Natural plant flavor biscuit for preventing and treating mastitis of lactating female livestock and preparation method of natural plant flavor biscuit

Abstract

The invention discloses natural plant flavor biscuits for preventing and treating mastitis of lactating female animals and a preparation method thereof, and belongs to the field of veterinary functional foods. The biscuit is prepared from astragalus membranaceus, honeysuckle, dandelion, cyperus rotundus, hydroxypropyl-beta-cyclodextrin and a biscuit base material according to a specific ratio, and the preparation method comprises the following steps: adding an ethanol extract concentrated solution into a cyclodextrin-containing functional inclusion mother solution for pre-reaction through a specific reaction sequence, then adding cyperus rotundus volatile oil for inclusion, and finally adding hydroxypropyl-beta-cyclodextrin into the inclusion mother solution for pre-reaction. Plant endogenous components are guided to synergistically self-assemble to form a core-shell type supramolecular compound anchored and reinforced by molecules. After the compound is mixed with a biscuit base material and baked at low temperature, the technical problems that volatile oil is poor in stability and uneven in distribution are effectively solved, and the retention rate and uniformity of active ingredients in a final product are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of veterinary functional food and feed additive technology, and in particular to a natural plant-flavored biscuit for preventing and treating mastitis in lactating animals and its preparation method. Background Technology

[0002] Mastitis is one of the most common diseases affecting lactating female animals such as cows and goats. Its high incidence not only seriously affects milk production and dairy product quality but also causes huge economic losses due to treatment and culling of infected animals. Currently, clinical prevention and treatment of mastitis mainly rely on antibiotics. However, the long-term and excessive use of antibiotics can easily lead to drug resistance in pathogens, and the issue of drug residues in milk and meat products is increasingly raising public concerns about food safety. Therefore, developing safe, effective, and residue-free antibiotic alternatives has become an urgent technological need in this field.

[0003] The use of natural plants, especially traditional Chinese herbal medicines with heat-clearing, detoxifying, swelling-reducing, and nodule-dispersing effects, as an alternative for the prevention and treatment of mastitis has shown broad application prospects. These plants typically contain active ingredients with multi-target and multi-pathway synergistic effects, making them less prone to drug resistance. However, applying these plant active ingredients to large-scale farms, especially in the preparation of palatable and easy-to-feed solid formulations (such as biscuits or block feed), presents significant technical challenges. Many key active ingredients, such as volatile oils in plants like Cyperus rotundus, are extremely unstable in physicochemical properties and sensitive to heat, light, and oxygen. In traditional physical mixing and heat processing (such as baking), these active ingredients are easily volatilized, degraded, or oxidized, leading to a significant reduction in their content in the final product and failing to guarantee the expected preventive and therapeutic effects. Furthermore, these oil-soluble components have poor compatibility with water-based dough matrices, making it difficult to achieve uniform dispersion, resulting in inconsistent active ingredient content between batches of products, affecting the accuracy of administration and the stability of efficacy. Therefore, how to effectively protect these unstable active components and ensure their uniform distribution in solid carriers is a pressing technical challenge that needs to be addressed in the development of functional animal foods using natural plants. Summary of the Invention

[0004] The purpose of this invention is to provide a natural plant-flavored biscuit for preventing and treating mastitis in lactating animals and its preparation method. This invention solves the technical problem of low retention rate and uneven distribution of active plant ingredients due to thermal instability and poor compatibility with the matrix when volatile oils and other unstable plant active ingredients are prepared into solid preparations.

[0005] In a first aspect, the present invention provides a natural plant-flavored biscuit for preventing and treating mastitis in lactating livestock, employing the following technical solution: A natural plant-flavored biscuit for preventing mastitis in lactating livestock, made from the following ingredients in parts by weight: Astragalus: 400-500 portions; Honeysuckle: 450-550 servings; Dandelion: 500-600 servings; Cyperus rotundus: 350-450 portions; Hydroxypropyl-β-cyclodextrin: 30-60 parts; Biscuit base: 1500-2500 portions.

[0006] By adopting the above technical solution, the present invention can effectively fix the volatile oil of Cyperus rotundus, which has unstable physicochemical properties, into the biscuit base, thus solving the technical problems of easy loss and uneven distribution of volatile oil under traditional physical mixing methods.

[0007] First, the functional inclusion complex mother liquor composed of honeysuckle and dandelion extracts provides a macromolecular environment rich in natural saponins, polyphenols, and polysaccharides. These endogenous components, acting as natural surfactants and stabilizers, provide a pre-emulsification and dispersion basis for the subsequent inclusion of volatile oils, and form an outer stabilizing layer around the subsequently formed inclusion complex.

[0008] Secondly, before adding the volatile oils, a concentrated ethanol extract of the astragalus and cyperus residues is introduced into the system. In this step, components such as astragaloside A play two key roles: Firstly, through competitive interaction with the hydrophobic cavity of hydroxypropyl-β-cyclodextrin, it creates conditions for the subsequent preferential entry and stable binding of more hydrophobic volatile oil molecules. Secondly, these components can be adsorbed or interspersed in the outer stabilizing layer, playing a role in molecular anchoring and thus strengthening the stability of the overall structure.

