Preparation and application of iron chelate for reducing degradation of lactoferrin in stomachs of dogs and cats

Abstract

The invention discloses preparation and application of an iron chelate for reducing degradation of lactoferrin in stomachs of dogs and cats, and relates to the technical field of metal chelate preparation. Polysaccharide extracts of okra and tremella are chelated with Fe < 3 + > to prepare the iron chelate, and the iron chelate can increase Fe < 3 + > concentration and liquid viscosity in a local environment around lactoferrin in the stomachs of dogs and cats to inhibit Fe < 3 + > from being separated from the lactoferrin, so that the structural stability of the lactoferrin in the stomachs of dogs and cats is improved, and degradation of the lactoferrin is reduced. The iron chelate provided by the invention can be applied to or matched with related products of lactoferrin.

Description

Technical Field

[0001] This invention relates to the field of metal chelate preparation technology, specifically to a method for preparing iron chelates that can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, and its application in dog and cat food. Background Technology

[0002] Lactoferrin is an iron-binding glycoprotein naturally found in mammalian milk. It possesses various biological activities, including enhancing immunity (such as promoting lymphocyte proliferation), regulating bone marrow cell growth, and exhibiting antibacterial and antiviral effects (see Research Progress on Lactoferrin Bioactive Peptides and Their Functional Mechanisms, Shi Pujie; Research Progress on the Biological Functions and Detection of Lactoferrin, Song Huimin). Currently, lactoferrin is widely used in canine and feline food, such as in canine and feline milk powder (a functional formula milk powder suitable for both cats and dogs and its preparation method, CN202411878863.9) and canine and feline staple foods (a kitten food without palatability enhancers and its preparation method, CN202410966672.1; a pet staple food that enhances canine disease resistance and regulates gut microbiota, CN201610906205.5).

[0003] Fe 3+ The presence of Fe3+ is fundamental to the functionality and stability of lactoferrin. However, in an acidic environment, lactoferrin's structure is unstable and expands, causing the Fe3+ ions encapsulated within it to easily escape and expose the internal structure. Therefore, lactoferrin is easily degraded by pepsin and loses its activity after entering the stomach of dogs and cats. Current research has explored various methods to reduce lactoferrin's retention rate in the stomach and inhibit its degradation. These include using caffeine to bind and modify lactoferrin in vitro (a method to inhibit lactoferrin digestion in humans, CN202310862516.6) to form a complex, or using porous starch to encapsulate lactoferrin in vitro (a method to inhibit lactoferrin digestion in humans, CN201610630888.6) to prepare lactoferrin microcapsules to inhibit hydrolysis and degradation in human gastric juice. However, these methods modify and encapsulate lactoferrin before ingestion and do not address the inherent instability, expansion, and Fe3+ ion release in acidic environments. 3+ The problem of it easily coming off.

[0004] Okra is an annual herbaceous plant belonging to the Malvaceae family. Its young fruits are rich in polysaccharides, dietary fiber, and flavonoids. Tremella, also known as white fungus or snow fungus, is a valuable edible fungus whose fruiting body is rich in polysaccharides, proteins, and minerals. Currently, there are no reports on studies using extracts prepared from okra and tremella, and then ingesting them together with lactoferrin to reduce the degradation of lactoferrin in the stomachs of dogs and cats. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an iron chelate that can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, and its application in dog and cat food.

[0006] In a first aspect, the present invention provides a method for preparing an iron chelate that reduces the degradation of lactoferrin in the gastric environment of dogs and cats, comprising the following steps: (1) Preparation of polysaccharide extract: Take dried okra fruit and dried tremella fruit body in proportion, pulverize them together, add distilled water for polysaccharide extraction and apply ultrasonic-assisted extraction, then add ethanol for alcohol precipitation, reconstitute the precipitate with distilled water, centrifuge and retain the supernatant, which is the polysaccharide extract.

[0007] (2) Preparation of iron chelate: Dissolve trisodium citrate in polysaccharide extract, adjust temperature and pH, add ferric chloride solution to carry out chelation reaction, dry after chelation reaction to obtain solid product, wash the solid product with ethanol solution and dry to obtain iron chelate.

