of the digested intestinal fluid using a gradient concentration method, then take 10 mL of the solution... 2 10 3 10 4 10 5 10 6 100 μL of gradient dilution solution was used for coating and counting.

[0089] Table 1. Composition of reserve liquid in each stage

[0090]

[0091] (10) Establishing animal models

[0092] Dextran sulfate sodium salt (DSS)-induced inflammatory bowel disease (IBD) animal models are currently the most widely used method for modeling IBD. This method is rapid, convenient, and dose-dependent, and can effectively simulate human symptoms. Forty male ICR mice, 7-8 weeks old and weighing (23±2) g, were housed in an animal center with a 12h:12h circadian rhythm, an ambient temperature of 23-25℃, and an ambient humidity of 40%-60%. After one week of acclimatization, the experiment officially began. The experimental groups were: normal control group, model group, free probiotic group, nanoparticle probiotic group, and gel-embedded group. From day 1 to day 7, except for the control group mice which had free access to sterile water, the other groups drank 3% DSS aqueous solution to establish an acute colitis model. From day 8 to day 14, all five groups drank sterile water.

[0093] (11) Administration

[0094] Mice in the control and model groups were administered 100 μL of PBS solution by gavage daily. Mice in the free probiotic group were administered 100 μL of bacterial suspension by gavage daily. Mice in the nanoparticle-encapsulated probiotic group were administered 100 μL of nanoparticles by gavage daily. Mice in the gel ink-encapsulated group were administered 1000 μL of probiotic ink by gavage daily (morning and afternoon).

[0095] (12) Mouse weight recording and colon analysis

[0096] The initial and final weights of 40 mice were recorded. The mice were fasted for 12 hours before weighing. The weight gain rate was calculated based on the initial and final weights using the following formula: Weight gain rate (%) = [(final weight - initial weight) / initial weight] × 100%.

[0097] (13) Enzyme-linked immunosorbent assay (ELISA)

[0098] Blood was collected from mouse eyeballs and placed obliquely on ice for 3 hours, then centrifuged at 3000 rpm for 10 minutes to separate serum. TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were used to detect their expression levels in serum according to the manufacturer's instructions. Each group was tested three times, and the average expression level for each group was calculated based on the standard curve.

[0099] Research Results

[0100] (1) Effect of pH on the zeta potential of the complex

[0101] The zeta potential is a physical quantity that characterizes the charged state of a particle surface. By measuring the velocity or electrophoretic mobility of particles in a dispersion system, the value of the zeta potential can be calculated, thereby assessing the surface charge properties of the particles and the type of interaction between particles. It is a method used to characterize the charge state and stability of particles or interfaces in a dispersion system. Figure 1 The zeta potentials of Cas, SA, and the complex at different pH values ​​were displayed. The results showed that the isoelectric point of casein is around pH 4.5. At pH 4.0, the zeta potential of casein alone was positive, while that of sodium alginate was negative, indicating that electrostatic adsorption occurs between the negatively charged sodium alginate and the positively charged casein at this pH. This adsorption is achieved by shielding water molecules with hydrophilic groups to form an ion cloud, thus creating a stable complex structure, reducing mutual repulsion and cohesion, thereby improving stability and inhibiting aggregation. When the pH is less than 4.0, as the pH decreases, the positive charge of casein increases, causing its potential to shift towards the positive direction. Simultaneously, the negative charge of sodium alginate decreases, causing its potential to shift towards the negative direction. The absolute value of the complex potential decreases, and the charge attraction weakens, which may lead to a decrease in the stability of the complex.

[153] This leads to the aggregation and precipitation of the complex, just as... Figure 2 As shown, the complex exhibited varying degrees of flocculation and precipitation at pH values ​​of 2.0 and 3.0. Therefore, based on the results, a complex with optimal stability was prepared at pH 4.0 for subsequent experiments.