[0009] Ultimately, the hydrophobic cavity of hydroxypropyl-β-cyclodextrin encapsulates the volatile oil of Cyperus rotundus. Under the synergistic effect of the aforementioned stabilizing system and anchoring molecules, a dense shell is formed around the core by polysaccharides, polyphenols, saponins, and other substances. This supramolecular structure, with the volatile oil-cyclodextrin core, plant macromolecules as the shell, and anchored and reinforced by specific components, significantly enhances the physical and thermal stability of the volatile oil, inhibiting its aggregation, degradation, and escape during dough mixing and baking. This ensures the uniformity and stability of the active ingredient content in the final biscuit product.

[0010] Preferably, the raw materials are in the following proportions by weight: 440 parts Astragalus membranaceus, 480 parts Lonicera japonica, 520 parts Taraxacum mongolicum, 400 parts Cyperus rotundus, 42 parts hydroxypropyl-β-cyclodextrin, and 2000 parts biscuit base.

[0011] By adopting the above technical solution, the formulation is an optimized result that comprehensively considers the synergistic effect of each component, cost, and palatability of the final product, and can achieve better volatile oil inclusion rate and system stability within this range.

[0012] Preferably, the raw materials for its preparation include an ethanol extract concentrate, which is obtained by ethanol extraction and concentration of the residue of Astragalus membranaceus and Cyperus rotundus after water extraction.

[0013] By adopting the above technical solution, this limitation clearly defines the alcohol extract of Astragalus membranaceus and Cyperus rotundus residue as an effective source of components to achieve the effect of "molecular anchoring and competitive substitution", and at the same time improves the comprehensive value of raw materials by utilizing the residue.

[0014] Preferably, the raw materials for its preparation include a functional inclusion mother liquor, which is obtained by water extraction and concentration of the honeysuckle and the dandelion.

[0015] By adopting the above technical solution, the limitation clearly defines that the water extracts of honeysuckle and dandelion are the material basis for constituting the "outer stabilizing layer". This measure utilizes endogenous plant components to replace exogenous synthetic surfactants.

[0016] Preferably, the biscuit base comprises flour, baking powder, powdered sugar, and cooked starch slurry.

[0017] By adopting the above technical solution, a biscuit base component suitable for adding the complex of the present invention is provided, ensuring the shapeability and taste of the final product.

[0018] Secondly, the present invention provides a method for preparing a natural plant-flavored biscuit for preventing and treating mastitis in lactating livestock, using the following technical solution: A method for preparing a natural plant-flavored biscuit includes the following steps: Step 1: Extract and concentrate honeysuckle and dandelion with water to obtain functional inclusion mother liquor; Step 2: Extract the volatile oil from Cyperus rotundus to obtain Cyperus rotundus volatile oil and water-extracted residue; Step 3: Take Astragalus membranaceus and the water extract residue from Step 2 for alcohol extraction and concentration to obtain a concentrated ethanol extract. Step 4: Dissolve hydroxypropyl-β-cyclodextrin in the functional inclusion mother liquor from Step 1, then add the concentrated ethanol extract from Step 3 for pre-reaction, and then add the Cyperus rotundus volatile oil from Step 2 for inclusion reaction to obtain a multi-component supramolecular complex. Step 5: Mix the multi-component supramolecular complex from Step 4 with the biscuit base to form dough; Step 6: Press the dough into shape and bake at a low temperature to obtain the final product.

[0019] By adopting the above technical solution, the core of this preparation method lies in the unique feeding sequence and reaction design in step four. This design actively guides the orderly self-assembly of each component in the system, thereby forming the stable supramolecular structure described in the first aspect.

[0020] Preferably, in step four, after adding the concentrated ethanol extract, the reaction is carried out at 38-42°C for 25-35 minutes.

[0021] By adopting the above technical solution, suitable kinetic and thermodynamic conditions are provided for the interaction between the components in the ethanol extract concentrate and the cyclodextrin and mother liquor system, ensuring the full progress of the pre-reaction.

[0022] Preferably, in step four, after adding Cyperus rotundus volatile oil, the reaction continues at 38-42°C for 210-230 minutes.

[0023] By adopting the above technical solution, it is ensured that the volatile oil molecules have enough time to diffuse, enter the cyclodextrin cavity and reach inclusion equilibrium, while also ensuring the formation and solidification of the external stabilizing layer.

[0024] Preferably, the water extraction in step one is performed using an ultrasonic-assisted extraction method.

[0025] By adopting the above technical solution, the mass transfer process is enhanced by utilizing the cavitation effect of ultrasound, which improves the extraction rate of effective components (polysaccharides, saponins, etc.) in honeysuckle and dandelion, thereby enhancing the synergistic stabilizing ability of the functional inclusion mother liquor.

[0026] Preferably, the low-temperature baking in step six is ​​carried out at a temperature of 80-85°C, and the baking endpoint is when the moisture content in the center of the biscuit is less than 5%.

[0027] By adopting the above technical solutions, low-temperature baking can minimize the secondary loss of volatile oils that are already protected by supramolecular structures during the heat treatment process, while controlling the final moisture content ensures the shelf-life stability of the product.