[0008] Preparation of the polysaccharide extract in step (1) above: The dried okra fruit is preferably obtained by naturally sun-drying or freeze-drying tender okra fruits harvested 8-12 days after flowering, or by using fresh tender okra fruits 8-12 days after flowering. The dried tremella fruiting body is preferably obtained by naturally sun-drying or freeze-drying mature tremella fruiting bodies harvested 5-15 days after the fruiting bodies have fully expanded, or by using fresh mature tremella fruiting bodies 5-15 days after the fruiting bodies have fully expanded. The dried okra fruit and dried tremella fruiting body are mixed in a specific mass ratio of 4:1 to 1:1.

[0009] Preparation of polysaccharide extract in step (1) above: When adding distilled water for polysaccharide extraction, the material-to-liquid ratio is 1:20~1:30 (g / mL); when performing ultrasonic-assisted extraction, the ultrasonic frequency is 20~60kHz, the ultrasonic power density is 20~40W / L, the ultrasonic time is 30~60min, and the ultrasonic extraction temperature is 40~60℃.

[0010] Preparation of polysaccharide extract in step (1) above: For alcohol precipitation, ethanol is added so that the volume ratio of ethanol in the final solution is 70%~90%, the alcohol precipitation temperature is 2~8℃, and the alcohol precipitation time is 12~24h.

[0011] Preparation of polysaccharide extract in step (1) above: When adding distilled water for redissolution, the final volume is 20%~25% of the volume of the extract before ultrasonic precipitation with ethanol.

[0012] In step (2) above, the preparation of iron chelates is as follows: trisodium citrate is added to the polysaccharide extract at a ratio of 1-4 g / L, the temperature is adjusted to 60-80℃, and the pH is adjusted to 5-6; during the chelation reaction, ferric chloride is added to the polysaccharide extract at a ratio of 5-10 g / L, and the chelation reaction is carried out for 40-80 min.

[0013] In step (2) above, the preparation of iron chelate is as follows: when washing with ethanol solution, the solid-to-ethanol solution ratio is 0.1~0.2g / mL, and the volume percentage of ethanol in the ethanol solution is 70%~90%.

[0014] A second aspect of the present invention provides the application of the iron chelate prepared by the above method in dog and cat food.

[0015] The method of application is to feed the iron chelate prepared by the above method together with lactoferrin to dogs and cats, with the mass ratio of iron chelate to lactoferrin being 1:5 to 1:10.

[0016] Furthermore, the above-mentioned feeding of dogs and cats together specifically refers to feeding the iron chelate and lactoferrin simultaneously, or feeding the iron chelate and lactoferrin sequentially within a short period of time.

[0017] Furthermore, the aforementioned iron chelates can be fed directly to dogs and cats, mixed into dog and cat food (or treats), or added as a formula component to dog and cat food (or treats).

[0018] Furthermore, the iron chelates prepared by the above method can be applied to other pets such as foxes, hamsters, and rabbits.

[0019] The present invention has the following beneficial effects: The reason why traditional lactoferrin products are easily degraded after consumption is that lactoferrin consists of two parts: the N-lobes (containing two subdomains, N1 and N2) and the C-lobes (containing two subdomains, C1 and C2). Each lobe binds an Fe... 3+ At this point, the two lobes close like two pincers, maintaining a stable, compact, spherical closed conformation. (Fe) 3+ This is the basis for the functionality and stability of lactoferrin. Lactoferrin has an isoelectric point (PI) of 8-9. In an acidic environment, the amino and carboxyl groups on lactoferrin protonate, resulting in a large positive charge and strong electrostatic repulsion. Furthermore, the acidic environment disrupts the hydrogen bonds and salt bridges that maintain structural stability in lactoferrin, ultimately causing the entire lactoferrin structure to expand and change from a closed conformation to a loose conformation. 3+ It easily separates from lactoferrin. It loses Fe. 3+ The core lactoferrin, with its internal structure further exposed, is readily degraded by pepsin in the canine and feline gastric environment. The iron chelate prepared using the method of this invention has the following advantages: (1) Reduce the degradation of lactoferrin in the gastric environment of dogs and cats Okra polysaccharides are mainly pectin polysaccharides (soft and curled), especially in young okra pods harvested 8-12 days after flowering, which are rich in pectin polysaccharides. Tremella polysaccharides are mainly long-chain mannans (with a rigid structure), especially in mature Tremella fruiting bodies harvested 5-15 days after full expansion, which have a high mannan content. After entering the gastric environment of dogs and cats, okra and Tremella polysaccharides can form a "skeleton-filler" complex network of polysaccharides, encapsulating lactoferrin and increasing the viscosity of the surrounding local fluid. This not only effectively intercepts and adsorbs free water, reduces molecular diffusion rate, inhibits electrostatic repulsion on lactoferrin, and slows lactoferrin swelling, but also reduces the movement speed of pepsin and its contact rate with lactoferrin, thereby reducing the degradation of lactoferrin by pepsin.