[0102] (2) Turbidity analysis and particle size analysis of the complex

[0103] The Cas-SA ratio determines the interaction between proteins and polysaccharides. Figure 3 The effect of different mass ratios on the turbidity of the complex was shown. Figure 4The appearance of the complex at different mass ratios is shown. At a total concentration of 0.5% and pH 4.0, when the Cas content was high, the unencapsulated Cas flocculated and precipitated in the solution. This is related to the transformation of Cas into insoluble micelles at its isoelectric point. This may be due to the increased number of Cas molecules in the solution, leading to increased micelle aggregation caused by hydrophobic interactions. This results in more Cas aggregating into larger aggregates, eventually forming visible flocs. As the SA concentration increased, the turbidity of the complex gradually decreased, and the Cas flocculation disappeared. This indicates that the addition of SA improved the self-aggregation of Cas at its isoelectric point, suggesting an interaction between the two. This interaction may be electrostatic interaction, leading to the formation of a stable complex with good dispersibility, preventing the self-aggregation of Cas molecules. When the SA and Cas concentrations were the same, the turbidity of the complex was the lowest. Further increasing the SA concentration increased the turbidity of the complex. This may be because the adsorption of Cas by SA reaches saturation at a mass ratio of 1:1. Therefore, to fully utilize both substances and obtain a better complex, a Cas:SA ratio of 1:1 (w / w) was chosen.

[0104] Particle size represents the dimensions of a complex. By measuring the particle size, we can understand the overall size of the complex, that is, the size range of the complex in three-dimensional space. This is very important for studying the functionality and stability of the complex in different applications. Figure 5 The particle size and particle size distribution peaks of a 1:1 mixture of casein and sodium alginate, as well as the mixture after adjusting the pH to 4.0, are shown. The particle size distribution peaks show a single peak at pH 4.0, indicating a uniform particle morphology and complete adsorption of sodium alginate and casein. However, under pH conditions, three peaks are observed, suggesting that sodium alginate and casein are not completely adsorbed. Furthermore, the particle size indicates that the composite has a particle size of approximately 200 nm, falling within the nanoparticle range.

[0105] (3) Probiotic encapsulation rate

[0106] The encapsulation efficiency of nanoparticles for probiotics is one of the important indicators for evaluating the encapsulation performance of nanoparticles. Using the plate count method, the viable count of *Lactobacillus plantarum* before and after encapsulation with nanoparticles both reached 10⁻⁶. 8 Based on the cfu / g, the encapsulation efficiency of *Lactobacillus plantarum* was calculated to be 80.13% ± 1.51%, which is considered satisfactory compared to other microcapsules encapsulating *Lactobacillus plantarum*. (Guo Chunyan et al.)

[155] Using acrylic resin and SA as a double-layer coating wall material, *Lactobacillus plantarum* was encapsulated, and the encapsulation rate reached 82.48%. In conclusion, the encapsulation rate of nanoparticles for probiotics reflects the effective preservation ability of nanoparticles during the encapsulation process. A high encapsulation rate means that more probiotics can be successfully encapsulated in the nanoparticles, thereby maximizing the stability and survival rate of probiotics.

[0107] (4) Laser confocal analysis

[0108] Syto9 is a commonly used nucleic acid dye, frequently used in biological and biomedical research. It is a green fluorescent dye that binds highly selectively to DNA, thus enabling the quantification and visualization of nucleic acids. This dye can penetrate living bacterial cells and bind to the bacterial DNA, causing it to fluoresce green. Using this dye, one can observe whether *Lactobacillus plantarum* is encapsulated by nanoparticles, thus indirectly assessing the encapsulation effect of *Lactobacillus plantarum*. Figure 6 As shown, the rod-shaped structure of *Lactobacillus plantarum* is clearly visible and evenly distributed within the field of view. Furthermore, the presence of incompletely encapsulated *Lactobacillus plantarum* was observed through the green-stained specimens, although their numbers were small, indicating a relatively good encapsulation effect.

[0109] (5) 3D Printing Analysis

[0110] The 3D printing effect of pet treats loaded with probiotic nanoparticles is as follows: Figure 7 As shown, the state of the 3D printing ink did not change significantly after being loaded with probiotics, and the printed samples all maintained the intended shape of the model after 3D printing. The extrusion effect of the printed lines was uniform and consistent. In the star model, the printing effect of the five corners was clear and distinct, and the angles were uniform. In addition, when stacking a 10mm high ring, the ink did not overflow or become discontinuous, and the ring model was printed well. In summary, it is evident that loading probiotic nanoparticles did not affect the 3D printing effect of the ink, which is of positive significance for further research and development of 3D printed products loaded with probiotic nanoparticles.

