A "milk-reducing" antibiotic-free dry milk composition and its preparation method

Abstract

The present invention provides an antibiotic-free drying composition comprising an effective amount of chitosan and baicalein, as well as an oily carrier, a suspending agent, a wetting agent, and an antioxidant. The drying composition promotes the repair of mammary tissue in dry cows, inhibits milk secretion, and enhances mammary immunity. It can accelerate mammary epithelial renewal, reduce milk production, and thus lower intra-milk pressure. It also exhibits a synergistic effect in promoting antioxidant activity and inhibiting the sensitivity of Staphylococcus aureus, Escherichia coli, and other bacteria to drugs. It can significantly reduce the milk leakage rate of dairy cows during the dry period, demonstrating a significant drying effect, particularly for early-stage drying.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture technology, and in particular to a "milk-reducing type" antibiotic-free dry milk composition. Background Art

[0002] Mastitis is a common and highly prevalent disease in dairy cows. Studies have found that 24.5% of high-yielding dairy cows leak milk during dry-off. The risk of mastitis and intramammary infection in udders that leak milk during the early stages of dry-off is twice as high and 1.5 times higher than in normal udders. Mastitis during the dry-off period can even lead to clinical mastitis 60 days postpartum. This period, when mammary acinar cell proliferation fluctuates significantly, represents the optimal time for prevention and treatment. Currently, 98.64% of dairy farms in China administer antibiotic-based dry-off treatment to the entire herd to reduce the risk of mammary gland infection during the dry-off period. However, the increasing problems of antibiotic resistance, antibiotic residues in animal products, and potential environmental pollution are forcing the dairy industry to seek new solutions. The UK officially banned herd-wide dry-off treatment in 2022, and EU regulations, which came into effect in June 2019, also completely prohibited it. Therefore, in the context of antibiotic reduction and replacement, the efficient and environmentally friendly way to promote dry-off is a new challenge facing the dairy industry.

[0003] Chitosan (CS) is a bioactive, biocompatible, biodegradable, and non-toxic hydrocolloid that can alter the secretory state of mammary cells, accelerating mammary gland involution. It also exhibits antibacterial and wound-healing properties. CN109310705A provides a composition for accelerating mammary gland involution and preventing intramammary infection during the drying-off period of lactating ruminants. This composition primarily leverages the temperature-sensitive properties of chitosan gel to achieve this goal through mammary perfusion. However, the excipient β-glycerophosphate is relatively expensive, and chitosan itself may gradually degrade into short chains in an acidic environment, affecting its efficacy. Furthermore, the agent has strict storage temperature requirements, which poses certain limitations in terms of pasture environment and profitability.

[0004] Baicalein (BAI) is one of the flavonoids with the highest content in Scutellaria baicalensis. It has been shown to reduce cerebral vascular resistance, improve cerebral circulation, increase cerebral blood flow, and inhibit platelet aggregation. It is clinically used to treat post-cerebrovascular paralysis. Furthermore, Scutellaria baicalensis has a broad antimicrobial spectrum, demonstrating inhibitory effects against Shigella dysenteriae, Corynebacterium diphtheriae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Pneumococcus, and meningococci in experimental studies.

[0005] Existing research includes the preparation of ophthalmic lipid nanoparticles by modifying baicalein with N-trimethylchitosan (CN110051654A), and the preparation of baicalein nanoparticles by loading baicalein onto a chitosan-cyclodextrin graft polymer (CN113546072A). In these studies, chitosan is primarily used as a drug carrier.

[0006] So far, there has been no research on the combined use of chitosan and baicalein for antibiotic-free dry milk in animals. Summary of the Invention

[0007] The object of the present invention is to provide an antibiotic-free dry milk composition.

[0008] The present invention has found that the combined use of chitosan and baicalein can promote the repair of mammary tissue and regulate mammary immunity in dry cows, accelerate mammary epithelial renewal, reduce milk production to reduce intra-milk pressure, and improve animal welfare. It also has a synergistic effect in promoting antioxidant activity and inhibiting the sensitivity of common mastitis bacteria such as Escherichia coli and Staphylococcus aureus to drugs. It can significantly reduce the rate of milk leakage during dry-off, and is particularly effective for early dry-off. Based on this, the present invention provides the following technical solutions:

[0009] The present invention first provides the use of chitosan and baicalein in preparing a dry milk composition, which is used for quickly drying milk, reducing milk leakage rate during the dry milk period and reducing mastitis.

[0010] Furthermore, the present invention provides a dry milk composition comprising an effective amount of chitosan and baicalein.

[0011] Furthermore, the composition also includes an oily carrier, a suspending agent, a wetting agent and an antioxidant.

[0012] Furthermore, the composition comprises the following ingredients in weight and volume percentages: chitosan 1-10%, baicalein 0.5-5%, suspending agent 0.1-1%, wetting agent 0.005-0.05%, antioxidant 0.001-0.01%, and an oily carrier is added to the volume. Furthermore, the composition comprises the following ingredients in weight and volume percentages: chitosan 2.5%, baicalein 1.25%, suspending agent 0.6%, wetting agent 0.01%, antioxidant 0.005%, and an oily carrier is added to the volume.