[0020] (2) Improve the structural stability of lactoferrin in the gastric environment of dogs and cats Okra polysaccharides are rich in galacturonic acid, while Tremella fruiting body polysaccharides are rich in glucuronic acid. These glucuronic acids are monosaccharides whose terminal hydroxymethyl groups have been oxidized to carboxyl groups. They can react with Fe through the carboxyl group. 3+ Iron chelates are formed through coordination. These chelates, along with the polysaccharide complex, approach lactoferrin as it encapsulates the lactoferrin, creating a locally rich Fe environment around the lactoferrin. 3+ Environment, this rich in Fe 3+ The environment can inhibit Fe in lactoferrin through the common ion concentration balance effect. 3+ The release of these proteins enhances the stability of the protein structure.

[0021] In the preparation of iron chelates, excess iron is added to initiate the chelation reaction, aiming to chelate with uronic acid in the solution as fully as possible. After chelation, an alcohol precipitation step is performed to obtain the iron chelate while removing unchelated Fe. 3+ It combines with uronic acid, thereby increasing the content of iron chelates in the product.

[0022] Electrophoresis images of centrifuged supernatant samples in simulated canine and feline gastric digestion experiments ( Figure 1 , Figure 2 The protein bands in lanes 6-8 (Examples 1-3) are relatively lighter in color than all the bands, indicating that the lactoferrin content in the supernatant is low. (Electrophoresis diagram of the centrifuged precipitate sample) Figure 3 , Figure 4The protein bands in lanes 6-8 (Examples 1-3) were relatively darker than all the bands, indicating a higher content of lactoferrin in the precipitate. This is because the iron chelate encapsulates the lactoferrin and increases the viscosity of the surrounding local liquid. After centrifugation, the protected lactoferrin remains in the precipitate. These results demonstrate that using iron chelates effectively encapsulates lactoferrin and protects its structural stability. This helps prevent excessive degradation of lactoferrin in the stomach and avoids premature loss of its active peptides, allowing for the release of active peptides at appropriate locations after entering the intestines. When the iron chelate prepared according to the method of this invention was co-processed with lactoferrin in a mixture simulating canine gastric digestion, the proliferation rate of canine mixed lymphocytes was 57.53-62.67% (Examples 1-3), an increase of 17.26%-77.33% compared to the control. The iron chelate prepared according to the method of the present invention was mixed with lactoferrin in a mixture that simulated feline gastric digestion. The proliferation rate of feline mixed lymphocytes was 52.21%~56.77% (Examples 1~3), which was 11.73%~69.36% higher than the control. The above results indicate that the iron chelate prepared by the method of the present invention can effectively protect lactoferrin in the gastric environment, so that it retains good functional activity after gastric digestion. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 The electrophoresis diagram simulates the supernatant of the mixture after digestion in dogs. Lane 1 is the marker, lanes 2 to 5 are the supernatant samples of the mixture after digestion in dogs of Control Examples 1 to 4, and lanes 6 to 8 are the supernatant samples of the mixture after digestion in dogs of Examples 1 to 3.

[0025] Figure 2 To simulate the electrophoresis diagram of the supernatant of the mixture after digestion in the cat's stomach, lane 1 is the marker, lanes 2 to 5 are the supernatant samples of the mixture after digestion in the cat's stomach in Control Examples 1 to 4, respectively, and lanes 6 to 8 are the supernatant samples of the mixture after digestion in the cat's stomach in Examples 1 to 3, respectively.

