'delactation type 'antibiotic-free dry milk composition and preparation method thereof

Abstract

The invention provides an antibiotic-free dry milk composition which comprises an effective amount of chitosan and baicalein, and further comprises a greasy carrier, a suspending aid, a wetting agent and an antioxidant. The dry milk composition disclosed by the invention has the effects of promoting dry cow mammary tissue repair, inhibiting milk secretion and enhancing milk region immunity, and can accelerate mammary epithelium renewal and reduce milk yield so as to reduce internal milk pressure; the synergistic effect is achieved in the aspects of promoting the antioxidant activity and inhibiting the sensitivity of staphylococcus aureus, escherichia coli and the like to drugs; the milk leakage rate of the dairy cows in the dry period can be obviously reduced, and the milk drying effect is obvious, especially the early-stage milk drying effect is obvious.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture technology, and particularly to a "lactation regression type" antibiotic-free dry cow composition. Background Art

[0002] Cow mastitis is a common and highly prevalent disease. It has been found that 24.5% of high-yielding dairy cows experience milk leakage during dry-off, and the risk of mastitis and intramammary infection in the mammary glands with milk leakage at the initial stage of dry-off is 2 times and 1.5 times that of normal mammary glands; moreover, mastitis during the dry period can even cause clinical mastitis 60 days after parturition. The proliferation of mammary alveolar cells in dairy cows fluctuates significantly during this period, which is also the best period for prevention and treatment. Currently, 98.64% of farms in China use dry cow drugs mainly composed of antibiotics for the whole herd to reduce the risk of intramammary infection during the dry period of dairy cows. However, the problems of antibiotic resistance, antibiotic residues in animal products, and potential environmental pollution are becoming increasingly severe, forcing the dairy farming industry to seek new solutions. Since 2022, the UK has officially prohibited whole-herd dry cow treatment. The EU regulations came into effect in June 2019 and have completely prohibited whole-herd dry cow treatment. Therefore, in the context of the overall reduction and substitution of antibiotics in aquaculture, how to promote dry-off in a green and efficient manner is a new challenge faced by the current dairy farming industry.

[0003] Chitosan (CS) is a bioactive, biocompatible, biodegradable, and non-toxic hydrocolloid that can change the secretion state of mammary cells to accelerate the process of mammary gland regression and has advantages such as antibacterial and promoting wound healing. CN109310705A provides a composition for accelerating mammary gland regression and preventing intramammary infection during dry-off of lactating ruminants, mainly relying on the thermosensitive properties of chitosan gel to achieve the goal of promoting dry-off by means of mammary perfusion. However, the price of the drug excipient β-glycerophosphate is relatively high. Chitosan itself may gradually degrade into short chains in an acidic environment, which affects the drug efficacy, and this agent has strict requirements for storage temperature, which has certain limitations in terms of the ranch environment and the ranch's profit requirements.

[0004] Baicalein (BAI) is one of the flavonoid compounds with the highest content in Scutellaria baicalensis. Baicalein has the effects of reducing cerebrovascular resistance, improving cerebral blood circulation, increasing cerebral blood flow, and antiplatelet aggregation. It is clinically used for the treatment of paralysis after cerebrovascular diseases. In addition, Scutellaria baicalensis has a broad antibacterial spectrum and has inhibitory effects on Shigella dysenteriae, Corynebacterium diphtheriae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus, Streptococcus pneumoniae, and Neisseria meningitidis in experiments.

[0005] In existing research, baicalein has been modified with N-trimethyl chitosan to prepare an ophthalmic lipid nanoparticle preparation (CN110051654A), and a baicalein nano-preparation prepared by loading baicalein on a chitosan-cyclodextrin graft polymer (CN113546072A). Here, chitosan is mainly used as a drug carrier.

[0006] So far, there has been no report on the combined use of chitosan and baicalein for antibiotic-free dry cow therapy. Summary of the Invention

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

[0008] The present invention has found through research that the combined use of chitosan and baicalein has the effects of promoting the repair of mammary gland tissue in dry cows and regulating mammary gland immunity, can accelerate the renewal of mammary epithelium, reduce milk production to decrease intramammary pressure, and improve animal welfare; has a synergistic effect in promoting antioxidant activity and inhibiting the sensitivity of common mastitis-causing bacteria such as Escherichia coli and Staphylococcus aureus to drugs; can significantly reduce the milk leakage rate during dry period; especially has a significant effect on early dry cow therapy. Based on this, the present invention provides the following technical solutions: The present invention first provides the use of chitosan and baicalein in the preparation of a dry cow composition. It is used for rapid dry cow therapy, reducing the milk leakage rate during the dry period and reducing mastitis.

[0009] Furthermore, the present invention provides a dry cow composition, which comprises an effective amount of chitosan and baicalein.

[0010] Furthermore, the composition further comprises an oily carrier, a suspending agent, a wetting agent and an antioxidant.