[0013] Furthermore, the oleaginous carrier can be selected from one or more of soybean oil for injection, peanut oil for injection, sesame oil for injection, castor oil for injection, olive oil for injection, isopropyl myristate, dimethicone, or medium-chain triglycerides. In one embodiment of the present invention, the oleaginous carrier is soybean oil for injection.

[0014] Furthermore, the suspending agent can be selected from one or more of hydrogenated castor oil, hydrogenated peanut oil, hydrogenated tea seed oil, polyvinyl pyrrolidone, soybean lecithin, white beeswax, 75% aluminum monostearate or hydrogenated sesame oil. In one embodiment of the present invention, the suspending agent is hydrogenated castor oil.

[0015] Furthermore, the wetting agent is selected from Span 80 or fatty acid glyceride. The wetting agent here is an oil-soluble wetting agent. The suspending effect can be achieved by using a suspending agent and a wetting agent. Of course, those skilled in the art can also select other suspending agents that can achieve a suspending effect.

[0016] Furthermore, the antioxidant is selected from one or more of vitamin E oil, ascorbyl palmitate, lipoic acid, coenzyme Q10, butylated hydroxyanisole or butylated hydroquinone. In one embodiment of the present invention, the antioxidant is vitamin E oil.

[0017] The present invention selects an oily carrier, a suspending agent, an oil-soluble wetting agent and an oil-soluble antioxidant, which can effectively address the difficult problems that baicalein and chitosan are difficult to dissolve in the solution and chitosan is easy to degrade.

[0018] Furthermore, the present invention provides a method for preparing the above-mentioned dry milk composition, comprising the following steps:

[0019] 1) Weigh the formulated amount of suspending agent and add it to an appropriate amount of oily carrier, heat it to completely dissolve it, and obtain a standby solution;

[0020] 2) Pour the remaining amount of the oily carrier into a ball mill, add the chitosan, wetting agent and reserve solution in the formula amount in sequence, and grind intermittently at 400-800 rpm for about 30-50 minutes;

[0021] 3) Slowly add the formulated amount of baicalin and continue grinding intermittently for 30-60 minutes;

[0022] 4) Inspection and packaging.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention does not contain any antibiotic ingredients, will not cause drug irritation or residue to the cow's body, and will not affect the quality of milk;

[0025] (2) The drug of the present invention is locally perfused into the breast, and the drug concentration on the surface of the breast is high, thereby maximizing its function;

[0026] (3) The active pharmaceutical ingredients of the present invention have no toxic side effects, are degradable and absorbable, and do not burden the mammary glands of dairy cows;

[0027] (4) The present invention can alleviate the occurrence of milk swelling and milk leakage in the early stage of dry milk by promoting the "regeneration and degeneration" of mammary epithelial cells, and provide assistance for the formation of keratin plugs and the prevention of new infections in the mammary gland;

[0028] (5) The present invention has a significant inhibitory and killing effect on Staphylococcus aureus, Escherichia coli, etc. that induce mastitis by adding Chinese herbal extracts to achieve synergistic antibacterial effects. At the same time, it can reduce the content of inflammatory factors in serum, enhance the anti-infection ability of mammary epithelium against pathogens, and improve the antioxidant function;

[0029] (6) The present invention is an oily preparation, which not only addresses the solubility problem of the preparation, but also can form a physical barrier, blocking the nipple pore in the form of an oil film, forming a natural "sealing" effect, and protecting the breast tissue;

[0030] (7) The active ingredients of the present invention are stable, and the addition of oil-soluble antioxidants can increase the clinical efficacy period;

[0031] (8) The preparation method of the composition of the present invention is simple and low in cost.

[0032] The present invention utilizes a suspension formulation, which facilitates the conversion of poorly soluble chitosan and baicalein into a liquid formulation for clinical application. After injection into milk, the suspension remains within the alveolar cavity, gradually releasing the drug due to diffusion from the oil system. The development of this compound formulation contributes to ensuring the safety of animal-derived food and increasing farmers' incomes. It has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown are the antioxidant status of CS / CAI;

[0034] Figure 2 shows the in vitro antibacterial effect of CS / CAI. Figure 2A and 2B It is the antibacterial status of Staphylococcus aureus. Figure 2C and 2D is the antibacterial effect of Escherichia coli, where the control group is the drug solvent, and the subscript is the effective concentration of the solvent in the solution, in mg / ml;

[0035] Figure 3 The figure shows the number of somatic cells of mammary gland degeneration indicators in different groups. Compared with the negative control group, * indicates a significant difference. P <0.05, *** indicates extremely significant difference P <0.001;

[0036] Figure 4 The figure shows the albumin level, an indicator of breast degeneration, in different groups. * indicates a significant difference compared with the negative control group. P <0.05, *** indicates extremely significant difference P <0.001;

[0037] Figure 5 The figure shows the lactate dehydrogenase level, an indicator of breast degeneration, in different groups. * indicates a significant difference compared with the positive control group. P <0.05, *** indicates extremely significant difference P <0.001;

[0038] Figure 6 The figure shows the lactoferrin level, an indicator of breast degeneration, in different groups. * indicates a significant difference compared to the positive control group. P <0.05, *** indicates extremely significant difference P <0.001;

[0039] Figure 7 Shown are photos of breast degeneration in different groups. Columns 1 to 6 are photos of the groups on days 0, 1, 2, 3, 5, and 7, respectively.