[0026] Figure 3 To simulate the electrophoretic image of the centrifuged precipitate of the mixture after digestion in a dog's stomach, lane 1 is the marker, lanes 2 to 5 are the centrifuged precipitate samples of the mixture after digestion in dogs of Control Examples 1 to 4, and lanes 6 to 8 are the centrifuged precipitate samples of the mixture after digestion in dogs of Examples 1 to 3.

[0027] Figure 4To simulate the electrophoresis diagram of the centrifuged precipitate of the mixed solution after digestion in a cat's stomach, lane 1 is the marker, lanes 2 to 5 are the samples of the centrifuged precipitate of the mixed solution after digestion in cats of Control Examples 1 to 4, respectively, and lanes 6 to 8 are the samples of the centrifuged precipitate of the mixed solution after digestion in cats of Examples 1 to 3, respectively. Detailed Implementation

[0028] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments to the invention based on the above description.

[0029] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used, if necessary, are indicated upon their first appearance, and subsequent use of the same reagents, unless otherwise specified, are identical to the initial indication.

[0030] In this embodiment and the control example, the plant extract was used to simulate the gastric digestion test method in dogs and cats. The test sample was dissolved in distilled water to prepare a solution (final lactoferrin concentration: 10 g / L). 10 mL of this solution was mixed with 10 mL of simulated canine or feline gastric juice and digested in a 37°C water bath (100 rpm) for 120 min. After digestion, the pH was adjusted to 7.0, and the sample was centrifuged. The supernatant and precipitate were then subjected to protein electrophoresis.

[0031] The simulated canine and feline gastric juices were prepared following the method of Liu et al. (In vitro simulated canine and feline gastrointestinal digestion of fructooligosaccharides and isomaltooligosaccharides and their effects on intestinal microbiota, 2023) with slight modifications. Pepsin (250 U / mg activity) was added to the simulated canine and feline gastric juices to a final concentration of 0.04 mg / mL. The pH of the simulated canine gastric juice was adjusted to 2.0, and the pH of the simulated feline gastric juice was adjusted to 1.5.

[0032] The protein electrophoresis assay method in this embodiment and the control example: Protein electrophoresis was performed using the method described by Wang Xiaojun et al. (Study on the stability of recombinant human lactoferrin in an in vitro simulated gastrointestinal environment, 2012) with slight modifications. The separating gel concentration was 12%, the stacking gel concentration was 5%, and Coomassie brilliant blue staining was used.

[0033] The method for preparing mixed lymphocytes in this embodiment and the control example: Fresh peripheral venous blood was collected from dogs and cats 12 hours after fasting, diluted with PBS buffer, and thoroughly mixed. The diluted blood samples were slowly stacked on lymphocyte separation medium, centrifuged at 2500 rpm for 20 min, and the middle white membrane layer was carefully collected. The cells were washed twice with PBS solution, centrifuged again, and the supernatant was discarded. Cell viability was assessed by trypan blue staining after cell counting. The cell concentration was adjusted to 2 × 10⁶ cells / mL using RPMI 1640 medium containing 20% ​​fetal bovine serum. 6 cells / mL.

[0034] The method for determining the proliferation rate of mixed lymphocytes in the simulated canine and feline gastric digestive mixture in this embodiment and the control example: Adjust the pH of the simulated canine / feline gastric digestion mixture to 7.4, and add an appropriate amount to RPMI 1640 medium to achieve a final lactoferrin concentration of 1 mg / mL. Filter the medium through a bacterial filter for sterilization. Resuspend the cultured mixed lymphocyte suspension in RPMI 1640 medium and adjust the cell density to 2 × 10⁻⁶ cells / mL. 6 Cells / mL: 0.1 mL of the mixed lymphocyte suspension was seeded into 96-well concave culture plates, with 6 wells per group. The control group received 0.1 mL of physiological saline solution, while the experimental group received 0.1 mL of filtered, sterilized sample solution per well. The culture plates were placed in an incubator at 37°C and 5% CO2 saturated humidity for 24 hours. After incubation, the culture medium was aspirated, and 200 µL of 5 mg / mL tetramethylazozid salt solution was added. The plates were incubated for another 6 hours, after which the supernatant was aspirated, and 150 µL of dimethyl sulfoxide was added to each well. The plates were mixed using a micromixer, and the absorbance of the 570 nm region in each well was measured using a microplate reader. The proliferation rate (%) of the mixed lymphocytes was calculated as: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0035] Compare with Example 1 Lactoferrin was prepared into a solution (final concentration of 1.0 g / L). 10 mL of this solution was mixed with 10 mL of simulated canine or feline gastric juice and subjected to a simulated canine or feline gastric digestion experiment. The mixture after digestion with the simulated canine or feline gastric juice was centrifuged, and the supernatant and precipitate were subjected to protein electrophoresis separately.

