Silymarin feed additive as well as electrostatic spinning preparation method and application thereof

Abstract

The invention relates to the technical field of feed additives, in particular to a silymarin feed additive and an electrostatic spinning preparation method and application thereof. The invention provides a silymarin feed additive. The silymarin feed additive comprises an electrostatic spinning fiber membrane and silymarin loaded on the surface of an electrostatic spinning fiber membrane material, the electrostatic spinning fiber membrane comprises pectin, sodium caseinate and laccase. The pectin, the sodium caseinate and the laccase are used as raw materials, the electrostatic spinning fiber membrane with a three-dimensional cross-linked network is prepared through an electrostatic spinning method, the electrostatic spinning fiber membrane serves as a carrier of the silymarin, the slow-release effect can be achieved, and the bioavailability of an organism to the silymarin is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a silymarin feed additive and its electrospinning preparation method and application. Background Technology

[0002] Fatty Liver Syndrome (FLS) is a common metabolic disease in poultry, with pathogenesis and pathological features highly similar to non-alcoholic fatty liver disease (NAFLD) in mammals. In recent years, the proportion of fat added to feed has been increasing to improve broiler growth performance and farming efficiency, but this has also increased the risk of FLS. In affected chickens, the liver becomes enlarged and brittle due to severe fat deposition, and may even rupture and bleed, leading to sudden death. Studies have shown that FLS has become a major cause of non-infectious mortality in caged broilers, accounting for approximately 75% of broiler deaths. The peak incidence is concentrated in broiler chicks aged 3-4 weeks, while outbreaks are less frequent before 11 days of age and after 32 days of age. The mortality rate is typically 5%, but can rise to 30% in severe cases, causing significant economic losses to the poultry industry.

[0003] Silymarin is a mixture of flavonoid lignans extracted from the dried, mature fruit of the milk thistle. Its main active components include silybin, isosilybin, silybinine, and silybinin. Pharmacological studies have shown that silymarin has significant hepatoprotective effects, antioxidant (its free radical scavenging ability is 1.5 times that of vitamin C), immunomodulatory effects, and lipid-lowering effects.

[0004] However, the application of silymarin has the following limitations: (1) it has a distinct bitter taste, affecting palatability; (2) it has poor stability and is easily oxidized and inactivated; (3) it is prone to aggregation after protonation in gastric acid, resulting in poor dissolution and low bioavailability; (4) ordinary formulations are difficult to withstand the high temperatures of feed processing. These drawbacks limit its application in poultry farming. Summary of the Invention

[0005] The present invention aims to overcome the deficiency of low bioavailability of silymarin in the prior art by providing a silymarin feed additive and its electrospinning preparation method and application to overcome the above-mentioned deficiency.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a silymarin feed additive, comprising an electrospun fiber membrane and silymarin loaded on the surface of the electrospun fiber membrane material; the electrospun fiber membrane comprises: pectin, sodium caseinate and laccase.

[0008] This invention uses electrospun fiber membranes as a sustained-release carrier for silymarin, which helps to achieve sustained release of silymarin during use and improves the bioavailability of silymarin to organisms.

[0009] Specifically, the hydrophobic microdomains of sodium caseinate strongly bind to the lipophilic flavonoid lignans in silymarin through hydrophobic interactions. The hydrophilic chains of pectin enhance the structural stability of the electrospun fiber membrane in the gastrointestinal environment, preventing its rapid disintegration. Laccase catalyzes pectin to construct an electrospun fiber membrane with a three-dimensional cross-linked network, reducing the risk of burst release and allowing more silymarin to be released into the gastrointestinal tract of the organism.

[0010] Preferably, the electrospun fiber membrane comprises, by weight, 5-15 parts pectin, 3-10 parts sodium caseinate and 0.01-0.1 parts laccase.

[0011] Preferably, the electrospun fiber membrane comprises, by weight, 3-8 parts pectin, 2-5 parts sodium caseinate and 0.01-0.1 parts laccase.

[0012] Preferably, the diameter of the electrospun fiber membrane is 100–500 nm.

[0013] A suitable electrospun fiber membrane size can be used to load a sufficient amount of silymarin, and allow the loaded silymarin to be evenly distributed for easy absorption by animals.

[0014] Preferably, the active ingredients of silymarin include multiple of silymarin, isosilymarin, silymarinine, and silymarinine.

[0015] Preferably, the degree of esterification of the pectin is ≥70%.