[0011] Furthermore, the composition comprises the following components in weight / volume percentage: chitosan 1-10%, baicalein 0.5-5%, suspending agent 0.1-1%, wetting agent 0.005-0.05%, antioxidant 0.001-0.01%, and the volume is made up to the mark with the oily carrier. Further, the composition comprises the following components in weight / volume percentage: chitosan 2.5%, baicalein 1.25%, suspending agent 0.6%, wetting agent 0.01%, antioxidant 0.005%, and the volume is made up to the mark with the oily carrier.

[0012] Furthermore, the oily 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, dimethyl silicone oil or medium-chain triglycerides. In one embodiment of the present invention, the oily carrier is soybean oil for injection.

[0013] Further, the suspending agent may be selected from one or more of hydrogenated castor oil, hydrogenated peanut oil, hydrogenated camellia seed oil, polyvinylpyrrolidone, 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.

[0014] Further, the wetting agent is selected from Span 80 or fatty acid glyceride. Here, an oil-soluble wetting agent is selected. The suspending effect can be achieved through the suspending agent and the wetting agent. Of course, those skilled in the art can also select other suspending agents that can achieve the suspending effect.

[0015] Further, the antioxidant is selected from one or more of vitamin E oil, ascorbyl palmitate, lipoic acid, coenzyme Q10, butylated hydroxyanisole, or tert-butylhydroquinone. In one embodiment of the present invention, the antioxidant is vitamin E oil.

[0016] The present invention selects an oily carrier, a suspending agent, an oil-soluble wetting agent, and an oil-soluble antioxidant, which can effectively solve the problems that baicalein and chitosan are difficult to dissolve in solution and chitosan is easily degraded.

[0017] Further, the present invention provides a method for preparing the above-mentioned dry cow composition, which comprises the following steps: 1) Weigh the formula amount of the suspending agent and add it to an appropriate amount of oily carrier, heat it to completely dissolve, and obtain a standby solution; 2) Take the remaining formula amount of the oily carrier and uniformly disperse it into a ball mill, and sequentially add the formula amount of chitosan, wetting agent, and standby solution, and intermittently grind at a speed of 400-800 rpm for about 30-50 min; 3) Slowly add the formula amount of baicalein and continue to intermittently grind for 30-60 min; 4) Check and package.

[0018] The beneficial effects of the present invention are: (1) The present invention does not contain any antibiotic components, will not cause drug irritation and residues to the cow body, and does not affect the milk quality; (2) The drug of the present invention is used for local perfusion in the mammary gland, and the drug concentration on the surface of the mammary gland is high, and the function is exerted to the greatest extent; (3) The effective drug components of the present invention are non-toxic and side-effect-free, biodegradable and absorbable, and will not cause a burden on the cow mammary gland; (4) The present invention can reduce the occurrence of milk swelling and milk leakage in the early stage of dry milk by promoting the "regeneration and degradation" of mammary epithelial cells, and provides help for the formation of horny plugs and the prevention of new infections in the mammary gland; (5) The present invention synergistically resists bacteria by adding a traditional Chinese medicine extract, and has obvious inhibitory and killing effects on Staphylococcus aureus, Escherichia coli, etc. that induce mastitis. At the same time, it can reduce the content of pro-inflammatory factors in the serum, enhance the anti-infection ability of mammary epithelium against pathogenic bacteria, and improve the antioxidant function; (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 to block the nipple pores in the form of an oil film, forming a natural "sealing" effect to protect the mammary tissue; (7) The active ingredients of the present invention are stable, and adding an oil-soluble antioxidant can increase the clinical shelf life; (8) The preparation method of the composition of the present invention is simple and has low cost.

[0019] The use of the suspension dosage form in the present invention is beneficial to preparing a liquid preparation from poorly soluble chitosan and baicalein for clinical application; after the suspension liquid is injected into the breast, it will remain in the alveolar cavity, and the drug is gradually released due to the diffusion of the oil-based system. The development of this compound preparation helps to ensure the food safety of animal origin and promote the increase of farmers' income. It has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the antioxidant situation of CS / CAI; Figure 2 shows the in vitro antibacterial situation of CS / CAI, where Figure 2A and 2B is the antibacterial situation of Staphylococcus aureus, Figure 2C and 2D is the antibacterial situation 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; Figure 3 shows the somatic cell count of the mammary gland regression index in different groups. In the figure, compared with the negative control group, * represents a significant difference P <0.05, *** represents a highly significant difference P <0.001; Figure 4 shows the albumin of the mammary gland regression index in different groups. In the figure, compared with the negative control group, * represents a significant difference P <0.05, *** represents a highly significant difference P <0.001; Figure 5 shows the lactate dehydrogenase of the mammary gland regression index in different groups; in the figure, compared with the positive control group, * represents a significant difference P <0.05, *** represents a highly significant difference P <0.001; Figure 6The figure shows the lactoferrin levels, an indicator of mammary gland involution, in different groups; compared with the positive control group in the figure, * indicates a significant difference P <0.05, *** indicates a highly significant difference P <0.001; Figure 7 The figure shows photos of mammary gland involution in different groups. The photos in the first to sixth columns are the photos of each group on days 0, 1, 2, 3, 5, and 7 respectively; Figure 8 The figure shows the trend of the intermammary distance in different groups. Compared with the antibiotic (positive control group) in the figure, * indicates a significant difference P <0.05, *** indicates a highly significant difference P <0.001; compared with the CAI group in the figure, # indicates a significant difference P <0.05, indicates a highly significant difference P <0.001. Detailed implementation manners

