Functional granular feed for preventing subclinical mastitis during pregnancy and lactation period of dairy cows and quality detection method thereof

Abstract

The application discloses a kind of functional granular feed for preventing latent mastitis of dairy cow during pregnancy lactation period and a quality detection method thereof.The functional natural plant composition of the application is composed of Eucommia ulmoides leaf, radix paeoniae alba, semen pharbitidis, dandelion, perilla leaf, white kidney bean, shen and licorice.The functional granular feed is prepared from the natural plant composition of the application and base feed such as soybean meal, corn, wheat bran and yeast powder.The detection method includes multi-component content determination and fingerprint determination.The application provides optimal chromatographic conditions screened by a large number of experiments, and the established detection method has the characteristics of convenient operation, reliable method and good reproducibility.The quality detection method of the natural plant composition of the application is an important means to ensure the stable and standardized production of the natural plant composition for preventing latent mastitis of dairy cow during pregnancy lactation period and to guarantee the function of the natural plant composition.

Description

Technical Field

[0001] This invention relates to the fields of traditional Chinese medicine and feed additives, and more specifically to a functional natural plant composition for preventing subclinical mastitis in dairy cows during pregnancy and lactation, a method for testing the quality of the same, and a method for testing the quality of functional pelleted feed containing the natural plant composition. Background Technology

[0002] Under the national policy and regulations prohibiting the addition of growth-promoting drug feed additives (excluding traditional Chinese medicine) to animal feed, and given the long-term production practice demonstrating the green, natural, safe, and pollution-free properties of traditional Chinese medicine, which also possess both nutritional and medicinal effects, the development of traditional Chinese medicine feed products is gradually becoming a research hotspot in the livestock industry. In the research and development of traditional Chinese medicine feed products, the quality of the products is a crucial factor for their healthy development. Therefore, the quality testing method of the natural plant composition involved in this invention is an important means to ensure the stability of the quality and function of the natural plant composition used in dairy cows during pregnancy and lactation to prevent subclinical mastitis.

[0003] One of the most prominent problems in the dairy farming industry is subclinical mastitis in dairy cows, characterized by infection of the udder by pathogenic microorganisms, leading to an elevated somatic cell count in milk, which in turn harms the health of dairy cows and affects milk yield and quality. This primarily occurs during the lactation and dry periods. Therefore, the development of traditional Chinese medicine (TCM) feed products to help prevent subclinical mastitis in pregnant and lactating dairy cows is crucial. Furthermore, the stable performance of these TCM feed products relies heavily on quality control. Currently, most enterprise standards for TCM feed products adhere to ordinary feed quality requirements, including moisture, ash, and microorganisms, lacking the determination of key indicative chemical components. This is a significant reason for the inconsistent quality of TCM feed products and has become a critical factor for the healthy development of TCM in the livestock industry. In addition, fingerprinting technology is an effective means of characterizing the relationship between the components of TCM and its quality, and has become an important quality evaluation model widely recognized both domestically and internationally. Therefore, establishing a rapid, efficient, and high-throughput characteristic fingerprinting method for feed TCM products is essential to comprehensively reflect the intrinsic quality of the products and provide a direct, simple, and effective means for quality control.

[0004] In summary, it is of great importance to provide a simple, rapid, easy-to-operate, and reproducible quality control method for functional natural plant-based feed compositions and their prepared medicated pellet feeds used to prevent subclinical mastitis in dairy cows during pregnancy and lactation. This is a crucial guarantee for the quality control of postpartum health care products for dairy cows. Summary of the Invention

[0005] The purpose of this invention is to provide a functional natural plant composition for preventing subclinical mastitis in dairy cows during pregnancy and lactation, the functional pelleted feed prepared therefrom, and a method for quality testing.

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

[0007] A functional pelleted feed for preventing subclinical mastitis in dairy cows during pregnancy and lactation includes the following steps:

[0008] (1) Take 10-50 parts of Eucommia ulmoides leaves, 5-25 parts of Paeonia lactiflora, 10-40 parts of Cuscuta chinensis, 10-40 parts of Taraxacum mongolicum, 5-20 parts of Perilla frutescens leaves, 10-30 parts of white hyacinth bean and 5-20 parts of Glycyrrhiza uralensis, mix them evenly, and then grind them with circulating water until they pass through a 20-300 mesh sieve to obtain raw material powder;

[0009] (2) Take the raw material powder prepared in step (1) and mix it evenly with the basic feed soybean meal, corn, wheat bran, yeast powder, flaxseed meal, salt and palm oil. After pelleting, functional pellet feed is obtained.