[0016] Using low-ester pectin can lead to poor uniformity of the spinning solution. The carboxyl groups strongly bind with the amino groups of sodium caseinate, easily causing the solution to gel and making it difficult to form continuous fibers. Furthermore, low-ester pectin readily reacts with calcium in gastric acid. 2+ Combined with swelling, this may lead to premature release of silymarin. Furthermore, when used as a feed additive, palatability for animals must be considered, necessitating particle size reduction. Using low-ester pectin can decrease the overall mechanical properties of the feed additive, making the electrospun fiber membrane more brittle and prone to dust generation during pulverization, thus affecting feed processing.

[0017] Preferably, the silymarin loading is 5-15 wt% of the mass of the electrospun fiber membrane.

[0018] Preferably, the silymarin loading is 8-12 wt% of the mass of the electrospun fiber membrane.

[0019] This invention provides a method for preparing a silymarin feed additive, comprising:

[0020] S1. Pectin and sodium caseinate are dissolved in an acidic buffer solution to obtain a spinning solution, which is then electrospun to obtain a precursor membrane.

[0021] S2. Spray laccase solution onto the surface of the precursor membrane, followed by ultraviolet crosslinking, to obtain an electrospun fiber membrane;

[0022] S3. The electrospun fiber membrane is immersed in a silymarin organic solution for silymarin loading, and then dried to obtain a silymarin feed additive.

[0023] Direct loading of silymarin onto precursor membranes prepared from pectin and sodium caseinate can easily lead to burst release problems, resulting in poor bioavailability. Therefore, adding laccase for cross-linking and curing can, on the one hand, construct an electrospun fiber membrane with a three-dimensional cross-linked network, significantly improving the mechanical properties of the electrospun fiber membrane; on the other hand, laccase can pre-oxidize silymarin, converting the phenolic hydroxyl groups in silymarin into quinones, enhancing the covalent binding strength with the amino groups in sodium caseinate, and reducing rapid release.

[0024] Preferably, in S1, the pH of the acidic buffer solution is 4.0 to 6.0.

[0025] Acidic buffer solutions, such as citrate buffer.

[0026] Preferably, in S1, the conditions for electrospinning are: voltage 10-20kV, receiving distance 10-20cm, and humidity <40%.

[0027] Preferably, in S2, the temperature of the laccase solution is 4–25°C, and the UV crosslinking time is 3–10 min.

[0028] Preferably, in S3, the silymarin organic solution contains 8-12 wt% silymarin.

[0029] Preferably, the organic solvent in the silymarin organic solution is at least one of acetone, ethanol, and methanol.

[0030] This invention provides a silymarin feed, comprising a silymarin feed additive and a feed; the silymarin feed additive has a particle size of 50-100 mesh, and the amount of the silymarin feed additive is 0.1-0.5 wt% of the feed.

[0031] Preferably, the feed is poultry feed or aquatic feed.

[0032] Preferably, the feed is chicken feed, pig feed, duck feed, fish feed, or goose feed.

[0033] Therefore, the present invention has the following beneficial effects:

[0034] (1) The present invention uses pectin, sodium caseinate and laccase as raw materials to prepare an electrospun fiber membrane with a three-dimensional cross-linked network by electrospinning. As a carrier of silymarin, it can achieve a sustained release effect and greatly increase the bioavailability of silymarin in organisms.

[0035] (2) The present invention utilizes laccase to pre-oxidize silymarin, converting the phenolic hydroxyl groups in silymarin into quinones, thereby enhancing the covalent bonding strength with the amino groups in sodium caseinate and reducing the risk of sudden release of silymarin after electrospun fiber membrane loading.

[0036] (3) The present invention uses high-ester pectin to avoid poor uniformity of spinning solution caused by low-ester pectin, which leads to fiber discontinuity and premature release of silymarin.

[0037] (4) The silymarin feed additive provided by the present invention can be widely used as poultry feed or aquatic feed. Attached Figure Description

[0038] Figure 1 Images showing the results of H&E staining and Oil Red O staining of the liver. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0040] In this section, all raw materials were sourced as follows: Citrus pectin, 70% esterification, purchased from Yuening Biotechnology; Citrus pectin, 50% esterification, purchased from Yuening Biotechnology; Sodium caseinate, purchased from Pinchuan Food Technology; Citric acid and sodium citrate, purchased from Shandong Yingxuan Industrial Co., Ltd.; Silymarin, purchased from Liaoning Fengrui Tiancheng Biotechnology Co., Ltd.

[0041]

Example

[0042] Example 1

[0043] 1.5g of citrus pectin (70% esterification) and 3g of sodium caseinate were dissolved in 100mL of citrate buffer (pH 5.0) and stirred for 2 hours to obtain a homogeneous spinning solution. The citrate buffer was prepared by citric acid and sodium citrate, and the pH was adjusted with hydrochloric acid.