[0111] (6) Gastrointestinal stability analysis of probiotics

[0112] The purpose of the in vitro simulated digestion test of probiotics is to explore the survival of probiotics under different conditions during the gastrointestinal digestion process, so as to verify the protective effect of nanoparticles and gel ink on probiotics. Figure 8 The figure shows the number of viable Lactobacillus plantarum bacteria after simulated digestion in vitro. At the end of the simulated gastric digestion, i.e., before the start of the intestinal digestion phase (0h) in the figure, the number of free Lactobacillus plantarum bacteria decreased to 10. 3The cfu / g concentration decreased by five orders of magnitude compared to before digestion. However, both the *Lactobacillus plantarum* encapsulated with nanoparticles and the *Lactobacillus plantarum* encapsulated with nanoparticles loaded with gel ink maintained high activity after simulated gastric digestion, with a viable count of 10cfu / g. 6 The CFU / g concentration decreased by two orders of magnitude compared to before digestion, indicating that the nanoparticle coating helps *Lactobacillus plantarum* resist the acidic environment of the stomach. After 2 hours of simulated intestinal digestion, the number of viable *Lactobacillus plantarum* samples varied significantly. No viable bacteria were detected in the free state, possibly due to the strong acidity of the simulated stomach and the high bile salt content of the simulated intestine, which damaged the cell wall of *Lactobacillus plantarum*, leading to its inactivation. Furthermore, the number of viable *Lactobacillus plantarum* coated with nanoparticles reached 10 after simulated intestinal digestion. 4 The cfu / g of *Lactobacillus plantarum* loaded with gel ink can achieve a viable count of 10 after simulated intestinal digestion. 5 CFU / g. In summary, encapsulating nanoparticles enhances the ability of *Lactobacillus plantarum* to resist adverse environments. Loading with gel ink does not affect the survival rate of *Lactobacillus plantarum* and even improves its resistance to the environment. This provides a reference for the development of 3D printed products loaded with probiotics and indicates that nanoparticles and gel ink can be used as a means to protect probiotics and improve their survival rate and stability during gastrointestinal digestion.

[0113] (7) Effects of Lactobacillus plantarum on mouse body weight under different conditions

[0114] According to the data in Table 2, the weight gain rate of the model group mice was significantly lower than that of the blank control group. Under different treatments involving *Lactobacillus plantarum* feeding, although the mice showed varying degrees of weight gain, they still failed to reach the level of the blank control group. Specifically, the weight gain rate of the model group mice was significantly inhibited, far lower than that of the uninduced blank control group. In contrast, the weight gain of mice treated with *Lactobacillus plantarum* improved, but still showed a trend of falling short of the blank control group. This indicates that although *Lactobacillus plantarum* has a certain positive regulatory effect on mouse weight, its weight gain effect has not yet fully recovered to the level of a healthy state.

[0115] Table 2. Changes in mouse body weight

[0116]

[0117] (8) Effects of Lactobacillus plantarum on disease symptom in mice with colitis under different conditions

[0118] Colon length, as a primary indicator for assessing the severity of colitis in mice, played a crucial role in this study. For example... Figure 9As shown, after two weeks of feeding with *Lactobacillus plantarum*, the colon length of mice in different treatment groups showed significant differences. The shortest colon length was 6.9 cm in the model group, significantly lower than the 8.16 cm in the control group. Compared to the model group, the colon length of mice fed with free probiotics was 7.43 cm, an increase of 0.53 cm, showing a significant increase. After feeding with nanoparticles or encapsulated probiotic pet treats, the colon length of mice was significantly higher than that of the model group, reaching 7.53 cm in the nanoparticle group and 7.93 cm in the probiotic pet treat group, which was closest to the control group. These results indicate that probiotic feeding can effectively improve colonic inflammation in mice, and the therapeutic effect is particularly significant after using nanoparticles or encapsulated probiotic pet treats.