[0021] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0022] 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 the amount having a pharmacological effect, for example, the amount that can effectively reduce the milk leakage rate during dry-off and has an obvious dry-off effect. The results can be referred to Example 6 of the present invention.

[0023] The volume make-up of the oily carrier described in the present invention means that the oily carrier is made up to 100% by volume as the solvent.

[0024] Example 1 Preparation of the compound preparation 1. The composition of the compound preparation is as follows according to the weight-volume ratio (W / V, g / ml * 100%): 1.1 Active ingredients: Chitosan (CS), white powder, 2.5% (W / V).

[0025] Baicalein (CAI), yellow powder, 1.25% (W / V).

[0026] 1.2 Oily carrier: Made up to 100% by volume Soybean oil for injection, light yellow transparent liquid.

[0027] 1.3 Suspending agent, oil-soluble wetting agent, antioxidant: Hydrogenated castor oil, colorless transparent liquid, 0.6% (W / V).

[0028] Span 80, a colorless and transparent liquid, 0.01% (W / V).

[0029] Vitamin E oil, a pale yellow powder, 0.005% (W / V).

[0030] The suspending agent is used to increase the viscosity of the dispersion medium to reduce the sedimentation rate of drug particles or increase the hydrophilicity of the particles.

[0031] The function of the antioxidant is to extend the shelf life of the compound pharmaceutical preparation.

[0032] 2. Preparation of the preparation (140 ml) 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil, heat it to completely dissolve, and obtain a stock solution. 2) Uniformly disperse the remaining soybean oil into the built-in ball mill tank of the YXQM-0.4L ball mill. Sequentially add the formulated amounts of CS, Span 80, and the stock solution, start the ball mill, and intermittently grind at a speed of 600 rpm for about 40 min. (Here, intermittent grinding is mainly to avoid overheating of the ball mill. According to the technical recommendation, grind for 10 - 15 min and then have a 10-min interval to prevent overheating of the ball mill. When the total time is a multiple of 10, it is a "10 + 10" cycle; when the total time contains a mantissa of 5, it is 10 + 10 ……, and finally, add a 5-min grinding after a 10-min interval). 3) Slowly add the formulated amount of CAI and continue intermittent grinding for 45 min.

[0033] 4) Check the particle fineness: The particles with a particle size of less than 15 μm should be no less than 90%, the particles with a particle size of less than 20 μm should be no less than 95%, and there should be no particles exceeding 50 μm. Stop grinding, mix well, and package.

[0034] 5) Sterilize. Use a 10 MeV, 20 - 25 kW electron linear accelerator to sterilize the packaged medicine to obtain the veterinary suspension containing CS and CAI in this example.

[0035] Example 2 Preparation of the compound preparation 1. The composition of the compound preparation is as follows according to the weight - volume ratio (W / V, g / ml * 100%): 1.1 Active ingredients: Chitosan (CS), a white powder, 5% (W / V).

[0036] Baicalein (CAI), a yellow powder, 2.5% (W / V).

[0037] 1.2 Oleaginous carrier: Make up to a volume of 100% Soybean oil for injection, a pale yellow transparent liquid.

[0038] 1.3 Suspending agent, oil-soluble wetting agent, antioxidant: Hydrogenated castor oil, a colorless and transparent liquid, 1% (W / V).

[0039] Span 80, a colorless and transparent liquid, 0.05% (W / V).

[0040] Vitamin E oil, a light yellow powder, 0.01% (W / V).

[0041] 2. Preparation of the preparation (140 ml) 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil, heat to completely dissolve it to obtain a stock solution; 2) Uniformly disperse the remaining soybean oil into the built-in ball mill tank of the YXQM-0.4L ball mill, sequentially add the formulated amounts of CS, Span 80 and the stock solution, start the ball mill, and intermittently grind at a speed of 600 rpm for about 30 min.

[0042] 3) Slowly add the formulated amount of CAI and continue intermittent grinding for 60 min.

[0043] 4) Check the particle fineness: The particles with a particle size of less than 15 μm are not less than 90%, the particles with a particle size of less than 20 μm are not less than 95%, and there are no particles exceeding 50 μm. Stop grinding, mix well, and package.