[0044] S2. The precursor membrane is obtained by electrospinning the spinning solution. The electrospinning conditions are: voltage 15kV, receiving distance 15cm, humidity 30%, and room temperature.

[0045] S3. Spray 0.05wt% laccase solution (4℃) onto the surface of the precursor membrane, and then perform UV crosslinking for 5 min to obtain an electrospun fiber membrane with a diameter of about 300nm.

[0046] S4. Immerse the electrospun fiber membrane in a silymarin ethanol solution (silymarin content 10wt%) overnight for silymarin loading. After soaking, remove the membrane and dry it at 80℃ for 12 hours. Control the amount of electrospun fiber membrane added and the amount of silymarin ethanol solution to finally obtain a 10wt% silymarin feed additive.

[0047] Comparative Example 1 (without laccase)

[0048] 1.5g of citrus pectin (70% esterification) and 3g of sodium caseinate were dissolved in 100mL of citrate buffer (pH 5.0) and stirred for 2 hours to obtain a homogeneous spinning solution. The citrate buffer was prepared by citric acid and sodium citrate, and the pH was adjusted with hydrochloric acid.

[0049] S2. The precursor membrane is obtained by electrospinning the spinning solution. The electrospinning conditions are: voltage 15kV, receiving distance 15cm, humidity 30%, and room temperature.

[0050] S3. The precursor membrane was immersed in a silymarin ethanol solution (silymarin content 10wt%) for silymarin loading overnight. After soaking, it was removed and dried at 80℃ for 12 hours. By controlling the amount of precursor membrane added and the amount of silymarin ethanol solution, a 10wt% silymarin feed additive was finally obtained.

[0051] Comparative Example 2 (low pectin esterification degree)

[0052] 1.5g of citrus pectin (50% esterification) and 3g of sodium caseinate were dissolved in 100mL of citrate buffer (pH 5.0) and stirred for 2 hours to obtain a homogeneous spinning solution. The citrate buffer was prepared by citric acid and sodium citrate, and the pH was adjusted with hydrochloric acid.

[0053] S2. The precursor membrane is obtained by electrospinning the spinning solution. The electrospinning conditions are: voltage 15kV, receiving distance 15cm, humidity 30%, and room temperature.

[0054] S3. Spray 0.05wt% laccase solution (4℃) onto the surface of the precursor membrane, and then perform UV crosslinking for 5 min to obtain an electrospun fiber membrane with a diameter of about 300nm.

[0055] S4. Immerse the electrospun fiber membrane in a silymarin ethanol solution (silymarin content 10wt%) overnight for silymarin loading. After soaking, remove the membrane and dry it at 80℃ for 12 hours. Control the amount of electrospun fiber membrane added and the amount of silymarin ethanol solution to finally obtain a 10wt% silymarin feed additive.

[0056] [Performance Testing]

[0057] 1. Gastrointestinal dissolution test

[0058] Artificial gastric and intestinal fluids were prepared in accordance with the requirements of the 2020 edition of the Chinese Veterinary Pharmacopoeia.

[0059] Artificial gastric fluid operation procedure: Take 0.2g each of raw milk thistle powder and milk thistle feed additives prepared in Examples 1 and Comparative Examples 1-2, accurate to 0.0002g, and place them in a dissolution vessel. Using a rotating basket, the apparatus is designed according to Method 1 (basket method) of Part I, 0931, of the 2020 edition of the Chinese Veterinary Pharmacopoeia. Add 400mL of artificial gastric fluid, set the temperature to 38℃, and the rotation speed to 100r / min. After rotating for 0.5h, 1h, 1.5h, and 2h, respectively, the residue content is determined using high-performance liquid chromatography (HPLC). After the 2h gastric fluid stage, transfer the entire rotating basket to a new dissolution vessel, rinse the basket three times with 10mL of 37℃ physiological saline, add 400mL of artificial intestinal fluid, set the temperature to 38℃, and the rotation speed to 100r / min. After rotating for 1h, 2h, 3h, and 4h, the residue content is determined using HPLC.

[0060] The test results recorded in artificial gastric fluid and human intestinal fluid are shown in Table 1.