[0036] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1As shown in lane 2, the band color is relatively darker than in all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 2, the band color is relatively light among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated canine gastric digestion mixture was 42.75%.

[0037] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 2, the band color is relatively darker than in all lanes; the electrophoresis results of the protein precipitate after simulated digestion by cat gastric juice are as follows. Figure 4 As shown in lane 2, the band color is relatively light among all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating cat gastric digestion was 39.84%.

[0038] Compare with Example 2 Prepare a lactoferrin solution and add ferric chloride to achieve a final lactoferrin concentration of 1.0 g / L and a final ferric chloride concentration of 0.1 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0039] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 3, the band color is relatively darker than in all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 3, the band color is relatively light among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated canine gastric digestion mixture was 35.34%.

[0040] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 3, the band color is relatively darker than in all lanes; the electrophoresis results of the protein precipitate after simulated feline gastric digestion are as follows. Figure 4 As shown in lane 3, the band color is relatively light among all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating cat gastric digestion was 33.52%.

[0041] Compare with Example 3 100g of dried okra pods were crushed, added to 2000mL of distilled water and stirred evenly. The mixture was then extracted with ultrasonic assistance (frequency 20kHz, power density 40W / L, temperature 40℃) for 60min. After extraction, the supernatant was collected by centrifugation. Ethanol was added to make the final solution 70% by volume. The ethanol precipitation temperature was 2℃ and the precipitation time was 12h. The precipitate was reconstituted with 500mL of distilled water, and 0.5g of trisodium citrate was added. The temperature was raised to 80℃, the pH was adjusted to 6, and then 2.5g of ferric chloride was added. After chelation reaction for 40min, the mixture was dried to obtain a solid. The solid was washed with ethanol solution (90% ethanol by volume) at a material-to-liquid ratio of 0.1g / mL and dried again to obtain the iron chelate.

[0042] Prepare a lactoferrin solution and add iron chelate to achieve a final lactoferrin concentration of 1.0 g / L and an iron chelate concentration of 0.1 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0043] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 4, the intensity of the band color is moderate among all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 4, the intensity of the band color is moderate among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated canine gastric digestion mixture is 47.78%.

[0044] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 4, the intensity of the band color is moderate among all lanes; the electrophoresis results of the protein precipitate after simulated feline gastric digestion are as follows. Figure 4 As shown in lane 4, the intensity of the band color is moderate among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated feline gastric digestion mixture is 46.73%.

[0045] Compare with Example 4 100g of dried Tremella fuciformis fruiting body was pulverized, added to 2000mL of distilled water and stirred evenly. The mixture was then extracted with ultrasonic assistance (frequency 20kHz, power density 40W / L, temperature 40℃) for 60min. After extraction, the supernatant was collected by centrifugation. Ethanol was added to make the final solution contain 70% ethanol by volume. The ethanol precipitation temperature was 2℃ and the precipitation time was 12h. The precipitate was reconstituted with 500mL of distilled water, and 0.5g of trisodium citrate was added. The temperature was raised to 80℃, the pH was adjusted to 6, and then 2.5g of ferric chloride was added. After chelation reaction for 40min, the mixture was dried to obtain a solid. The solid was washed with ethanol solution (ethanol volume fraction 90%) at a material-to-liquid ratio of 0.1g / mL and dried again to obtain the iron chelate.

[0046] Prepare a lactoferrin solution and add iron chelate to achieve a final lactoferrin concentration of 1.0 g / L and an iron chelate concentration of 0.1 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0047] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 5, the intensity of the band color is moderate among all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 5, the intensity of the band color is moderate among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated canine gastric digestion mixture is 49.06%.