[0010] As a preferred option, the functional pelleted feed for preventing subclinical mastitis in pregnant and lactating dairy cows described above consists of 25 parts Eucommia ulmoides leaves, 15 parts Paeonia lactiflora, 20 parts Cuscuta chinensis, 20 parts Taraxacum mongolicum, 15 parts Perilla frutescens leaves, 10 parts Dolichos lablab and 10 parts Glycyrrhiza uralensis.

[0011] A quality testing method for functional pelleted feed used to prevent subclinical mastitis in pregnant and lactating dairy cows includes the determination of chemical components and the establishment of fingerprint spectra. The specific steps are as follows:

[0012] (1) Preparation of test solution: Accurately weigh Eucommia ulmoides leaves, white peony root, dodder seed, dandelion, perilla leaves, white hyacinth bean and licorice, place them in a stoppered conical flask, add 25% methanol, weigh, let stand, extract by ultrasonication, cool to make up the weight loss, shake well, take an appropriate amount, filter through a 0.45μm microporous membrane, and obtain the filtrate as the test solution;

[0013] (2) Preparation of reference solutions: Accurately weigh chlorogenic acid, paeoniflorin, chicoric acid, perilla ketone and glycyrrhizic acid, add them to methanol to prepare a mixed mother liquor, and dilute stepwise to prepare 7 reference solutions of different concentrations.

[0014] (3) Content determination method: Accurately pipette 2 μL of the test solution in step (1) and the five reference solutions of different concentrations in step (2) into the liquid chromatograph, plot the standard curve with peak area as the ordinate and concentration as the abscissa, and then calculate the content of the main chemical components in the test sample based on the standard curve.

[0015] (4) Establishment of fingerprint spectrum: The test solution from step (1) and the reference solution from step (2) were precisely pipetted into the liquid chromatograph and measured. The chromatogram was recorded at an absorption wavelength of 232 nm. The chromatogram results were imported into the software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" for analysis. The "median method" was used, and the time width was set to 0.2 min. Multi-point correction and automatic matching were performed on the chromatographic peaks of each fingerprint spectrum to generate a common pattern of fingerprint spectrum. The fingerprint spectrum was then obtained and similarity analysis was performed.

[0016] As a preferred option, the above-described method for quality testing of functional pelleted feed for preventing subclinical mastitis in pregnant and lactating dairy cows includes the following steps: (1) Preparation of the test solution: Accurately weigh Eucommia ulmoides leaves, Paeonia lactiflora, Cuscuta chinensis, Taraxacum mongolicum, Perilla frutescens leaves, Dolichos lablab and Glycyrrhiza uralensis, place them in a stoppered conical flask, add 25 mL of 80% methanol, weigh, let stand for 0.5 h, extract by ultrasonication for 1 h, cool to make up for weight loss, shake well, take an appropriate amount, filter through a 0.45 μm microporous membrane, and obtain the filtrate as the test solution.

[0017] As a preferred option, the quality testing method for the functional pelleted feed used to prevent subclinical mastitis during the lactation period of dairy cows described above includes the preparation of the reference solution in step (2): accurately weigh chlorogenic acid, paeoniflorin, chicoric acid, perilla ketone, and glycyrrhizic acid, add them to methanol to prepare mixed mother liquors with concentrations of 100.3 μg / mL, 180 μg / mL, 99.5 μg / mL, 100.2 μg / mL, and 98.5 μg / mL, respectively, and dilute them stepwise to prepare 7 reference solutions of different concentrations.

[0018] As a preferred option, the quality testing method for the functional pelleted feed used to prevent subclinical mastitis during the pregnancy and lactation period of dairy cows described above, the liquid chromatography and chromatographic conditions in step (3) are as follows: Chromatographic system: Waters 2695 high performance liquid chromatography system, Waters 2998 PDA detector, Empower TM Chromatography workstation software; using ACQUITY TM UPLC BEH C 18 Chromatographic column: 100 mm × 2.1 mm, 1.7 μm; column temperature: 30 ℃; injection volume: 2 μL; mobile phase: acetonitrile-0.1% phosphoric acid water, gradient elution; flow rate: 0.4 mL / min; detection wavelength: 232 nm.

[0019] The quality testing method for the functional feed natural plant composition used to prevent subclinical mastitis in dairy cows during pregnancy and lactation, as described above, includes the following gradient elution conditions:

[0020] Time (min) Flow rate (mL / min) Acetonitrile (%) 0.1% phosphoric acid solution (%) 0 0.4 5 95 10 0.4 12 88 18 0.4 16 84 22 0.4 27 73 30 0.4 60 40 31 0.4 95 5 32 0.4 5 95 .