[0028] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention significantly improves the stability of volatile oils. By constructing a multi-level supramolecular complex with volatile oil-cyclodextrin as the core, functional inclusion complexes containing macromolecular components in the mother liquor as the outer physical barrier, and molecular anchoring by components in the ethanol extract, the thermal and physical stability of Cyperus rotundus volatile oil is effectively improved. This significantly reduces its degradation and loss during subsequent processing such as biscuit mixing, molding, and hot air baking, ensuring the retention rate of active ingredients in the final product.

[0029] 2. This invention improves the uniformity of active ingredient distribution in the final product. The prepared multi-component supramolecular complex is a homogeneous or micro-homogeneous liquid system, which, when mixed with biscuit base ingredients such as flour, can be uniformly dispersed at the microscale throughout the entire dough matrix. This solves the technical problem of uneven distribution of oil-soluble components such as volatile oils due to poor compatibility with aqueous dough systems, ensuring the relative consistency of active ingredient content in each finished biscuit.

[0030] 3. This invention achieves comprehensive utilization of plant raw materials and synergistic effects among components. This method not only uses the aqueous extracts of honeysuckle and dandelion as functional inclusion mother liquors for constructing a stable system, but also utilizes the residue from the aqueous extraction of Cyperus rotundus combined with Astragalus membranaceus for alcohol extraction. This design not only improves the comprehensive utilization rate of raw materials, but more importantly, it enables each plant component to functionally support each other and jointly participate in the construction of a stable supramolecular structure, producing a technical effect greater than the sum of the individual effects of each component. Detailed Implementation

[0031] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0032] Hydroxypropyl-β-cyclodextrin, CAS No.: 128446-35-5. The product used in this invention is pharmaceutical excipient grade.

[0033] Ethanol, CAS No.: 64-17-5. The product used in this invention is of analytical grade, with a volume concentration of 90.0%-95.0%.

[0034] The flour is commercially available food-grade high-gluten wheat flour.

[0035] Baking powder is a commercially available food-grade compound leavening agent. Its main components include acidic salts (such as potassium hydrogen tartrate and calcium dihydrogen phosphate), alkaline salts (sodium bicarbonate, CAS No.: 144-55-8), and starch fillers.

[0036] The sugar powder is commercially available food grade, and its main ingredient is sucrose (CAS number: 57-50-1).

[0037] Cooked starch slurry is made by gelatinizing commercially available food-grade corn starch with water.

[0038] Lemon peel is the chopped outer peel of commercially available food-grade dried lemons.

[0039] Vanillin, chemically known as 4-hydroxy-3-methoxybenzaldehyde, CAS number: 121-33-5, is a commercially available food-grade product. Preparation Example 1: Take 480g of honeysuckle and 520g of dandelion, mix them, and pulverize them until they can pass through a 30-mesh sieve. Place the pulverized mixture in an extraction tank, add 10 times the total weight of deionized water (10000ml), and soak at room temperature (25℃) for 2 hours. Turn on the ultrasonic generator (1000W power, 30kHz frequency) and perform the first ultrasonic-assisted extraction at a controlled temperature of 50℃ for 50 minutes. After extraction, filter through a 200-mesh filter cloth and collect the filtrate. Add another 10 times the amount of deionized water to the residue in the extraction tank, repeat the ultrasonic extraction and filtration process, and collect the second filtrate. Combine the two collected filtrates and transfer them to a vacuum concentration tank. Concentrate under reduced pressure at a vacuum degree of -0.07MPa and a jacket temperature of 60℃ until the total liquid volume is 4000ml. This concentrate is the "functional inclusion mother liquor" (custom component A), which should be sealed and refrigerated at 4-8℃ for later use.

[0040] Preparation Example 2: Take 400g of Cyperus rotundus and grind it until it can pass through a 15-mesh sieve. Place it in a multi-functional extraction vessel and add 5000ml of water. Extract the volatile oil using vacuum distillation, controlling the vacuum degree of the system at -0.05MPa, the jacket heating temperature at 60℃, and the distillation time at 80 minutes. Separate and collect the upper light yellow oily liquid through an oil-water separator, which is the Cyperus rotundus volatile oil (component B), and weigh and record the yield. After distillation, retain all the residue and aqueous phase liquid in the vessel for subsequent steps.

[0041] Preparation Example 3: Take 440g of Astragalus membranaceus, grind it to 30 mesh, and combine it with the Cyperus rotundus residue and aqueous phase liquid retained in Preparation Example 2. Add deionized water to make the total water volume 8000ml. Extract with ultrasound (1000W) at 60℃ for 45 minutes. Filter and collect the filtrate. Add another 8000ml of water to the residue, repeat the ultrasound extraction once, and combine the two filtrates to obtain "Astragalus membranaceus aqueous extract". Add 9 times the wet weight of the residue to the mixed residue after water extraction, add 90% ethanol, and extract with ultrasound (1000W) at 50℃ for 50 minutes. Filter and collect the filtrate. Extract the residue again with ethanol under the same conditions once. Combine the two ethanol filtrates and recover the ethanol under vacuum of -0.07MPa and 50℃ until there is no obvious alcohol odor, obtaining a viscous liquid with a volume of about 500ml, which is the "ethanol extract concentrate" (custom component C), and seal for later use.