[0044] 5) Sterilize. Use a 10 MeV, 20 - 25 kW electron linear accelerator to sterilize the packaged medicine to obtain the veterinary suspension containing CS and CAI in this example.

[0045] Example 3 Preparation of the compound preparation 1. The composition of the compound preparation is as follows according to the weight - volume ratio (W / V, g / ml * 100%): 1.1 Active ingredients: Chitosan (CS), a white powder, 10% (W / V).

[0046] Baicalein (CAI), a yellow powder, 0.5% (W / V).

[0047] 1.2 Oleaginous carrier: Make up the volume to 100% Soybean oil for injection, a light yellow transparent liquid.

[0048] 1.3 Suspending agent, wetting agent, antioxidant: Hydrogenated castor oil, a colorless and transparent liquid, 0.1% (W / V).

[0049] Span 80, a colorless and transparent liquid, 0.005% (W / V).

[0050] Vitamin E oil, light yellow powder, 0.001% (W / V).

[0051] 2. Preparation of the preparation (140 ml) 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil, heat to completely dissolve it to obtain a stock solution; 2) Uniformly disperse the remaining soybean oil into the internal ball milling tank of the YXQM-0.4L ball mill, sequentially add the formulated amounts of CS, span 80 and the stock solution, start the ball mill, and intermittently grind at a speed of 600 rpm for about 40 min.

[0052] 3) Slowly add the formulated amount of CAI and continue intermittent grinding for 45 min.

[0053] 4) Check the particle fineness: The particles with a particle size of less than 15 μm are not less than 90%, the particles with a particle size of less than 20 μm are not less than 95%, and there are no particles exceeding 50 μm. Stop grinding, mix well, and package.

[0054] 5) Sterilize. Use a 10 MeV, 20 - 25 kW electron linear accelerator to sterilize the packaged medicine to obtain the veterinary suspension containing CS and CAI in this example.

[0055] Example 4 Preparation of the compound preparation 1. Composition of the compound preparation is as follows according to the weight - volume ratio (W / V, g / ml * 100%): 1.1 Active ingredients: Chitosan (CS), white powder, 1% (W / V).

[0056] Baicalein (CAI), yellow powder, 5% (W / V).

[0057] 1.2 Oleaginous carrier: Make up to a volume of 100% Soybean oil for injection, light yellow transparent liquid.

[0058] 1.3 Suspending agent, wetting agent, antioxidant: Hydrogenated castor oil, colorless transparent liquid, 0.5% (W / V).

[0059] Span 80, colorless transparent liquid, 0.01% (W / V).

[0060] Vitamin E oil, light yellow powder, 0.005% (W / V).

[0061] 2. Preparation of the preparation (140 ml) 1) Weigh the formulated amount of hydrogenated castor oil and add it to 40 mL of soybean oil, heat to completely dissolve it to obtain a stock solution; 2) Pour the remaining soybean oil evenly into the built-in ball mill jar of the YXQM-0.4L ball mill, add the prescribed amounts of CS, Span 80 and the reserve solution in sequence, start the ball mill, and intermittently grind at a speed of 600 rpm for about 40 min.

[0062] 3) Slowly add the prescribed amount of CAI, and continue intermittent grinding for 45 min.

[0063] 4) Check the particle fineness: The particles with a particle size of less than 15 μm are not less than 90%, the particles with a particle size of less than 20 μm are not less than 95%, and there are no particles exceeding 50 μm. Stop grinding, mix well, and package.

[0064] 5) Sterilize. Use a 10 MeV, 20-25 kW electron linear accelerator to sterilize the packaged medicine to obtain the veterinary suspension containing CS and CAI in this example.

[0065] In vitro experiments of the preparation in Example 5 1. Combined antioxidant experiment Weigh a certain amount of CS and CS / CAI samples respectively. After dissolving them with absolute ethanol or dimethyl sulfoxide (DMSO), prepare preparations with a concentration of 0.05-15 mg / mL in 4 mL. Mix them with 1 mL of methanol solution containing 0.05 mg / mL DPPH (2,2-Diphenyl-1-picrylhydrazyl). Shake the mixture at a speed of 1000 rpm in the dark for 30 minutes, and then measure the absorbance of each sample solution at 517 nm. At the same time, add the same amount of ascorbic acid and absolute ethanol as positive control and negative control respectively, and measure them under the same conditions. Measure in parallel for 3 times, draw the scavenging rate curve, and calculate the half-inhibitory concentration (IC50) value of free radicals according to the following formula.

[0066] 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.