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The quality of Chinese herbal medicine (TCM) feed products is crucial for their healthy development. Currently, most TCM feed products on the market use feed raw materials as their quality standard, including the determination of indicators such as moisture, ash content, and microorganisms. However, as TCM products, their efficacy relies on a strong material basis, and conventional TCM feed raw materials are insufficient to reflect the quality of the material benchmark. As a standard reference for measuring whether the actual product corresponds to the material benchmark, its quality should be strengthened through specific identification and multi-component, overall quality control. The quality control method of this invention, including the determination of the content of major chemical components and fingerprint identification, can better ensure its effectiveness.

[0023] This natural plant composition, composed of various natural fodder plants, boasts a rich variety of functional components and requires the detection of numerous chemical components. This invention, through optimization of chromatographic conditions and system applicability, establishes a high-performance liquid chromatography (HPLC) method for the simultaneous determination of five compounds, including chlorogenic acid, paeoniflorin, chicoric acid, perillaldehyde, and glycyrrhizic acid. Simultaneously, this invention establishes a rapid, efficient, and high-throughput ultraviolet (UV) characterization method for feed-grade herbal products, comprehensively reflecting their intrinsic quality and providing a direct, simple, and effective means for quality control. Furthermore, the quality testing method of this invention not only includes methods for testing the natural plant composition but also provides a method for detecting the effective components in pelleted feed prepared using this natural plant composition. This detection method exhibits strong specificity and good reproducibility, truly reflecting safety, effectiveness, and controllable quality.

[0024] The quality testing method of the functional feed natural plant composition involved in this invention is an important means to ensure the stable quality, standardized production, and effective functioning of the feed natural plant composition for preventing subclinical mastitis during the pregnancy and lactation period of dairy cows. Attached Figure Description

[0025] Figure 1 Chromatograms at 232 nm for mixed reference standards, natural plant compositions, and functional pelleted feed. (A represents mixed reference standards, B represents natural plant compositions, and C represents functional pelleted feed; peak 1 represents chlorogenic acid, peak 3 represents paeoniflorin, peak 8 represents chicoric acid, peak 11 represents perillaldehyde, and peak 15 represents glycyrrhizic acid).

[0026] Figure 2 Fingerprint patterns of 10 batches of natural plant compositions.

[0027] Figure 3 Fingerprint spectrum of natural plant composition. Specific Implementation

[0028] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific process conditions and results described in the following embodiments are only for illustrating the present invention, and the implementation of the present invention is not limited to the following embodiments. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0029] Example 1

[0030] 1. A functional feed-grade natural plant composition for preventing subclinical mastitis in dairy cows during pregnancy and lactation, made from the following raw materials:

[0031] Eucommia ulmoides leaves 50kg, white peony root 30kg, dodder seed 40kg, dandelion 40kg, perilla leaves 30kg, white hyacinth bean 20kg, and licorice root 20kg, totaling 240kg.

[0032] 2. A method for preparing functional pelleted feed for preventing subclinical mastitis in dairy cows during pregnancy and lactation includes the following steps:

[0033] (a) After mixing all the above 240kg of Chinese medicinal materials evenly, grind them using circulating water cooling until they pass through a 40-mesh sieve to obtain a natural plant composition powder for later use.

[0034] (b) Mix 460 kg of corn, 140 kg of soybean meal and 130 kg of flaxseed meal evenly and then crush them through a 10-mesh sieve to obtain material 1 for later use.

[0035] (c) Mix 130 kg of wheat bran, 59 kg of yeast powder, 14 kg of salt, and 27 kg of palm oil evenly to obtain material 2 for later use. Mix material 2 evenly with the natural plant composition powder, and then mix it evenly with material 1 for later use.

[0036] (d) The mixture obtained in step (c) is pressed into pellets using a pellet mill. Pellet feed containing a functional feed natural plant composition is obtained.

[0037] 3. Determination of the content of various components in natural plant compositions and their prepared functional pelleted feeds:

[0038] (1) Preparation of test solution ①: Accurately weigh 1g of the natural plant composition powder that has passed through a 40-mesh sieve, place it in a stoppered conical flask, add 25mL of 80% methanol, weigh, let stand for 0.5h, sonicate for 1h (250W, 40kHz), cool and make up for the weight loss, shake well, take an appropriate amount, filter through a 0.45μm microporous membrane, and obtain the filtrate as test solution ①, repeat 3 times.