[0042] Preparation Example 4: Preparation of Astragalus Extract The "Astragalus aqueous extract" obtained in Preparation Example 3 was concentrated under reduced pressure at 60°C and a vacuum degree of -0.07 MPa until the relative density of the concentrate reached 1.08 when measured at 50°C. The resulting product is "Astragalus Clear Extract" and is used for later use.

[0043] Example 1: This embodiment provides a natural plant-flavored biscuit for preventing mastitis in lactating animals and its preparation method. The process parameters used are the midpoints of various ranges, specifically including: Raw material preparation: Weigh out 450g of Astragalus membranaceus, 500g of Lonicera japonica, 550g of Taraxacum mongolicum, 400g of Cyperus rotundus, and 2000g of roasted flour.

[0044] Intermediate preparation: Preparation of the functional inclusion complex mother liquor: Take 500g of honeysuckle and 550g of dandelion, mix and pulverize them, add 10 times the amount (10500ml) of water, and extract twice by ultrasonication at 50℃, 50 minutes each time. Combine the filtrates, concentrate under reduced pressure to 4200ml to obtain the "functional inclusion complex mother liquor", and refrigerate for later use.

[0045] Preparation of Cyperus rotundus volatile oil: Take 400g of Cyperus rotundus, crush it, add 5000ml of water, and extract the volatile oil by vacuum distillation to obtain about 6g of Cyperus rotundus volatile oil (estimated based on a yield of 1.5%). Retain the residue and the aqueous phase liquid.

[0046] Preparation of the concentrated ethanol extract: Take 450g of Astragalus membranaceus, combine it with the Cyperus rotundus residue and aqueous phase liquid from the previous step, add water to make up to 8000ml, and perform ultrasonic water extraction twice at 60℃. The residue after water extraction is ultrasonically extracted twice with 90% ethanol, the ethanol extracts are combined, the ethanol is recovered and concentrated to about 500ml to obtain the "concentrated ethanol extract".

[0047] Preparation of Astragalus Extract: The above-mentioned "Astragalus aqueous extract" was concentrated under reduced pressure to a relative density of 1.08 (measured at 50℃) to obtain "Astragalus Extract".

[0048] Preparation of supramolecular complexes and biscuit shaping: Step 1: Weigh 45g of hydroxypropyl-β-cyclodextrin (approximately 7.5 times the weight of the volatile oil, corresponding to 30-60 parts in the claims). Add the middle to upper range of the range to the "functional inclusion mother liquor" and stir at 40℃ and 200r / min until dissolved.

[0049] Step 2: Add "ethanol extract concentrate" and react at 40℃ and 200r / min for 30 minutes.

[0050] Step 3: Add approximately 6g of Cyperus rotundus volatile oil slowly at a rate of 2.0ml / min, and continue the reaction for 220 minutes after the addition is complete.

[0051] Step 4: The mixture is refrigerated at 4-8℃ and allowed to stand for 22 hours to obtain a "multi-component supramolecular complex".

[0052] Steps 5 to 9: Combine the above complex with "Astragalus Clearing Paste", then mix with 2000g of roasted flour and other biscuit ingredients (added in proportion), knead into a dough, press into 4mm thick biscuit blanks, and bake at 82℃ until the center moisture content is less than 5%.

[0053] Example 2: This embodiment provides a natural plant-flavored biscuit for preventing mastitis in lactating animals and its preparation method. The process parameters used are the lower limits of each range, specifically including: Raw material preparation: Weigh out 400g of Astragalus membranaceus, 450g of Lonicera japonica, 500g of Taraxacum mongolicum, 350g of Cyperus rotundus, and 1500g of roasted flour.

[0054] Intermediate preparation and biscuit shaping: The above raw materials were processed according to the method of Example 1: A "functional inclusion mother liquor" (approximately 3800 ml) was prepared from honeysuckle and dandelion.

[0055] Extract volatile oil from Cyperus rotundus (approximately 5.25g), retaining the dregs.

[0056] Astragalus and Cyperus rotundus residue were used to prepare "ethanol extract concentrate" and "Astragalus extract paste".

[0057] Take 32g of hydroxypropyl-β-cyclodextrin (approximately 6 times the weight of the volatile oil, corresponding to the lower limit of the range of 30-60 parts in the claims) and prepare a "multi-component supramolecular complex" with the above intermediate according to the process parameters of Example 1 (40°C, 200r / min, etc.).

[0058] Finally, mix with 1500g of flour and the corresponding proportion of auxiliary ingredients to make cookies.

[0059] Example 3: This embodiment provides a natural plant-flavored biscuit for preventing mastitis in lactating livestock and its preparation method. The process parameters used are the upper limits of each range, specifically including the following: Raw material preparation: Weigh out 500g of Astragalus membranaceus, 550g of Lonicera japonica, 600g of Taraxacum mongolicum, 450g of Cyperus rotundus, and 2500g of roasted flour.

[0060] Intermediate preparation and biscuit shaping: The above raw materials were processed according to the method of Example 1: A "functional inclusion mother liquor" (approximately 4600 ml) was prepared from honeysuckle and dandelion.

[0061] Extract volatile oil from Cyperus rotundus (approximately 6.75g), retaining the dregs.

[0062] Astragalus and Cyperus rotundus residue were used to prepare "ethanol extract concentrate" and "Astragalus extract paste".