[0067] As Figure 1As shown, Vc has strong antioxidant capacity, and the free radical scavenging rate reaches 96.96±0.06% even at 0.5mg / mL. In the range of 0.05 - 15mg / mL, the scavenging rate of CS alone is between 7.56±0.85% and 21.02±14.64%. The DPPH scavenging ability of the CS / CAI mixed preparation increases with the increase of concentration, from 4.55±2.56% to 91.49±0.18%. Through prism software analysis, the IC50 values of the mixed system and Vc for scavenging DPPH free radicals are 0.68mg / mL and 0.12mg / mL respectively. The experimental results show that the positive control ascorbic acid exhibits strong free radical scavenging efficiency, significantly higher than that of chitosan. The CS mixed system added with CAI has significantly better free radical scavenging efficiency than CS alone, and the addition of CS does not affect the antioxidant function of CAI itself.

[0068] 2. Joint antibacterial experiment According to the K-B agar method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) of the United States, the inhibition zones of CS and CAI used in combination against Staphylococcus aureus and Escherichia coli were measured. Both strains were wild strains preserved in the Large Animal Clinical Medicine Center of China Agricultural University. Prepare commercial filter paper discs with a diameter of 5mm. After sterilization and drying, drop 1.25 - 5mg / ml of CS and CAI sample solutions (the preparation of the sample solutions is the same as that for joint antioxidant) dissolved in dilute acetic acid and DMSO respectively to make antibacterial paper discs. Under aseptic conditions, pour the culture medium into the petri dish and spread it evenly quickly, with a thickness of about 5mm. After cooling and solidifying, use a sterile cotton swab to evenly coat the surface of the culture medium with a 0.5 McFarland turbidity bacterial suspension, and rotate the plate about 60° and repeat the coating operation 3 times. After the bacterial liquid on the plate is completely absorbed by the agar, use sterile forceps to gently place the antibacterial paper disc flat on the inoculated plate, invert the plate, incubate at 37°C for 18 - 24 h and take it out, use a vernier caliper to measure the diameter of the inhibition zone, measure the diameter of the inhibition zone with an accuracy of 0.01mm from the back of the plate and record it. The edge of the inhibition zone is limited to the point where no obvious bacterial growth can be seen with the naked eye. Among them, an equal amount of mixed solution of dilute acetic acid and DMSO is used as the control. The experiment is repeated 3 times and the average value is taken.

[0069] See Figure 2, where the control group is the solvent, and the subscript is the effective concentration of the solvent in the solution, with the unit of mg / ml.

[0070] From Figure 2A and 2BIt can be seen that when using standard concentrations of 2.5 mg / ml CS and CAI alone, the average diameters of the inhibition zones for strains NRSA-S01 and ATCC-Q01 are 8.46, 8.12 and 8.86, 8.55 mm respectively. Compared with the control group, both drugs alone have certain antibacterial effects. When using CS alone, there is no difference in the diameters of the inhibition zones for the two strains at CS concentrations of 1.25 - 5 mg / ml; while within this concentration range, with the increase in the CAI concentration, the diameters of the inhibition zones for the two strains both show an increasing trend. Both strains are sensitive to the combined use of each group of the two drugs. When using 2-fold and 1-fold standard concentrations of CAI in combination with 1-fold standard concentration of CS, the diameters of the inhibition zones increase significantly ( P <0.05), and the diameters of the inhibition zones for strains NRSA and ATCC reach 12.32, 13.96 and 11.39, 13.95 mm respectively, and the antibacterial effect is significantly improved. Among them, the antibacterial effect on the ATCC strain shows a positive correlation with the concentration; when different concentrations of CS are combined with 1-fold standard concentration of CAI, the diameters of the inhibition zones range from 11.50 - 12.32 mm and 9.92 - 11.39 mm. There is no obvious difference in the diameters of the inhibition zones for the two strains when each concentration of CS is combined with CAI, indicating that the CS concentration does not contribute much to the antibacterial performance of the combined system. Based on the above analysis, the CS / CAI combination has a synergistic antibacterial effect. This effect mainly comes from the antibacterial effect of CAI, and with the increase in the CAI concentration, the sensitivity of strains MRSA-S01 and ATCC-Q01 to the mixed preparation is significantly enhanced.