[0048] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 5, the intensity of the band color is moderate among all lanes; the electrophoresis results of the protein precipitate after simulating feline gastric digestion are as follows. Figure 4 As shown in lane 5, the intensity of the band color is moderate among all lanes; the proliferation rate of mixed lymphocytes treated with the simulated feline gastric digestion mixture is 44.51%.

[0049] Example 1 Take 80g of dried okra pods and 20g of dried tremella fruiting bodies, crush them, add 2000mL of distilled water and stir evenly. Extract with ultrasonic assistance (frequency 20kHz, power density 40W / L, temperature 40℃) for 60min. After extraction, centrifuge and collect the supernatant. Add ethanol to make the final solution 70% by volume. Precipitation temperature is 2℃, and precipitation time is 12h. Redissolve the precipitate in 500mL of distilled water, add 0.5g of trisodium citrate, heat to 80℃, adjust pH to 6, then add 2.5g of ferric chloride. After chelation reaction for 40min, dry to obtain a solid. Wash the solid with ethanol solution (90% ethanol by volume) at a material-to-liquid ratio of 0.1g / mL, and dry again to obtain the iron chelate.

[0050] Prepare a lactoferrin solution and add iron chelate to achieve a final lactoferrin concentration of 1.0 g / L and an iron chelate concentration of 0.1 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0051] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 6, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 6, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating canine gastric digestion was 57.53%.

[0052] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 6, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated feline gastric digestion are as follows. Figure 4 As shown in lane 6, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating cat gastric digestion was 52.21%.

[0053] Example 2 Take 50g of dried okra pods and 50g of dried tremella fruiting bodies, crush them, add 2000mL of distilled water and stir evenly. Extract with ultrasonic assistance (frequency 60kHz, power density 20W / L, temperature 60℃) for 30min. After extraction, centrifuge and collect the supernatant. Add ethanol to make the final solution 90% by volume. Precipitation temperature is 8℃, and precipitation time is 24h. Dissolve the precipitate in 400mL of distilled water, add 2.0g of trisodium citrate, heat to 60℃, adjust pH to 5, then add 5.0g of ferric chloride. After chelation reaction for 80min, dry to obtain a solid. Wash the solid with ethanol solution (70% by volume) at a material-to-liquid ratio of 0.2g / mL, then dry again to obtain the iron chelate.

[0054] Prepare a lactoferrin solution and add iron chelate to achieve a final lactoferrin concentration of 1.0 g / L and an iron chelate concentration of 0.2 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0055] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 7, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 7, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture of simulated canine gastric digestion was 62.67%.

[0056] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 7, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated feline gastric digestion are as follows. Figure 4 As shown in lane 7, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating cat gastric digestion was 55.15%.

[0057] Example 3 Take 60g of dried okra pods and 40g of dried tremella fruiting bodies, crush them, add 2000mL of distilled water and stir evenly. Extract with ultrasonic assistance (frequency 40kHz, power density 30W / L, temperature 50℃) for 45min. After extraction, centrifuge and collect the supernatant. Add ethanol to make the final solution 80% by volume. Ethanol precipitation temperature is 5℃ and precipitation time is 18h. Take the precipitate and redissolve it in 450mL of distilled water. Add 1.0g of trisodium citrate, heat to 70℃, adjust pH to 5.5, and then add 4.0g of ferric chloride. After chelation reaction for 60min, dry to obtain solid. Wash the solid with ethanol solution (ethanol volume fraction 80%) at a material-to-liquid ratio of 0.15g / mL and dry again to obtain iron chelate.

[0058] Prepare a lactoferrin solution and add iron chelate to achieve a final lactoferrin concentration of 1.0 g / L and an iron chelate concentration of 0.15 g / L. Mix 10 mL of this solution with 10 mL of simulated canine or feline gastric juice and perform a simulated canine or feline gastric digestion experiment. Centrifuge the mixture after digestion with the simulated canine or feline gastric juice, and perform protein electrophoresis on the supernatant and precipitate separately.

[0059] Electrophoresis results of protein supernatant after simulated canine gastric digestion are as follows: Figure 1 As shown in lane 8, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated canine gastric digestion are as follows. Figure 3 As shown in lane 8, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture of simulated canine gastric digestion was 60.49%.