[0040] Figure 8 The trend of milk median distance in different groups is shown. Compared with the antibiotic (positive control group), * indicates significant difference. P <0.05, *** indicates extremely significant difference P <0.001; # indicates significant difference compared with the CAI group P <0.05, ### represents extremely significant difference P <0.001. DETAILED DESCRIPTION

[0041] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0042] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The effective amount in the present invention refers to an amount that has a pharmacological effect, for example, can effectively reduce the rate of dry milk leakage and have a significant dry milk effect. The results can be referred to Example 6 of the present invention.

[0043] The constant volume replenishment of the grease carrier described in the present invention means that the grease carrier is used as a solvent to replenish the volume to 100%.

[0044] Example 1 Preparation of compound preparation

[0045] 1. The composition of the compound preparation according to weight-to-volume ratio (W / V, g / ml*100%) is as follows:

[0046] 1.1 Active ingredients:

[0047] Chitosan (CS), white powder, 2.5% (W / V).

[0048] Baicalein (CAI), yellow powder, 1.25% (W / V).

[0049] 1.2 Grease carrier: Add to 100% of the volume

[0050] Soybean oil for injection, light yellow transparent liquid.

[0051] 1.3 Suspending agents, oil-soluble wetting agents, antioxidants:

[0052] Hydrogenated castor oil, colorless transparent liquid, 0.6% (W / V).

[0053] Span 80, colorless and transparent liquid, 0.01% (W / V).

[0054] Vitamin E oil, light yellow powder, 0.005% (W / V).

[0055] Suspending agents are used to increase the viscosity of the dispersion medium to reduce the sedimentation rate of drug particles or increase the hydrophilicity of the particles.

[0056] The role of antioxidants is to extend the shelf life of compound drug preparations.

[0057] 2. Preparation (140ml)

[0058] 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil. Heat until completely dissolved to obtain a standby solution.

[0059] 2) Evenly disperse the remaining soybean oil into the built-in milling tank of a YXQM-0.4L ball mill. Add the prescribed amount of CS, Span 80, and reserve solution in sequence. Start the ball mill and intermittently grind at 600 rpm for approximately 40 minutes. (This intermittent grinding is primarily to prevent overheating of the ball mill. According to technical recommendations, grind for 10-15 minutes, with 10-minute intervals to prevent overheating. If the total time is a multiple of 10, repeat the "10+10" cycle. If the total time contains a decimal of 5, repeat the "10+10" cycle, with a final 5-minute interval between each 10-minute interval.)

[0060] 3) Slowly add the prescribed amount of CAI and continue intermittent grinding for 45 minutes.

[0061] 4) Check the particle fineness: no less than 90% of the particles are below 15μm, no less than 95% of the particles are below 20μm, and there are no particles larger than 50μm. Stop grinding, mix, and package.

[0062] 5) Sterilization: sterilize the encapsulated medicine using a 10 MeV, 20-25 kW electron linear accelerator to obtain the veterinary suspension containing CS and CAI of this example.

[0063] Example 2 Preparation of compound preparation

[0064] 1. The composition of the compound preparation according to weight-to-volume ratio (W / V, g / ml*100%) is as follows:

[0065] 1.1 Active ingredients:

[0066] Chitosan (CS), white powder, 5% (W / V).

[0067] Baicalein (CAI), yellow powder, 2.5% (W / V).

[0068] 1.2 Grease carrier: Add to 100% of the volume

[0069] Soybean oil for injection, light yellow transparent liquid.

[0070] 1.3 Suspending agents, oil-soluble wetting agents, antioxidants:

[0071] Hydrogenated castor oil, colorless transparent liquid, 1% (W / V).

[0072] Span 80, colorless and transparent liquid, 0.05% (W / V).

[0073] Vitamin E oil, light yellow powder, 0.01% (W / V).

[0074] 2. Preparation (140ml)

[0075] 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil. Heat until completely dissolved to obtain a standby solution.

[0076] 2) Evenly disperse the remaining soybean oil into the built-in ball mill of the YXQM-0.4L ball mill, add the prescribed amount of CS, Span 80 and reserve liquid in sequence, start the ball mill, and intermittently grind at 600 rpm for about 30 minutes.

[0077] 3) Slowly add the prescribed amount of CAI and continue intermittent grinding for 60 minutes.