[0063] Take 58g of hydroxypropyl-β-cyclodextrin (approximately 8.6 times the weight of the volatile oil, corresponding to the upper limit of the range of 30-60 parts in the claims) and prepare a "multi-component supramolecular complex" with the above intermediate according to the process parameters of Example 1 (40℃, 200r / min, etc.).

[0064] Finally, mix with 2500g of flour and the corresponding proportion of auxiliary ingredients to make cookies.

[0065] Comparative Example 1: Compared to Example 1, the difference lies in that the inclusion process of the volatile oil is not carried out in the "functional inclusion mother liquor," but in an equal volume of pure water, and the "ethanol extract concentrate" is not added beforehand. The "functional inclusion mother liquor" obtained in Preparation Example 1 and the "ethanol extract concentrate" obtained in Preparation Example 3 are physically mixed together with the inclusion compound and Astragalus extract in subsequent steps. Everything else is the same.

[0066] Comparative Example 2: The difference from Example 1 is that the "ethanol extract concentrate" obtained in Preparation Example 3 is not added for pre-reaction in step 2. This "ethanol extract concentrate" is physically mixed with the inclusion complex and Astragalus extract in subsequent steps. Everything else is the same.

[0067] Comparative Example 3: Compared to Example 1, the difference lies in that the inclusion process of the volatile oil (steps 1-3) is not carried out in the "functional inclusion mother liquor," but in an equal volume of pure water, although the step of adding the "ethanol extract concentrate" beforehand is retained. The "functional inclusion mother liquor" obtained in Preparation Example 1 is then physically mixed with the obtained inclusion compound and Astragalus extract in subsequent steps. Everything else is the same.

[0068] Comparative Example 4: Similar to Comparative Example 1, this comparative example involved the inclusion of volatile oils in pure water, except that 0.1% (w / v) of Tween-80 was added to the pure water as an emulsifier before inclusion. All other plant extract components (functional inclusion mother liquor, concentrated ethanol extract, and Astragalus extract) were physically mixed with the resulting inclusion complex in subsequent steps. Everything else was the same.

[0069] Test Example 1: Gas chromatography was used to quantitatively determine the inclusion rate of Cyperus rotundus volatile oil in the intermediates (multi-component supramolecular complexes or inclusion complexes) obtained in Examples 1-3 and Comparative Examples 1-4, in order to evaluate the inclusion capacity of different preparation processes for volatile oil.

[0070] Experimental methods: Determination of free volatile oil (surface extraction method) Accurately transfer 10.0 mL each of the complex / inclusion complex liquids prepared in Examples 1-3 and Comparative Examples 1-4 and cooled to room temperature into 50 mL stoppered centrifuge tubes. Accurately add 10.0 mL of n-hexane to each tube, tighten the stopper, and vortex for 2 minutes to ensure sufficient contact between the organic and aqueous phases. Then, centrifuge at 4000 rpm for 5 minutes. Carefully aspirate the upper n-hexane extract and filter it through a syringe fitted with a 0.45 μm hydrophobic filter membrane. Collect the filtrate in a gas chromatograph vial. This filtrate represents the unencapsulated free volatile oil.

[0071] Determination of total volatile oil (steam distillation method): Accurately transfer 20.0 mL each of the complex / inclusion complex liquids prepared in Examples 1-3 and Comparative Examples 1-4 and cooled to room temperature into a 1000 mL round-bottom flask, and dilute with water to approximately 500 mL. Proceed with the volatile oil determination method, heating and distilling for 4 hours. After distillation, read the volume of the oil layer in the volatile oil analyzer, rinse the inner wall of the analyzer with a small amount of n-hexane, combine the rinse water with the rinse water and transfer it to a 10 mL volumetric flask, dilute to the mark with n-hexane, and mix well. This solution represents the total volatile oil in the system.

[0072] Gas chromatography (GC) conditions: Chromatographic column: HP-5ms quartz capillary column (30m×0.25mm, 0.25μm).

[0073] Carrier gas: high-purity nitrogen, flow rate 1.0 mL / min.

[0074] Inlet temperature: 250℃.

[0075] Detector: Flame Ionization Detector (FID), temperature 280℃.

[0076] Column temperature program: Initial temperature 60℃, hold for 2 minutes; increase temperature to 180℃ at a rate of 10℃ / min, hold for 5 minutes; then increase temperature to 250℃ at a rate of 20℃ / min, hold for 5 minutes.

[0077] Injection volume: 1 μL.

[0078] Split ratio: 20:1.

[0079] Quantitative method: External standard method. α-cyperone was used as the quantitative indicator. The peak area of ​​α-cyperone in the solutions obtained in 2.1 and 2.2 was measured and compared with the standard curve to calculate its content.

[0080] Experimental data: Table 1. Volatile oil inclusion rates of the complexes / inclusion compounds obtained in each example and comparative example (n = 3, mean ± SD). Group Total (mg / mL) Free (mg / mL) Inclusion rate (EE,%) Example 1 2.15±0.08 0.12±0.03 94.42±1.25 Example 2 2.11±0.11 0.18±0.04 91.47±1.58 Example 3 2.18±0.09 0.14±0.02 93.58±0.81 Comparative Example 1 2.09±0.13 0.83±0.06 60.29±2.44 Comparative Example 2 2.12±0.10 0.35±0.05 83.49±2.11 Comparative Example 3 2.13±0.12 0.71±0.07 66.67±2.87 Comparative Example 4 2.10±0.09 0.29±0.04 86.19±1.70

[0081] The data in Table 1 show that the volatile oil inclusion rate of Examples 1-3 using the technical solution of the present invention is significantly higher than that of the comparative examples.