[0071] Example 6 In vivo verification of the effectiveness of the preparation a. Intramammary administration - Intramammary involution, inflammation and oxidative stress status According to the principles of parallel, randomized, and blinded trials, it was determined that the trial would be conducted in a dairy farm with a perfect dry-cow process and a sufficient number of captive dairy cows. A positive control group of the established dry-cow medicine (antibiotic) provided by the farm, a solvent negative control group (a drug-free solvent ball-milling group, configured and prepared in the same way as in Example 1 but without adding CS and CAI), a CS group (a ball-milling group without adding CAI, configured and prepared in the same way as in Example 1 but without adding CAI), a CAI group (a ball-milling group without adding CS, configured and prepared in the same way as in Example 1 but without adding CS), and a test group of this CS / CAI mixed preparation (double-drug ball-milling, prepared according to Example 1) were set up, with a total of 5 groups (n = 5). Each group was administered the drug by intramammary infusion, once on day D0, and the trial ended after observing for 5 days after administration in each group. Milk (mammary gland secretion) samples were manually collected from the test dairy cows at each time point of D-2, D-1, D0, D1, D3, and D5. The somatic cell count of the samples was measured and recorded in a timely manner, and the milk and somatic cells were separated by centrifugation (1000×g, 4°C, 20 minutes). The milk aliquots after separating the somatic cells were stored at -20°C until the concentrations of serum albumin (BSA), lactate dehydrogenase (LDH) activity, and lactoferrin (LTF) were determined; blood was collected from the base of the tail on days D-3, D-1, D1, and D3 for detecting the occurrence of inflammation and oxidative stress. The milk somatic cell count (SCC), the concentrations of degradation markers such as BSA, LDH, and LTF in each sample, the inflammation indicators such as IL-6 and TNF-α, and the oxidative stress indicators such as superoxide dismutase (SOD) and malondialdehyde (MDA) were measured and recorded in a timely manner.

[0072] (1) Inflammatory and oxidative stress conditions Table 1 Determination results of serum pro-inflammatory factors in dairy cows of each group

[0073] Note: Superscripts are for comparison in the same column, and subscripts are for comparison in the same row As can be seen from Table 1: The serum IL-6 and TNF-α in the 5 groups showed an increasing trend after dry-cow. Among them, the serum IL-6 in the negative control group was significantly higher than that in the positive control group on the 1st and 3rd days ( P <0.01, P <0.01), the CAI group ( P <0.01, P <0.01), and the 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 the 1st day; similarly, the serum TNF-α concentration in the negative control group was extremely significantly higher than that in the positive control group on the 1st day ( P <0.01) and the CAI group ( P<0.01), and no differences were observed between the first day and the third day and the CS group. There were no differences in the serum IL-6 content between the CS group and the CS / CAI mixed preparation group on the first day compared with the positive control group and the CAI group. On the third day, the serum TNF-α concentration was slightly higher than that of the positive control group, but the difference in IL-6 content was not significant. No significant differences in the content of pro-inflammatory factors were observed between the CS group and the CS / CAI mixed preparation group 2 after dry-off. It shows that inflammation will occur during the dry-off process. Compared with the negative control group, the use of the four drugs in the mammary gland all has a certain effect on reducing the secretion level of pro-inflammatory factors in dairy cows at the beginning of dry-off.

[0074] Table 2 Determination results of serum oxidative stress indexes of dairy cows in each group

[0075] Note: Superscripts are for comparison in the same column, and subscripts are for comparison in the same row As can be seen from Table 2: Compared with other groups, the SOD expression in the CS / CAI mixed preparation group ( P <0.01) increased extremely significantly on the first day after dry-off. On the third day, except for the higher SOD content in the negative control group, there were no obvious differences among the groups; no obvious differences were observed in MDA among the groups on the first day after dry-off. On the third day, the MDA content in the negative control group was significantly higher than that in the CS / CAI mixed preparation group ( P <0.05) and the positive drug group ( P <0.05). There were no differences in serum SOD and MDA among the CS / CAI mixed preparation group, the positive drug group and the CS drug group during each observation period. In summary, the CS / CAI mixed preparation can accelerate the production of SOD after dry-off, and has a similar effect to the positive control group and the CAI group in slowing down the increase in MDA content, proving that the treatment with the CS / CAI mixed preparation can promote the antioxidant effect of dairy cows on the first day after administration and reduce subsequent lipid peroxidation damage.

[0076] (2) Mammary gland involution As Figure 3 , Figure 4 , Figure 5 , Figure 6 shown, the concentration of mammary gland involution markers in all groups increased from the dry-off day (day 0) to day 5. The SCC concentration in the CS group and the CS / CAI mixed preparation group from the first day to the fifth day was extremely significantly higher than that of the remaining three groups ( P <0.01, P<0.01) ( Figure 3 ); compared with the two control groups, the BSA concentration in the mammary gland secretions of dairy cows treated with the CS / CAI mixed preparation group was higher on the first day and the fifth day ( P <0.01, P <0.01) ( Figure 4). In addition, on the 1st and 3rd days, the CS group and the negative control group showed a significant increase in LDH. Among them, on the 3rd day, the LDH content in the negative control group was higher than that in the CS / CAI mixture group and the positive control group. On the 5th day, there was no difference in the parameters of all groups ( Figure 5 ). Similarly, the LTF concentration showed varying degrees of content differences relative to other groups from the 1st day to the 5th day ( Figure 6 ). It has been reported that within 3 days after the start of mammary gland involution, the number of white blood cells in the mammary gland secretions increases rapidly, followed by an increase in macrophage infiltration. In addition, apoptotic mammary epithelial cells accumulate, jointly constituting the increased somatic cell count; damaged or apoptotic epithelial cells also secrete more LDH, and white blood cells are also involved in the increase in the LDH activity in milk during mammary gland inflammation. In addition, due to the relaxation of the tight junctions of mammary gland cells, blood-derived proteins (such as BSA, LTF, immunoglobulins) enter the mammary gland. In summary, the infusion of the CS / CAI mixture significantly accelerated the increase in the concentrations of mammary gland involution markers such as SCC, BSA, and LTF in the mammary gland secretions after dry-off, which is in line with the trend of changes in the milk internal environment during the mammary gland involution process in dairy cows; in addition, the LDH in the negative group was significantly higher in the first 3 days, which may be related to the relatively strong mammary gland inflammation in the mammary gland.