[0061] Table 1 Release rate (%) in simulated gastric and intestinal fluids

[0062]

[0063] As shown in Table 1, the formulation design of Example 1 is significantly superior to that of the comparative examples and the original powder. In simulated gastric fluid, Example 1 exhibits excellent sustained-release characteristics, effectively avoiding protonated aggregation; while Comparative Examples 1 and 2 show significant burst release from gastric fluid, indicating potential aggregation. Upon entering simulated intestinal fluid, Example 1 releases rapidly and completely, while Comparative Examples 1 and 2 experience limited release due to prior aggregation. The original powder exhibits poor gastrointestinal release due to its natural aggregation characteristics. The results indicate that Example 1 successfully solved the aggregation problem by optimizing the formulation process, achieving more efficient release and absorption.

[0064] 2. Loading rate, active ingredient retention rate, and feed intake experiments

[0065] Loading rate (%): A known mass W0 (g) of blank electrospun fiber membrane (Example 1: membrane prepared by steps S1-S3; Comparative Example 1: membrane prepared by steps S1-S2 only; Comparative Example 2: membrane prepared by steps S1-S3) and a known mass W1 (g) of corresponding silymarin-loaded fiber membrane (i.e., the final product membrane after loading silymarin and drying after completing steps S1-S4 or S1-S3 respectively) were taken. Subsequently, silymarin extraction was performed. The silymarin-loaded membrane W1 (g) was placed in an appropriate amount of ethanol, and the silymarin loaded in the membrane was completely dissolved by ultrasonic extraction. The extract was collected and diluted to a volume V (mL). Next, silymarin in the extract was quantitatively analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column, mobile phase methanol-water (70:30, v / v), and detection wavelength 288 nm. A standard curve was plotted using silymarin standards, and its concentration C (μg / mL) was calculated based on the peak area of ​​the extract. Finally, the loading rate was calculated using the formula:

[0066] Load factor (%) = [(C×V) / (0.1×M)]×100%

[0067] In the formula: C: concentration of silymarin in the extract obtained by high performance liquid chromatography (μg / mL); V: volume of the extract (mL); M: mass of the corresponding blank fiber membrane W0 (g); 0.1: theoretical silymarin-carrying ratio.

[0068] Activity retention rate (%): Three portions each of Example 1, Comparative Example 1, Comparative Example 2, and the original powder sample were taken, precisely heated in a 75℃ oven for 5 minutes, and then extracted with 70% ethanol by ultrasonication. The content (g) of silymarin was determined by HPLC (C18 column, methanol-0.1% phosphoric acid water 70:30, 288nm). With the unheated group as the control, the activity retention rate (%) was calculated as (content after heat treatment / initial content) × 100%.

[0069] Feed intake (g): Each group of silymarin preparations (0.1wt%) was evenly added to the basal diet of broilers. The daily feed intake (feed amount - remaining amount) of each bird was recorded for 7 consecutive days starting from 21 days of age, and the average value was calculated (n=30 / group).

[0070] Table 2 Performance Comparison Table

[0071] Load rate, % Activity retention rate, % Feed intake, g control group / 58.3 103.5 Example 1 95.2 94.1 122.8 Comparative Example 1 88.6 80.4 112.6 Comparative Example 2 92.1 86.9 117.3

[0072] As shown in Table 2, Example 1 showed the best performance in terms of loading rate, activity retention rate and feed intake, which was significantly better than Comparative Examples 1-2 and the control group. This indicates that the synergistic effect of laccase crosslinking and high esterification degree pectin can not only improve drug loading efficiency and thermal stability, but also effectively mask bitterness.

[0073] 3. Animal experiments

[0074] Three hundred and sixty 14-day-old AA broilers (initial weight 450g ± 10g) were randomly divided into three groups (n = 6 replicates / group, 20 birds / replica): control group (basal diet), milk thistle powder group (basal diet + 300mg / kg milk thistle powder), and Example 1 group (basal diet + 300mg / kg milk thistle feed additive). The broilers were raised until 35 days of age with free access to feed and water. The composition and nutrient levels of the basal diet are shown in Table 3.

[0075] Table 3. Feed formulation and nutrient levels (air-dried basal)

[0076]

[0077] 1 The premix provides the following per kilogram of 1-21 day old feed: Fe 40mg, Cu 8mg, Mn 60.0mg, Zn 55.3mg, Se 0.4mg, I 0.5mg, VA 12000IU, VB1 3mg, VD3 3000IU, VB6 1.2mg, VB2 8.0mg, VB12 0.01mg, VK3 3.0mg, VE 10mg, folic acid 2.0mg, niacin 10mg, pantothenic acid 12mg, and biotin 0.10mg.

[0078] 2 The premix provides the following per kilogram of diet for 22-35 day old animals: Fe 49mg, Cu 10mg, Mn 74mg, Zn 68mg, Se 0.4mg, I 0.6mg, VA 14720IU, VB1 4mg, VD3 3680IU, VB6 1.5mg, VB2 9.8mg, VB12 0.01mg, VK3 3.7mg, VE 12.3mg, folic acid 2.5mg, niacin 12.3mg, pantothenic acid 14.7mg, and biotin 0.12mg.