[0060] Electrophoresis results of protein supernatant after simulated feline gastric digestion are as follows: Figure 2 As shown in lane 8, the band color is relatively lighter than in all lanes; the electrophoresis results of the protein precipitate after simulated feline gastric digestion are as follows. Figure 4 As shown in lane 8, the band color is relatively darker than in all lanes; the proliferation rate of mixed lymphocytes treated with a mixture simulating cat gastric digestion was 56.77%.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an iron chelate that reduces the degradation of lactoferrin in the gastric environment of dogs and cats, characterized in that... Follow these steps: (1) Preparation of polysaccharide extract: Take dried okra fruit and dried tremella fruit and crush them together. When extracting them with water, apply ultrasonic-assisted extraction. After ethanol precipitation, the precipitate is redissolved with water. After centrifugation, the supernatant is the polysaccharide extract. (2) Preparation of iron chelate: Dissolve trisodium citrate in polysaccharide extract, adjust temperature and pH, add ferric chloride solution for chelation reaction, dry after chelation reaction to obtain solid product, wash the solid product with ethanol solution and dry to obtain iron chelate that can reduce the degradation of lactoferrin in the stomach environment of dogs and cats.

2. The method for preparing an iron chelate that reduces the degradation of lactoferrin in the gastric environment of dogs and cats according to claim 1, characterized in that... In step (1): the dried okra fruit is preferably obtained by naturally sun-drying or freeze-drying tender okra fruits harvested 8-12 days after flowering, or it can be replaced by fresh tender okra fruits 8-12 days after flowering. The dried tremella fruiting body is preferably obtained by naturally sun-drying or freeze-drying mature tremella fruiting bodies harvested 5-15 days after the fruiting bodies have fully expanded, or it can be replaced by fresh mature tremella fruiting bodies 5-15 days after the fruiting bodies have fully expanded; the dried okra fruit and dried tremella fruiting body are mixed in a specific mass ratio of 4:1 to 1:

1. In step (1): during water extraction, the material-to-liquid ratio is 1:20~1:30 (g / mL); In step (1): when performing ultrasonic-assisted extraction, the ultrasonic frequency is 20~60kHz, the ultrasonic power density is 20~40W / L, the ultrasonic time is 30~60min, and the ultrasonic extraction temperature is 40~60℃. In step (1): the alcohol precipitation involves adding ethanol to make the volume percentage of ethanol in the final solution 70%~90%, the alcohol precipitation temperature 2~8℃, and the alcohol precipitation time 12~24h. In step (1): the final volume when adding distilled water for reconstitution is 20% to 25% of the volume of the extract before ultrasonic precipitation with ethanol.

3. The method for preparing an iron chelate that reduces the degradation of lactoferrin in the gastric environment of dogs and cats according to claim 1, characterized in that... In step (2): add trisodium citrate to the polysaccharide extract at a ratio of 1~4 g / L, adjust the temperature to 60~80℃, and adjust the pH to 5~6; In step (2): Ferric chloride is added to the polysaccharide extract at a ratio of 5~10g / L to the liquid, and the chelation reaction lasts for 40~80min; In step (2): When washing with ethanol solution, the solid-to-ethanol solution ratio is 0.1~0.2g / mL, and the volume percentage of ethanol in the ethanol solution is 70%~90%.

4. The application of the iron chelate prepared by the method described in claims 1-3, which can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, is to be used by feeding it to dogs and cats together with lactoferrin, wherein the mass ratio of the iron chelate to lactoferrin is 1:5 to 1:

10.

5. The application of the iron chelate prepared by the method of claims 1-3, which can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, wherein the iron chelate and lactoferrin are fed to dogs and cats simultaneously, or fed to dogs and cats sequentially for a short period of time.

6. The iron chelate prepared by the method described in claims 1-3, which can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, can be fed to dogs and cats by direct feeding, mixing into dog and cat food (or snacks), or added as a formulation component to dog and cat food (or snacks).

7. The application of the iron chelate prepared by the method described in claims 1-3, which can reduce the degradation of lactoferrin in the gastric environment of dogs and cats, to other pets such as foxes, hamsters and rabbits.

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