[0119] (9) Serum TNF-α and IL-6 expression level analysis

[0120] After measuring the levels of inflammatory cytokines in mouse serum using ELISA, it was found that the serum levels of pro-inflammatory cytokines IL-6 and TNF-α in the model group mice were significantly elevated. The IL-6 level was 33.33 pg / mL, an increase of 12.14 pg / mL compared to the control group; the TNF-α level was 178.81 pg / mL, an increase of 39.95 pg / mL compared to the control group, indicating a significant intestinal inflammatory state. However, after intervention with *Lactobacillus plantarum* under different conditions, the elevated cytokine levels were significantly alleviated. The probiotic pet snack group showed relatively better relief, most closely resembling the control group, with an IL-6 level of 23.99 pg / mL, a decrease of 9.34 pg / mL compared to the model group; and a TNF-α level of 143.68 pg / mL, a decrease of 35.13 pg / mL compared to the model group. Figure 10 As shown in the figure, these results indicate that *Lactobacillus plantarum* effectively improved the intestinal immune barrier in mice by reducing the levels of pro-inflammatory cytokines. This regulatory effect may help alleviate the inflammatory response and maintain the stability of intestinal health.

Claims

1. A probiotic-loaded pet treat, characterized in that... It is prepared from the following raw materials in parts by weight: 1-20 parts lotus root starch Xanthan gum 0.5-2 parts, 0.5-2 parts whole egg powder, 1-10 parts probiotic nanoparticles Make up the total to 100 portions with water; The preparation method of the probiotic nanoparticles is carried out according to the following steps: (1) Prepare protein-polysaccharide nanoparticles for later use; (2) Centrifuge the Lactobacillus plantarum culture medium at 8000 r / min for 15 min to obtain bacterial sludge; (3) Then add sterile distilled water to the obtained bacterial sludge, wherein the volume ratio of sterile distilled water to the above Lactobacillus plantarum culture medium is 1:1, and mix thoroughly. (4) Centrifuge according to the centrifugation conditions in step (2) to obtain the washed bacterial sludge; (5) Add an equal volume of the protein-polysaccharide nanoparticle solution from step (1) to the washed bacterial mud and mix thoroughly. (6) Seal with sealing film to obtain probiotic-loaded nanoparticles; The preparation method of the protein-polysaccharide nanoparticles is carried out according to the following steps: (1) Casein was dispersed in pure water at a concentration of 1 wt%. (2) Sodium alginate was dispersed in purified water at a concentration of 1 wt%. (3) Mix casein and sodium alginate in a 1:1 ratio and stir thoroughly to ensure even mixing; (4) Adjust the pH of the mixed solution in step (3) to 4.0; (5) The complex from step (4) is loaded into a dispenser.

2. The probiotic-loaded pet treat according to claim 1, characterized in that... It is prepared from the following raw materials in parts by weight: 15 parts lotus root starch 1.5 parts xanthan gum 1 portion of whole egg powder 10 portions of probiotic nanoparticles Make up the total to 100 portions with water.

3. A method for preparing a probiotic-loaded pet treat according to claim 1 or 2, characterized in that... Follow these steps: (1) Lotus root starch was dispersed in purified water at a concentration of 15% (w / v); (2) Add xanthan gum and whole egg powder to the starch solution and stir thoroughly until completely dissolved; (3) Place the mixed solution from step (2) into a water bath to gelatinize; (4) Cool the gelatinized composite starch paste from step (3) to 40°C, add probiotic nanoparticles, and mix them evenly for later use. (5) After cooling the pet treat ingredients in the hopper in step (4) to room temperature, use a 3D printer to perform 3D printing behavior analysis; (6) The printed product obtained in step (5) is dried and then processed.

4. The method for preparing a probiotic-loaded pet treat according to claim 3, characterized in that... The concentration of whole egg powder in step (2) is 1% (w / v); the concentration of xanthan gum is 1.5% (w / v); and the gelatinization conditions in step (3) are 80℃ for 40 min.

5. The method for preparing a probiotic-loaded pet treat according to claim 3, characterized in that... The amount of probiotic nanoparticles added in step (4) is 10%.

6. The method for preparing a probiotic-loaded pet treat according to claim 3, characterized in that... The 3D printer parameters in step (5) are: pressure 35 PSI, syringe size 18 G, and printing speed 30 mm / s; the drying method in step (6) is: freeze drying.

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