[0079] At 28 days of age, two broiler chickens were randomly selected from each replicate. After fasting for 12 hours, blood was collected and serum was separated to measure biochemical indicators such as ALT and AST. At the end of the experiment (35 days of age), two broiler chickens were randomly selected from each replicate. After fasting for 12 hours, blood was collected and serum was separated to measure biochemical indicators such as ALT and AST. Tissue from the middle of the left lobe (1 cm) was also collected. 3 The sample was fixed in 40 g / L paraformaldehyde for 48 h, dehydrated, embedded in paraffin, and then sectioned into 4 μm sections for H&E staining and Oil Red O staining. The results are as follows: Figure 1 As shown in Table 4.

[0080] Table 4. Effects of milk thistle on serum biochemical parameters of broiler chickens at 28 and 35 days of age.

[0081]

[0082] Figure 1 The experimental results in Table 4 show that silymarin feed additives have a significant effect on improving liver health in broilers. As shown in Table 3, compared with the control group, at 28 days of age, the serum ALT and AST levels in the Example 1 group decreased by 22.7% (P<0.01) and 24.3% (P<0.01), respectively, and the TG and TC levels decreased by 41.4% (P<0.01) and 30.6% (P<0.01), respectively; by 35 days of age, these indicators remained significantly improved (ALT decreased by 26.1%, AST by 27.5%, TG by 34.1%, and TC by 22.7%, all P<0.01). More importantly, the improvement effect of the Example 1 group was significantly better than that of the original silymarin powder group (P<0.05). Liver pathological examination results ( Figure 1 Further confirmation showed that the hepatocytes in the Example 1 group had intact structures, and Oil Red O staining revealed a significant reduction in fat deposition area and pathological score compared to the control group. These results fully demonstrate that the silymarin feed additive prepared in this invention, using an electrospun fiber membrane with a three-dimensional cross-linked network as a carrier, can achieve intestinal-targeted release of silymarin, significantly improving the bioavailability of silymarin and exhibiting significantly better effects than the original powder formulation in improving broiler liver health and reducing fat deposition.

Claims

1. A silymarin feed additive, characterized in that, Includes an electrospun fiber membrane and silymarin loaded on the surface of the electrospun fiber membrane material; The electrospun fiber membrane comprises: pectin, sodium caseinate, and laccase.

2. The silymarin feed additive as described in claim 1, characterized in that, The electrospun fiber membrane comprises, by weight, 5-15 parts pectin, 3-10 parts sodium caseinate and 0.01-0.1 parts laccase.

3. The silymarin feed additive as described in claim 1 or 2, characterized in that, The diameter of the electrospun fiber membrane is 100–500 nm.

4. The silymarin feed additive as described in claim 1 or 2, characterized in that, The degree of esterification of the pectin is ≥70%.

5. The silymarin feed additive as described in claim 1 or 2, characterized in that, The silymarin loading is 5 to 15 wt% of the mass of the electrospun fiber membrane.

6. A method for preparing a silymarin feed additive as described in any one of claims 1 to 5, characterized in that, include: S1. Pectin and sodium caseinate are dissolved in an acidic buffer solution to obtain a spinning solution, which is then electrospun to obtain a precursor membrane. S2. Spray laccase solution onto the surface of the precursor membrane, followed by ultraviolet crosslinking, to obtain an electrospun fiber membrane; S3. The electrospun fiber membrane is immersed in a silymarin organic solution for silymarin loading, and then dried to obtain a silymarin feed additive.

7. The preparation method according to claim 6, characterized in that, In S1, the pH of the acidic buffer solution is 4.0 to 6.

0.

8. The preparation method according to claim 6, characterized in that, In S1, the conditions for electrospinning are: voltage 10-20kV, receiving distance 10-20cm, and humidity <40%. In S2, the temperature of the laccase solution is 4–25℃, and the UV cross-linking time is 3–10 min.

9. The preparation method according to claim 6, characterized in that, In S3, the silymarin organic solution contains 8-12 wt% silymarin.

10. A milk thistle feed, characterized in that, The feed includes silymarin feed additive as described in any one of claims 1 to 5 or silymarin feed additive prepared by any one of claims 6 to 9, and feed; the particle size of the silymarin feed additive is 50 to 100 mesh, and the amount of the silymarin feed additive is 0.1 to 0.5 wt% of the feed.

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