[0077] Intramammary administration - Gross mammary gland involution status, inflammation Take the CS / CAI mixture obtained from Experiment 1, select 25 dry-period dairy cows in a large-scale cattle farm, and randomly divide them into 5 groups for the experiment (n = 5). Set up a positive control group with the established dry-off drug (antibiotic) in the pasture, a solvent negative control group, a CS group, a CAI group, and an experimental group of this CS / CAI mixture. After administration to each group, the experiment was ended after observing for 7 days, and the dry-off day was recorded as D0. At each time point of D0, D1, D2, D3, D5, and D7, image collection of the gross appearance of the mammary gland of the experimental dairy cows was carried out, and the distance in the milk was measured. The milk leakage situation and the occurrence of mastitis of each nipple were observed and recorded daily, and the mammary gland inflammation score was carried out according to the method of the mammary gland chart created by Rambeaud et al.

[0078] (1) Gross mammary gland status As Figure 7As shown, the udders of the five groups of dairy cows were rosy when dry and their milk production was normal. After administration to each group, the mammary glands all showed a process of regression, first swelling and then shrinking, and there was no obvious redness, swelling, heat or pain visually. Among them, the udders reached the maximum volume on the 1st to 3rd days after CS / CAI administration, shrank significantly on the 3rd to 5th days, and showed a significant decrease in volume on the 7th day; the negative control group, the CS group and the CAI group began to shrink significantly on the 7th day; the udder volume of the positive control group still showed no obvious difference visually from that on the 1st day after dry-off on the 5th to 7th days. It is reported that when dairy cows stop milking during dry-off, the accumulation of milk will cause the expression of pro-apoptotic factors (such as transforming growth factor β1), and mammary epithelial cells will undergo changes of apoptosis and proliferation, changing from the lactating state to the non-lactating state. The inevitable process of milk swelling during this process will seriously affect the animal welfare of dairy cows, resulting in a shorter lying time of dairy cows and a significant increase in the frequency of turning back and kicking the abdomen, fighting and howling. Promoting the natural renewal of mammary epithelial cells in dairy cows and accelerating the regression of mammary gland volume undoubtedly benefits the animal welfare of dairy cows. In summary, the CS / CAI mixture can have a positive effect on the regression of the mammary gland volume of dairy cows, does not affect the health of the udders of dairy cows, and can relieve the discomfort of dairy cows in the early stage of dry-off.

[0079] (2) Changes in the mid-udder distance Table 3 Values of changes in the mid-udder distance

[0080] Note: Negative values indicate that the mid-udder distance on the next day is higher than that on the previous day; the superscript is for comparison in the same column As Figure 8 shown in and Table 3, the regression of the mammary glands of dairy cows was quantified by the mid-udder distance, and all 5 groups of dairy cows showed a decrease in the mid-udder distance. There was no difference in the mid-udder distance values of each group on the first day of dry-off; on the 3rd day, the mid-udder distance of the experimental group was significantly lower than that of the antibiotic group and the CAI group (13.34 ± 0.64 cm vs 15.44 ± 0.29 cm, P <0.05; 13.34 ± 0.64 cm vs 16.34 ± 0.76 cm, P <0.05), and there was no significant difference from the CS group and the negative control group; on the 7th day, the mid-udder distance of the experimental group was slightly lower than that of the antibiotic group (11.66 ± 0.56 cm vs 13.70 ± 0.43 cm, P <0.05), and there was no difference between the other days among the groups; the experimental group, the negative group and the CS group all showed an increase in the mid-udder distance on the first day after dry-off. In addition, the experimental group had the largest decrease in the mid-udder distance on the 3rd day after dry-off compared with the 1st day after dry-off, reaching 1.86 cm, which was significantly higher than the other groups ( P<0.05), and the CS group had the second-best effect of the change within the day. Similar decreases were recorded in the other groups on the 5th and 7th days. The chart results further show that compared with the widely used intramammary antibacterial drugs, the addition of CS can promote the reduction of the udder interval, which is consistent with the trend of the general state of the mammary gland. It further reflects that the CS / CAI mixed preparation can have a positive effect on the early lactation function degeneration and mammary gland atrophy of dry cows.