[0078] 4) Check the particle fineness: no less than 90% of the particles are below 15μm, no less than 95% of the particles are below 20μm, and there are no particles larger than 50μm. Stop grinding, mix, and package.

[0079] 5) Sterilization: sterilize the encapsulated medicine using a 10 MeV, 20-25 kW electron linear accelerator to obtain the veterinary suspension containing CS and CAI of this example.

[0080] Example 3 Preparation of compound preparation

[0081] 1. The composition of the compound preparation according to weight-to-volume ratio (W / V, g / ml*100%) is as follows:

[0082] 1.1 Active ingredients:

[0083] Chitosan (CS), white powder, 10% (W / V).

[0084] Baicalein (CAI), yellow powder, 0.5% (W / V).

[0085] 1.2 Grease carrier: Add to 100% of the volume

[0086] Soybean oil for injection, light yellow transparent liquid.

[0087] 1.3 Suspending agents, wetting agents, antioxidants:

[0088] Hydrogenated castor oil, colorless transparent liquid, 0.1% (W / V).

[0089] Span 80, colorless and transparent liquid, 0.005% (W / V).

[0090] Vitamin E oil, light yellow powder, 0.001% (W / V).

[0091] 2. Preparation (140ml)

[0092] 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil. Heat until completely dissolved to obtain a standby solution.

[0093] 2) Evenly disperse the remaining soybean oil into the built-in ball mill of the YXQM-0.4L ball mill, add the prescribed amount of CS, Span 80 and reserve liquid in sequence, start the ball mill, and intermittently grind at 600 rpm for about 40 minutes.

[0094] 3) Slowly add the prescribed amount of CAI and continue intermittent grinding for 45 minutes.

[0095] 4) Check the particle fineness: no less than 90% of the particles are below 15μm, no less than 95% of the particles are below 20μm, and there are no particles larger than 50μm. Stop grinding, mix, and package.

[0096] 5) Sterilization: sterilize the encapsulated medicine using a 10 MeV, 20-25 kW electron linear accelerator to obtain the veterinary suspension containing CS and CAI of this example.

[0097] Example 4 Preparation of compound preparation

[0098] 1. The composition of the compound preparation according to weight-to-volume ratio (W / V, g / ml*100%) is as follows:

[0099] 1.1 Active ingredients:

[0100] Chitosan (CS), white powder, 1% (W / V).

[0101] Baicalein (CAI), yellow powder, 5% (W / V).

[0102] 1.2 Grease carrier: Add to 100% of the volume

[0103] Soybean oil for injection, light yellow transparent liquid.

[0104] 1.3 Suspending agents, wetting agents, antioxidants:

[0105] Hydrogenated castor oil, colorless transparent liquid, 0.5% (W / V).

[0106] Span 80, colorless and transparent liquid, 0.01% (W / V).

[0107] Vitamin E oil, light yellow powder, 0.005% (W / V).

[0108] 2. Preparation (140ml)

[0109] 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil. Heat until completely dissolved to obtain a standby solution.

[0110] 2) Evenly disperse the remaining soybean oil into the built-in ball mill of the YXQM-0.4L ball mill, add the prescribed amount of CS, Span 80 and reserve liquid in sequence, start the ball mill, and intermittently grind at 600 rpm for about 40 minutes.

[0111] 3) Slowly add the prescribed amount of CAI and continue intermittent grinding for 45 minutes.

[0112] 4) Check the particle fineness: no less than 90% of the particles are below 15μm, no less than 95% of the particles are below 20μm, and there are no particles larger than 50μm. Stop grinding, mix, and package.

[0113] 5) Sterilization: sterilize the encapsulated medicine using a 10 MeV, 20-25 kW electron linear accelerator to obtain the veterinary suspension containing CS and CAI of this example.

[0114] Example 5 In vitro experiments of the preparation

[0115] 1. Combined antioxidant experiment

[0116] A certain amount of CS and CS / CAI samples were weighed and dissolved in anhydrous ethanol or dimethyl sulfoxide (DMSO) to prepare 4 mL of a 0.05-15 mg / mL preparation. The mixture was then mixed with 1 mL of a methanol solution containing 0.05 mg / mL DPPH (2,2-Diphenyl-1-picrylhydrazyl). The mixture was shaken at 1000 rpm in the dark for 30 minutes, and the absorbance of each solution was measured at 517 nm. Simultaneously, equal amounts of ascorbic acid and anhydrous ethanol were added as positive and negative controls and assayed under the same conditions. Three replicates were performed, and scavenging curves were plotted. The half-inhibition rate (IC50) of free radicals was calculated as follows:

[0117] DPPH scavenging ability (%) = [(ΔA517 对照 − ΔA517 样品 ) / ΔA517 对照 ] × 100%. List the formula and calculate the IC50 value (mg / mL) according to the formula.