[0082] The inclusion rates of Examples 1-3 (91.47%-94.42%) compared to Comparative Example 1 (60.29%) confirm the superiority of inclusion in the functional inclusion mother liquor. This is attributed to the synergistic emulsifying effect of endogenous surfactants such as natural saponins in the mother liquor during the inclusion process, which increases the contact interface between volatile oils and cyclodextrin molecules, thereby improving the inclusion efficiency.

[0083] The comparison between Example 1 (94.42%) and Comparative Example 2 (83.49%) demonstrates the necessity of pre-adding the ethanol extract concentrate. The competitive displacement mechanism introduced by this step allows the more hydrophobic volatile oil to preferentially and more stably occupy the cyclodextrin cavity, thereby further improving the inclusion rate.

[0084] The inclusion rates of Examples 1-3 were also higher than those of Comparative Example 4 (86.19%). This result indicates that the synergistic inclusion system constructed using endogenous plant components in this invention has a better overall effect than conventional methods that add industrial-grade surfactants (Tween-80), highlighting the uniqueness and effectiveness of the technical solution of this invention. The result of Comparative Example 3 (66.67%) also indirectly confirms that the synergistic effect of the functional inclusion mother liquor is the key factor in improving the inclusion rate; without this synergistic effect, the improvement in effect is limited by only a competitive displacement step.

[0085] In summary, the data from this test case confirm that the present invention can effectively improve the inclusion rate of volatile oils through the synergistic and competitive substitution mechanism of endogenous components.

[0086] Test Example 2: The physical stability of products obtained from different preparation processes was evaluated by measuring the turbidity change of the complex / inclusion complex liquid during static storage. Systems with high physical stability are less prone to particle aggregation or sedimentation, and their turbidity changes less over time.

[0087] Experimental methods: Sample preparation: Accurately transfer 50.0 mL of each of the complex / inclusion complex liquids prepared in Examples 1-3 and Comparative Examples 1-4 and cooled to room temperature, and place them into clean, dry 100 mL stoppered glass bottles, then seal them. All samples were stored at room temperature (25℃±2℃) and protected from light.

[0088] Turbidity measurement: The absorbance value (A) was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer with deionized water as a blank reference. 600 The supernatant was used as an indicator of sample turbidity. On days 0, 1, 3, 7, and 14 of storage, supernatant was gently aspirated from each sample vial (1 cm below the liquid surface) for measurement. The sample vials were not shaken before sampling to allow for natural sedimentation. Each sample was measured three times at each time point.

[0089] Experimental data: Table 2. Turbidity (A) of the complexes / inclusion compounds obtained in each example and comparative example during storage. 600 Variation (n=3, mean±SD) The data in Table 2 show that the turbidity of the complex systems obtained in Examples 1-3 changed slowly during the 14-day storage period, and the rate of change of turbidity (3.75%-8.30%) was significantly lower than that of all comparative groups.

[0090] The turbidity of Comparative Example 1 (TCR 80.34%) decreased sharply during storage, indicating severe aggregation and sedimentation of the inclusion complex prepared in pure water. In contrast, the stability of Example 1 (TCR 4.80%) demonstrates that the introduction of the functional inclusion mother liquor is crucial for maintaining system stability. This supports the mechanism of the invention, namely that macromolecules such as polyphenols and polysaccharides in the mother liquor form a steric hindrance and electrostatic repulsion layer around the inclusion complex, effectively preventing particle aggregation.

[0091] The stability of Example 1 was also superior to that of Comparative Example 2 (TCR 25.64%). This indicates that the step of pre-adding the ethanol extract concentrate not only improved the inclusion rate but also had a positive impact on the final physical stability of the system. Mechanistically, the displaced astragaloside A and other molecules participate in the stabilizing network around the inclusion complex through non-covalent interactions, playing a role in molecular anchoring and further enhancing the stability of the entire supramolecular structure.

[0092] Although Comparative Example 3 (TCR 64.73%) also incorporated an alcohol extract, its stability was far inferior to that of the Example Group due to the lack of synergistic effect of the functional inclusion mother liquor, which once again confirmed the key role of the functional inclusion mother liquor.

[0093] The stability of Examples 1-3 was also superior to that of Comparative Example 4 (TCR 26.18%). Although Tween-80 provided initial emulsification, the stable layer it formed was not as effective in long-term storage as the complex stable network constructed by the present invention using multiple endogenous components.

[0094] In summary, the data from this test case confirms that the technical solution of this invention can prepare supramolecular complexes with significantly better physical stability than products obtained by conventional methods. This is mainly due to the secondary stabilizing effect provided by the functional inclusion mother liquor and the molecular anchoring effect introduced by the ethanol extract. Test Example 3: Purpose: The protective effects of the complexes / inclusion complexes obtained in Examples 1-3 and Comparative Examples 1-4 on the volatile oil of Cyperus rotundus were evaluated through accelerated heating experiments. The ability of different preparation processes to maintain the stability of the volatile oil under thermal stress was assessed by measuring the retention rate of the volatile oil in the samples after heating.