[0081] (3) Breast inflammation score: Table 4 Mastitis score

[0082] Note: Subscripts are for peer comparison As shown in Table 4, the breast inflammation score of the CS / CAI test group was relatively the highest within 0-24 hours after treatment, which was significantly different from the positive control group; except for the negative control group, different treatments did not affect the breast inflammation score after 24 hours (P<0.1). This proves that the breast inflammation response in the early stage of dry-off may not be alleviated without the application of anti-inflammatory or antibacterial substances during dry-off; and the CS / CAI test group is consistent with the situation proposed by S. Lanctôt et al. that the use of CS hydrogel alone can induce moderate recruitment of immune cells to the mammary gland and accelerate degeneration, but will not cause acute inflammatory symptoms, indicating that the use of CS / CAI mixed preparations can also achieve similar effects.

[0083] Table 5 Breast status

[0084] As shown in Table 5, no clinical mastitis occurred in each group of dairy cows within the observation period; the nipple leakage rates of the mixed preparation group and the CS single-drug use group were relatively low on the 3rd day, and the nipple leakage rate of the negative control group was the highest. Among them, within 3 days after dry-off, the mixed preparation group could reduce the nipple leakage rate by 10% and 6.66% respectively compared with the negative control group and the positive control group; in addition, during the follow-up of the experimental dairy cows, it was found that not using relevant dry-off drugs during dry-off (such as only using negative drugs or CAI single-drug) might be related to the risk of blind udder in dairy cows. Research shows that even when milking stops, high-yield dairy cows still secrete a large amount of milk, and the increase in milk pressure will cause milk leakage and interfere with the formation of the horny plug in the nipple orifice. The early opening of the nipple duct provides conditions for the entry of environmental pathogens, easily causes intramammary infection, and increases the risk of blind udder treatment or even culling; in addition, during the early regression process, the concentrations of antibacterial components and immune cells in milk are relatively low. Currently, the drugs that simulate the horny plug mainly use sealants (bismuth), and the commonly used dry-off drugs are mainly antibiotics. The two are mostly used in combination for physical sealing and intramammary antibacterial, with a relatively high cost. The CS / CAI mixed preparation provides a new idea for the dry period. CS promotes the renewal of mammary epithelial cells, CAI alleviates the problem of intramammary infection, and relatively has a certain effect of reducing the risk of milk leakage. The raw material price is low, and it has certain potential.

[0085] Summary During the dry period, the mammary alveoli of dairy cows fluctuate abnormally, are prone to intramammary infection, and it is also the best period for the prevention and control of mastitis. In the experiment of the present invention, the CS / CAI mixed preparation combines the function of CS interfering with the tight junction of mammary epithelium and the antioxidant and antibacterial properties of CAI. After intramammary infusion, it can significantly increase the concentrations of the regression markers SCC and BSA in the mammary secretions after dry-off on the first day of administration, and accelerate the reduction of the mammary gland volume of dairy cows within 3 days, showing a relatively low milk leakage incidence in the early dry period. It not only reduces the risk of mastitis caused by incomplete closure of the nipple orifice due to milk leakage in the early dry period, but also shortens the process of mammary gland distension and pain in dairy cows after milking stops, reduces the risk of blind udder, and is beneficial to dairy cow welfare. The results of the present invention show that intramammary infusion of the CS / CAI mixed preparation during dry-off can accelerate the early dry-off regression process and does not affect mammary gland health. Compared with the currently used antibiotic dry-off drugs that only have a bactericidal function, it is expected to become an effective dry-off drug for promoting the renewal of mammary epithelium itself.

Claims

1. Application of chitosan and baicalein in preparing dry milk composition.

2. A dry milk composition comprising an effective amount of chitosan and baicalein.

3. The composition according to claim 2, characterized in that It also includes an oily vehicle, a suspending agent, a wetting agent and an antioxidant.

4. The composition according to claim 3, characterized in that The invention comprises the following components 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 is used to make up the volume.

5. The composition according to claim 4, characterized in that 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 is used to make up the volume.

6. The composition according to any one of claims 3 to 5, characterized in that 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 triglyceride.

7. The composition according to any one of claims 3 to 5, characterized in that 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.

8. The composition according to any one of claims 3 to 5, characterized in that The wetting agent is selected from Span 80 or fatty acid glyceride.

9. The composition according to any one of claims 3 to 5, characterized in that The antioxidant is selected from one or more of vitamin E oil, ascorbyl palmitate, lipoic acid, coenzyme Q10, butylated hydroxyanisole or tert-butylhydroquinone.

10. A method for preparing the dry milk composition according to any one of claims 2 to 9, comprising the following steps: 1) Weigh the amount of the suspending agent in the formula 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 evenly into a ball mill, add the chitosan, wetting agent, antioxidant and reserve solution in the formula amount in sequence, and grind intermittently at a speed of 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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