[0118] like Figure 1 As shown, vitamin C exhibits strong antioxidant capacity, achieving a free radical scavenging rate of 96.96 ± 0.06% at 0.5 mg / mL. CS alone exhibited a scavenging rate ranging from 7.56 ± 0.85% to 21.02 ± 14.64% within the 0.05–15 mg / mL range. The DPPH scavenging capacity of the CS / CAI mixture increased with increasing concentration, from 4.55 ± 2.56% to 91.49 ± 0.18%. Analysis using Prism software revealed that the IC50 values ​​for DPPH scavenging for the mixture and vitamin C were 0.68 mg / mL and 0.12 mg / mL, respectively. The experimental results demonstrate that the positive control, ascorbic acid, exhibited strong free radical scavenging efficiency, significantly exceeding that of chitosan. The CS mixture supplemented with CAI exhibited significantly better free radical scavenging efficiency than CS alone, and the addition of CS did not affect the antioxidant activity of CAI itself.

[0119] 2. Combined antibacterial experiment

[0120] The inhibition zones of the combined use of CS and CAI against Staphylococcus aureus and Escherichia coli were determined using the KB agar method recommended by the National Committee for Clinical Standards (NCCLS). Both strains were obtained from wild strains maintained at the Large Animal Clinical Medicine Center of China Agricultural University. Commercially available 5 mm diameter filter paper discs were sterilized and dried. 1.25-5 mg / ml of CS and CAI solutions dissolved in dilute acetic acid and DMSO, respectively, were then added dropwise (the sample preparations were the same as for the combined antioxidant). Under sterile conditions, the culture medium was poured into a Petri dish and quickly spread evenly to a thickness of approximately 5 mm. After cooling and solidification, a 0.5 McFarland's turbidimetric suspension was evenly applied to the surface of the culture medium using a sterile cotton swab. The plate was rotated approximately 60° and the coating process was repeated three times. After the bacterial solution on the plate has been completely absorbed by the agar, use sterile tweezers to place the antibacterial paper flatly on the inoculated plate. Invert the plate and incubate at 37°C for 18–24 hours. Remove the plate and measure the diameter of the inhibition zone with a vernier caliper. Measure the diameter of the inhibition zone from the back of the plate to an accuracy of 0.01 mm and record the diameter. The edge of the inhibition zone is defined as the point where no visible bacterial growth is observed. A mixture of equal volumes of dilute acetic acid and DMSO was used as a control. Repeat the experiment three times, and the average value is calculated.

[0121] See Figure 2, where the control group is the solvent, and the subscript is the effective concentration of the solvent in the solution, in mg / ml.

[0122] Depend on Figure 2A and 2B It can be seen that when CS and CAI at a standard concentration of 2.5 mg / ml were used alone, the average diameters of the inhibition zones for strains NRSA-S01 and ATCC-Q01 were 8.46 and 8.12 mm, and 8.86 and 8.55 mm, respectively. Compared with the control group, both drugs had a certain antibacterial effect when used alone. When used alone, there was no difference in the diameters of the inhibition zones of the two strains when CS at a concentration of 1.25-5 mg / ml was used; within this concentration range, the diameters of the inhibition zones of the two strains increased with the increase in CAI concentration. Both strains were sensitive to the combined use of the two drugs in each group. When 2 times and 1 times the standard concentration of CAI were used in combination with 1 times the standard concentration of CS, the diameters of the inhibition zones increased significantly ( P<0.05), the inhibition zone diameters against NRSA and ATCC strains reached 12.32 and 13.96 mm, and 11.39 and 13.95 mm, respectively, demonstrating significant antibacterial efficacy. The inhibitory effect against the ATCC strain was positively correlated with the concentration of the compound. When CS concentrations were combined with 1× the standard concentration of CAI, the inhibition zone diameters ranged from 11.50 to 12.32 mm and 9.92 to 11.39 mm, respectively. There were no significant differences in the inhibition zone diameters between the two strains at each CS concentration combined with CAI, indicating that CS concentration did not contribute significantly to the antibacterial activity of the combined system. These analyses suggest that the CS / CAI combination exhibits a synergistic antibacterial effect, primarily attributable to the antibacterial activity of CAI. Furthermore, increasing CAI concentration significantly enhanced the susceptibility of strains MRSA-S01 and ATCC-Q01 to the combined formulation.

[0123] Example 6 In vivo verification of formulation effectiveness

[0124] a. Intramammary administration - mammary gland involution, inflammation, and oxidative stress