[0095] Experimental methods: Sample heat treatment: Accurately transfer 10.0 mL each of the untreated complex / inclusion complex liquids prepared in Examples 1-3 and Comparative Examples 1-4 into 20 mL headspace vials, and immediately seal them with PTFE / silicone septa and aluminum caps. Place the sealed vials in a constant temperature oven at 60℃±1℃, horizontally, and heat for 24 hours. After heating, remove the vials and cool to room temperature for later use.

[0096] For the determination of initial volatile oil content, take another sample of the untreated complex / inclusion complex liquid and determine its initial volatile oil content (calculated as α-cyperone) according to the method for determining total volatile oil in Section 2.2 of Test Example 1.

[0097] Determination of volatile oil content after heating: After heat treatment and cooling, transfer all samples to a round-bottom flask. Rinse the headspace vial with a small amount of water, and combine the washings into the flask. Following the method for determining total volatile oil in Section 2.2 of Test Example 1, determine the remaining volatile oil content after heating, denoted as W_final.

[0098] All samples were measured three times.

[0099] Table 3. Retention rate of volatile oil in each embodiment and comparative sample after heat treatment. Group W_initial (mg / mL) W_final (mg / mL) Retention rate (RR, %) Example 1 2.14±0.09 1.95±0.11 91.12±2.65 Example 2 2.12±0.10 1.83±0.13 86.32±3.01 Example 3 2.17±0.08 2.02±0.09 93.09±1.98 Comparative Example 1 2.08±0.11 0.51±0.08 24.52±3.42 Comparative Example 2 2.11±0.09 1.58±0.12 74.88±2.88 Comparative Example 3 2.14±0.12 1.03±0.10 48.13±3.91 Comparative Example 4 2.11±0.10 1.72±0.11 81.52±2.54 The data in Table 3 show that the volatile oil retention rates (86.32%-93.09%) of the complexes obtained in Examples 1-3 after heating at 60°C for 24 hours were significantly higher than those of all comparative groups. This indicates that the supramolecular structure constructed by the technical solution of the present invention provides effective thermal protection for volatile oils.

[0100] The significant difference between Example 1 (RR 91.12%) and Comparative Example 1 (RR 24.52%) confirms that simple physical mixing cannot protect volatile oils from degradation or escape under thermal stress, while the inclusion structure of the present invention is the basis for achieving thermal stability.

[0101] The comparison between Example 1 and Comparative Example 2 (RR 74.88%) highlights the contribution of the molecular anchoring effect introduced by the ethanol extract to thermal stability. Thermodynamically, this multi-layered non-covalent network increases the energy barrier for volatile oil molecules to escape from the cyclodextrin cavity, thereby effectively reducing their loss rate under heating conditions.

[0102] The comparison between Example 1 and Comparative Example 3 (RR 48.13%) demonstrates that the synergistic effect of the functional inclusion mother liquor is indispensable for constructing thermally stable structures. The physical barrier formed by the macromolecules in the mother liquor around the inclusion compound is the first line of defense against thermal shock.

[0103] Furthermore, the retention rates of Examples 1-3 were all higher than those of Comparative Example 4 (RR 81.52%), which used a standard emulsifier. This indicates that the "core-outer wall-anchored molecule" three-level protection system synergistically constructed by endogenous components in this invention has better thermodynamic stability than simple micelles or stabilizing layers formed by a single surfactant.

[0104] In summary, the data from this test example confirm that the preparation method of the present invention can form a supramolecular complex with excellent thermal stability. This stability originates from the multiple synergistic effects of the core protection of cyclodextrin inclusion, the outer barrier of the functional inclusion mother liquor, and the molecular anchoring of the alcohol extract.

[0105] Test Example 4: The influence of different preparation processes on the uniformity of active ingredient distribution in a solid matrix was evaluated by measuring the content distribution of volatile oils in the same batch of final product (biscuits). The relative standard deviation (RSD) was used as an evaluation index for uniformity; a smaller RSD value indicates a more uniform component distribution.

[0106] Experimental methods: Sample collection and preparation: Ten cookies were randomly selected from each batch of finished cookies prepared in Examples 1-3 and Comparative Examples 1-4. Each cookie was individually ground into a fine powder that could pass through a 60-mesh sieve in a mortar, and then placed into sealed bags and numbered as samples to be tested.

[0107] Extraction of volatile oils (ultrasound-assisted solvent extraction) Accurately weigh approximately 2.0 g of each biscuit powder and place it in a 50 mL stoppered Erlenmeyer flask. Accurately add 20.0 mL of n-hexane and tighten the stopper. Place the Erlenmeyer flask in an ultrasonic cleaner (250 W, 40 kHz) and ultrasonically extract for 30 minutes at 25°C in a water bath. After extraction, centrifuge the mixture at 4000 rpm for 10 minutes. Collect the supernatant and filter it through a 0.45 μm hydrophobic membrane. Collect the filtrate in a gas chromatograph vial for analysis.

[0108] Gas chromatography analysis and content calculation: GC conditions were the same as in Test Example 1. The external standard method was used, with α-cyperone as the quantitative indicator, and the volatile oil content (mg / g) in each biscuit sample was calculated based on the standard curve.