[0125] Following the principles of a parallel, randomized, blinded trial, the trial was conducted on a dairy farm with a well-established dry-off process and a sufficient number of dairy cows. Five groups (n=5) were set up: a positive control group (provided by the farm) with the designated dry-off medication (antibiotics), a negative solvent control group (drug-free solvent ball milling group, prepared and configured as in Example 1, but without CS and CAI), a CS group (ball milling group without CAI, prepared and configured as in Example 1, but without CAI), a CAI group (ball milling group without CS, prepared and configured as in Example 1, but without CS), and a test group for the CS / CAI mixed formulation (ball milling with the dual-drug preparation, prepared as in Example 1). Each group received medication via intramammary infusion, once on D0. Each group was observed for 5 days after dosing before the trial ended. Milk (mammary secretion) samples were manually collected from the experimental cows at each time point (D-2, D-1, D0, D1, D3, and D5). The somatic cell count (SCC) of the samples was measured and recorded. The milk and somatic cells were separated by centrifugation (1000 × g, 4°C, 20 minutes). After somatic cell separation, milk aliquots were stored at -20°C until the determination of serum albumin (BSA) concentration, lactate dehydrogenase (LDH) activity, and lactoferrin (LTF) concentration. Blood was collected from the tail root on D-3, D-1, D1, and D3 to monitor inflammation and oxidative stress. The milk somatic cell count (SCC), degeneration markers such as BSA, LDH, and LTF, inflammatory markers such as IL-6 and TNF-α, and oxidative stress markers such as superoxide dismutase (SOD) and malondialdehyde (MDA) were measured and recorded in each sample.

[0126] (1) Inflammation and oxidative stress

[0127] Table 1 Determination results of pro-inflammatory factors in serum of dairy cows in each group

[0128]

[0129] Note: Superscripts are for comparison in the same column, subscripts are for comparison in the same industry

[0130] As shown in Table 1, the serum IL-6 and TNF-α levels in the five groups increased after the milk was dried off. Among them, the serum IL-6 level in the negative control group was significantly higher than that in the positive control group on the first and third days ( P <0.01, P <0.01), CAI group ( P <0.01, P <0.01) and CS / CA mixed preparation group ( P <0.05, P <0.01), no difference was observed between the negative control group and the CS group on day 1; similarly, the serum TNF-α concentration in the negative control group was significantly higher than that in the positive control group on day 1 ( P <0.01) and CAI group ( P <0.01), with no differences observed between the CS group and the CS / CAI mixed group on days 1 and 3. Serum IL-6 levels in the CS group and the CS / CAI mixed group did not differ from those in the positive control group or the CAI group on day 1. Serum TNF-α concentrations were slightly higher on day 3 compared with the positive control group, but IL-6 levels were not significantly different. After dry-off, no significant differences in proinflammatory cytokine levels were observed between the CS group and the CS / CAI mixed group. This suggests that the dry-off process can produce inflammation. Compared with the negative control group, intramammary administration of the four drugs demonstrated a certain effect in reducing the secretion of proinflammatory cytokines in dairy cows during the initial dry-off period.

[0131] Table 2 Determination results of serum oxidative stress indicators in each group of dairy cows

[0132]

[0133] Note: Superscripts are for comparison in the same column, subscripts are for comparison in the same industry

[0134] As shown in Table 2, compared with other groups, the CS / CAI mixed preparation group ( P <0.01) SOD expression increased significantly on the first day after drying up, and on the third day, except for the negative control group with higher SOD content, there was no significant difference among the groups; MDA was not significantly different among the groups on the first day after drying up, and on the third day, the MDA content of the negative control group was significantly higher than that of the CS / CAI mixed preparation group ( P <0.05) and positive drug group ( P<0.05). There were no differences in serum SOD and MDA levels among the CS / CAI mixture, positive drug, and CS drug groups on any observation day. In summary, the CS / CAI mixture accelerated SOD production after dry-off and, similar to the positive control and CAI groups, slowed the increase in MDA levels. This suggests that treatment with the CS / CAI mixture can enhance antioxidant function in dairy cows on the first day after administration and reduce subsequent lipid peroxidation damage.

[0135] (2) Breast degeneration

[0136] like Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 As shown in Figure 2, the concentrations of mammary gland involution markers increased in all groups from the dry day (day 0) to day 5. The SCC concentrations in the CS group and the CS / CAI mixed preparation group from day 1 to day 5 were significantly higher than those in the other three groups ( P <0.01, P <0.01) ( Figure 3 ); Compared with the two control groups, the BSA concentration in the mammary secretions of cows treated with the CS / CAI mixed preparation group was higher on days 1 and 5 ( P <0.01, P <0.01) ( Figure 4 In addition, the CS group and the negative control group showed a significant increase in LDH on the first and third days. The LDH content of the negative control group on the third day was higher than that of the CS / CAI mixed preparation group and the positive control group. There was no difference in the parameters of all groups on the fifth day ( Figure 5 ). Similarly, LTF concentrations showed different degrees of content differences compared with other groups from day 1 to day 5 ( Figure 6 It has been reported that within three days of the onset of mammary gland involution, the number of white blood cells in mammary gland secretions increases rapidly, followed by an increase in macrophage infiltration and accumulation of apoptotic mammary epithelial cells, which together contribute to the elevated somatic cell count. Damaged or apoptotic epithelial cells also secrete increased amounts of LDH, and white blood cells also contribute to the increase in milk LDH activity during mammary gland inflammation. Furthermore, due to the relaxation of tight junctions between mammary cells, blood-derived proteins (such as BSA, LTF, and immunoglobulins) enter the mammary gland. In summary, the infusion of a CS / CAI mixture significantly accelerated the increase in the concentrations of mammary gland involution markers such as SCC, BSA, and LTF in mammary gland secretions after dry-off, which is consistent with the trend of changes in the milk environment during mammary gland involution in dairy cows. Furthermore, LDH levels in the negative group were significantly elevated during the first three days, which may be related to the relatively strong mammary inflammation within the mammary gland.