[0109] Experimental data Table 4 shows the uniformity of volatile oil content in the biscuits obtained from each example and comparative example (n = 10, mean ± SD).

[0110] The data in Table 4 clearly show that the RSD values ​​(2.51%-4.15%) of the biscuits prepared in Examples 1-3 are much lower than those of all comparative groups, indicating that the technical solution of the present invention can ensure a highly uniform distribution of active ingredients in the final solid product.

[0111] This superior homogeneity is a direct manifestation of the core technology of this invention. The "multi-component supramolecular complex" liquid obtained in the examples is itself a highly stable homogeneous or micro-homogeneous system (as shown in Test Example 2). When this stable liquid is mixed with flour, the active components can be uniformly dispersed at the microscale throughout the dough matrix and maintained during subsequent shaping and baking processes.

[0112] Conversely, due to the poor physical stability of its liquid formulation, the inclusion complex particles aggregated and settled before or during mixing with flour in the comparative group. This resulted in some doughs acquiring high concentrations of active ingredient clumps, while others contained very little. This heterogeneity was solidified in the final biscuit product, resulting in extremely high RSD values ​​(28.71% for Comparative Example 1). The RSD values ​​of Comparative Examples 2, 3, and 4 were also significantly higher than those of the examples, further confirming the crucial role of the secondary stabilization of the functional inclusion mother liquor and the molecular anchoring effect of the alcohol extract in preparing a homogeneous final product. In summary, this test case demonstrates that the uniformity of the distribution of active ingredients in the final product directly depends on the physical stability of the intermediate formulation. This invention successfully solves the technical challenge of uniformly introducing liquid active components into a solid matrix by constructing a stable supramolecular complex.

Claims

1. A natural plant-flavored biscuit for preventing and treating mastitis in lactating livestock, characterized in that, Made from the following ingredients in parts by weight: Astragalus: 400-500 portions; Honeysuckle: 450-550 servings; Dandelion: 500-600 servings; Cyperus rotundus: 350-450 portions; Hydroxypropyl-β-cyclodextrin: 30-60 parts; Biscuit base: 1500-2500 portions.

2. The natural plant-flavored biscuit for preventing and treating mastitis in lactating animals according to claim 1, characterized in that, The weight parts of the raw materials are: Astragalus: 440 portions; Honeysuckle: 480 portions; Dandelion: 520 samples; Cyperus rotundus: 400 portions; Hydroxypropyl-β-cyclodextrin: 42 parts; Biscuit base: 2000 servings.

3. The natural plant-flavored biscuit for preventing and treating mastitis in lactating animals according to claim 1, characterized in that, The raw materials include an ethanol extract concentrate, which is obtained by ethanol extraction and concentration of the residue from water extraction of Astragalus membranaceus and Cyperus rotundus.

4. The natural plant-flavored biscuit for preventing and treating mastitis in lactating animals according to claim 1, characterized in that, The raw materials include a functional inclusion complex mother liquor, which is obtained by water extraction and concentration of the honeysuckle and the dandelion.

5. The natural plant-flavored biscuit for preventing and treating mastitis in lactating animals according to claim 1, characterized in that, The biscuit base contains flour, baking powder, powdered sugar, and cooked starch slurry.

6. A method for preparing a natural plant-flavored biscuit for preventing and treating mastitis in lactating animals, according to any one of claims 1-5, characterized in that... Includes the following steps: Step 1: Extract and concentrate honeysuckle and dandelion with water to obtain functional inclusion mother liquor; Step 2: Extract the volatile oil from Cyperus rotundus to obtain Cyperus rotundus volatile oil and water-extracted residue; Step 3: Take Astragalus membranaceus and the water extract residue from Step 2 for alcohol extraction and concentration to obtain a concentrated ethanol extract. Step 4: Dissolve hydroxypropyl-β-cyclodextrin in the functional inclusion mother liquor from Step 1, then add the concentrated ethanol extract from Step 3 for pre-reaction, and then add the Cyperus rotundus volatile oil from Step 2 for inclusion reaction to obtain a multi-component supramolecular complex. Step 5: Mix the multi-component supramolecular complex from Step 4 with the biscuit base to form dough; Step 6: Press the dough into shape and bake at a low temperature to obtain the final product.

7. The method for preparing natural plant-flavored biscuits for preventing and treating mastitis in lactating animals according to claim 6, characterized in that, In step four, after adding the concentrated ethanol extract, the mixture is reacted at 38-42°C for 25-35 minutes.

8. The method for preparing natural plant-flavored biscuits for preventing and treating mastitis in lactating animals according to claim 6, characterized in that, In step four, after adding Cyperus rotundus volatile oil, the reaction continues at 38-42℃ for 210-230 minutes.

9. The method for preparing natural plant-flavored biscuits for preventing and treating mastitis in lactating animals according to claim 6, characterized in that, The water extraction in step one uses an ultrasonic-assisted extraction method.

10. The method for preparing natural plant-flavored biscuits for preventing and treating mastitis in lactating animals according to claim 6, characterized in that, The low-temperature baking in step six is ​​carried out at a temperature of 80-85℃, and the baking endpoint is when the moisture content in the center of the biscuit is less than 5%.

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