[0137] Intramammary administration - gross mammary gland degeneration and inflammation

[0138] The CS / CAI mixture obtained in Experiment 1 was used to test 25 dry-off cows on a large-scale dairy farm. The cows were randomly divided into five groups (n=5). A positive control group was administered the established dry-off medication (antibiotic), a negative control group was administered with the solvent, a CS group, a CAI group, and a test group using the CS / CAI mixture. Each group was observed for 7 days after dosing, with the dry-off date designated as D0. Gross mammary gland images and mid-tea distance measurements were taken on D0, D1, D2, D3, D5, and D7. Milk leakage from each teat and the occurrence of mastitis were observed and recorded daily. Mammary inflammation scores were calculated using the mammary gland chart developed by Rambeaud et al.

[0139] (1) Gross condition of the breast

[0140] like Figure 7 As shown, the udders of the five groups of cows were rosy and milk production was normal at dry-off. After drug administration, all groups exhibited a mammary gland involution process, initially swelling and enlarging, followed by shrinkage and reduction. None of the groups presented with visually abnormal redness, swelling, heat, or pain. The udders reached their maximum volume between days 1 and 3 after CS / CAI administration, then significantly shrank between days 3 and 5, with a significant decrease on day 7. The negative control, CS, and CAI groups began to experience significant shrinkage on day 7. On days 5 and 7, the udder volume of the positive control group remained similar to that on day 1 after dry-off. It has been reported that when cows stop milking and accumulate milk during dry-off, it triggers the expression of pro-apoptotic factors (such as transforming growth factor-β1), causing mammary epithelial cells to undergo apoptosis and proliferation, shifting from a lactating to a non-lactating state. The inevitable swelling during this process can severely impact the welfare of dairy cows, leading to decreased lying time and significantly increased frequency of back-kicking, fighting, and howling. Promoting the natural renewal of the mammary epithelium and accelerating the regression of mammary gland volume in dairy cows is undoubtedly beneficial to animal welfare. In summary, the CS / CAI blend can have a positive effect on the regression of mammary gland volume in dairy cows without affecting udder health and can alleviate the discomfort of cows in the early stages of dryness.

[0141] (2) Changes in breast mid-distance

[0142] Table 3 Changes in breast distance

[0143]

[0144] Note: Negative values ​​mean that the breast median distance on the next day is higher than that on the previous day; superscripts refer to comparisons within the same column

[0145] like Figure 8 As shown in Table 3, the mammary gland degeneration of dairy cows was quantified by the mid-length distance between the milk glands. All five groups of cows showed a decrease in mid-length distance between the milk glands. There was no difference in mid-length distance between the groups on the first day of dry-off. On day 3, the mid-length distance between the milk glands of the experimental group was significantly lower than that of the antibiotic and CAI groups (13.34±0.64cm vs 15.44±0.29cm, P<0.05; 13.34±0.64cm vs 16.34±0.76cm, P <0.05), and there was no significant difference between the CS group and the negative control group; on day 7, the milk-mid-distance in the experimental group was slightly lower than that in the antibiotic group (11.66±0.56cm vs 13.70±0.43cm, P <0.05), and there was no difference between the groups on the other days; the experimental group, negative group and CS group all showed an increase in the milk center distance on the first day after drying off. In addition, the experimental group had the largest decrease in milk center distance on the third day after drying off compared with the first day after drying off, reaching 1.86cm, which was significantly higher than that of the other groups ( P <0.05), with the CS group showing the second-largest reduction in this change within a day. Similar reductions were observed in the remaining groups on days 5 and 7. The results in the chart further demonstrate that, compared to currently used intramammary antibacterial drugs, the addition of CS can promote a reduction in udder separation, consistent with the trend in gross mammary gland status. This further demonstrates that the CS / CAI mixture can have a positive effect on early lactation deterioration and mammary gland atrophy in dry cows.

[0146] (3) Mastitis score:

[0147] Table 4 Mastitis score

[0148]

[0149] Note: Subscripts refer to peer comparisons

[0150] As shown in Table 4, the CS / CAI treatment group had the highest mammary inflammation score between 0 and 24 hours after treatment, significantly different from the positive control group. With the exception of the negative control group, the different treatments did not affect the mammary inflammation score after 24 hours (P < 0.1). This suggests that the absence of anti-inflammatory or antibacterial substances during dry-off may not alleviate the early stages of mammary inflammation. Furthermore, the CS / CAI treatment group is consistent with the findings of S. Lanctôt et al. that the use of CS hydrogel alone can moderately recruit immune cells to the mammary gland and accelerate involution without causing acute inflammatory symptoms, suggesting that the use of a CS / CAI mixture can achieve similar results.

[0151] Table 5 Breast status

[0152]

[0153] As shown in Table 5, no clinical mastitis occurred in any of the groups during the observation period. The three-day teat leakage rate was lower in the combined formulation and CS monotherapy groups, with the negative control group showing the highest rate. Within three days after dry-off, the combined formulation group reduced teat leakage by 10% and 6.66%, respectively, compared to the negative and positive controls. Furthermore, follow-up of experimental cows revealed that not using relevant dry-off medications (such as using only negative medications or CAI alone) during dry-off may be associated with the risk of milk loss. Studies have shown that even after milking, high-yielding cows continue to secrete large quantities of milk. Increased milk pressure can cause milk loss and interfere with the formation of keratin plugs within the teat orifice. Early teat duct opening facilitates the entry of environmental pathogens, predisposing to intramammary infection and increasing the risk of milk loss requiring treatment or even culling. Furthermore, during early involution, milk contains lower concentrations of antimicrobial components and immune cells. Currently, drugs that mimic keratin plugs primarily consist of bismuth sealants, while commonly used dry-off medications primarily consist of antibiotics. These two are often used in combination for physical sealing and intramammary antibacterial measures, resulting in high costs. CS / CAI blends offer a new approach for the dry-off period. CS promotes mammary epithelial cell renewal, while CAI alleviates intramammary infection and, to a lesser extent, reduces the risk of milk leakage. These low-cost raw materials offer considerable potential.

[0154] Summarize

[0155] During the dry period, cows experience abnormal fluctuations in their mammary alveoli, making them susceptible to intramammary infection and the optimal period for mastitis prevention and control. In the present invention's experiments, a CS / CAI mixture combines the ability of CS to disrupt mammary epithelial tight junctions with the antioxidant and antibacterial properties of CAI. Intramammary infusion significantly increased the concentrations of SCC and BSA, markers of degeneration, in mammary secretions after dry-off on the first day after administration. It also accelerated the reduction in udder volume within three days, resulting in a lower incidence of milk leakage in the early stages of dry-off. This reduces the risk of mastitis caused by incomplete nipple closure caused by milk leakage in the early stages of dry-off, shortens the period of udder pain after milking cessation, reduces the risk of blindness, and improves cow welfare. The results of the present invention demonstrate that intramammary infusion of a CS / CAI mixture during dry-off can accelerate the early degeneration process without affecting mammary gland health. Compared to current antibiotic-based dry-off medications that only have bactericidal properties, this formulation is expected to become an effective dry-off medication specifically designed to promote mammary epithelial renewal.

Claims

1. Application of chitosan and baicalein in the preparation of a dry milk composition, the dry milk composition comprising the following ingredients in weight and volume percentages: 1-10% chitosan, 0.5-5% baicalein, 0.1-1% suspending agent, 0.005-0.05% wetting agent, 0.001-0.01% antioxidant, and an oily carrier to make up the volume.

2. A dry milk composition comprising the following ingredients in weight and volume percentages: 1-10% chitosan, 0.5-5% baicalein, 0.1-1% suspending agent, 0.005-0.05% wetting agent, 0.001-0.01% antioxidant, and an oily carrier to make up the volume.

3. The composition according to claim 2, wherein The invention comprises the following components in weight and volume percentages: 2.5% chitosan, 1.25% baicalein, 0.6% suspending agent, 0.01% wetting agent, 0.005% antioxidant, and an oily carrier to make up the volume.

4. The composition according to claim 2 or 3, wherein The oily carrier is selected from one or more of soybean oil for injection, peanut oil for injection, sesame oil for injection, castor oil for injection, olive oil for injection, isopropyl myristate, dimethyl silicone oil or medium chain triglycerides.

5. The composition according to claim 2 or 3, wherein The suspending agent is selected from one or more of hydrogenated castor oil, hydrogenated peanut oil, hydrogenated tea seed oil, polyvinyl pyrrolidone, soybean lecithin, white beeswax, 75% aluminum monostearate or hydrogenated sesame oil.

6. The composition according to claim 2 or 3, wherein The wetting agent is selected from Span 80 or fatty acid glyceride.

7. The composition according to claim 2 or 3, wherein The antioxidant is selected from one or more of vitamin E oil, ascorbyl palmitate, lipoic acid, coenzyme Q10, butylated hydroxyanisole or tert-butylhydroquinone.

8. A method for preparing the dry milk composition according to any one of claims 2 to 7, comprising the following steps: 1) Weigh the formulated amount of suspending agent and add it to an appropriate amount of oily carrier, heat it to completely dissolve it, and obtain a standby solution; 2) Pour the remaining amount of the oily carrier into a ball mill, add the chitosan, wetting agent, antioxidant and reserve solution in the formula amount in sequence, and grind intermittently at 400-800 rpm for about 30-50 minutes; 3) Slowly add the formulated amount of baicalin and continue grinding intermittently for 30-60 minutes; 4) Inspection and packaging.

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