[0039] (2) Preparation of test solution ②: Accurately weigh 1g of functional granular feed powder that has passed through a 40-mesh sieve, place it in a stoppered conical flask, add 25mL of 80% methanol, weigh, let stand for 0.5h, sonicate for 1h (250W, 40kHz), cool and make up for the weight loss, shake well, take an appropriate amount, filter through a 0.45μm microporous membrane, take 10mL of filtrate, centrifuge and concentrate to dryness, redissolve with 2mL of 80% methanol to obtain test solution ②.

[0040] (2) Preparation of reference solutions: Accurately weigh chlorogenic acid, paeoniflorin, chicoric acid, perilla ketone and glycyrrhizic acid and add them to methanol to prepare mixed stock solutions with concentrations of 100.3 μg / mL, 180 μg / mL, 99.5 μg / mL, 100.2 μg / mL and 98.5 μg / mL, respectively. Then, dilute stepwise to prepare 7 reference solutions with different concentrations.

[0041] (3) Liquid chromatography and chromatographic conditions: Chromatographic system: Waters 2695 high performance liquid chromatography system, Waters 2998 PDA detector, Empower TM Chromatography workstation software; ACQUITY™ UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm); column temperature: 30 ℃; injection volume: 2 μL; mobile phase: acetonitrile-0.1% phosphoric acid water, gradient elution details are shown in Table 1; flow rate: 0.4 mL / min; detection wavelength: 232 nm; theoretical plate number calculated based on paeoniflorin peak should not be less than 3000.

[0042] Table 1. Gradient conditions of the mobile phase

[0043] Time (min) Flow rate (mL / min) Acetonitrile (%) 0.1% phosphoric acid solution (%) 0 0.4 5 95 10 0.4 12 88 18 0.4 16 84 22 0.4 27 73 30 0.4 60 40 31 0.4 95 5 32 0.4 5 95

[0044] (5) Determination method: Accurately pipette 2 μL each of the test solution and reference solutions of different concentrations and inject them into the liquid chromatograph. Plot a standard curve with peak area as the ordinate and concentration as the abscissa to calculate the content of each component in the test sample. Chromatograms of natural plant compositions and functional pelleted feeds are shown below. Figure 1 The test results are shown in Table 2.

[0045] Table 2. Determination of compound content (mg / g) in natural plant compositions and functional pelleted feeds

[0046]

[0047] Example 2

[0048] Fingerprint mapping of natural plant compositions and generation of control fingerprints:

[0049] (1) Preparation of test solution: Three batches of samples were collected for each Chinese medicinal material. Ten batches of natural plant compositions were randomly matched according to the proportion in Example 1. The ten batches of natural plant compositions were ground through a 40-mesh sieve. 1g of sample was accurately weighed from each of the ten batches of sample powder and placed in a stoppered conical flask. 25mL of 80% methanol was added, weighed, and allowed to stand for 0.5h. The mixture was then sonicated for 1h (250W, 40kHz). After cooling, the weight loss was made up. The mixture was shaken well and an appropriate amount was taken and filtered through a 0.45μm microporous membrane to obtain the filtrate as the ten batches of test solution.

[0050] (2) Preparation of reference solutions: Accurately weigh chlorogenic acid, paeoniflorin, chicorylic acid, perilla ketone and glycyrrhizic acid and add them to methanol to prepare mixed reference solutions with concentrations of 100.3 μg / mL, 180 μg / mL, 99.5 μg / mL, 100.2 μg / mL and 98.5 μg / mL, respectively.

[0051] (3) Liquid chromatography and chromatographic conditions: Chromatographic system: Waters 2695 high performance liquid chromatography system, Waters 2998 PDA detector, Empower TM Chromatography workstation software; ACQUITY™ UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm); column temperature: 30 ℃; injection volume: 2 μL; mobile phase: acetonitrile-0.1% phosphoric acid water, gradient elution details are shown in Table 1; flow rate: 0.4 mL / min; detection wavelength: 232 nm; theoretical plate number calculated based on paeoniflorin peak should not be less than 3000.

[0052] Table 3. Mobile phase gradient conditions

[0053] Time (min) Flow rate (mL / min) Acetonitrile (%) 0.1% phosphoric acid solution (%) 0 0.4 5 95 10 0.4 12 88 18 0.4 16 84 22 0.4 27 73 30 0.4 60 40 31 0.4 95 5 32 0.4 5 95

[0054] (4) Determination method: Accurately pipette 10 μL each of the 10 test solutions from step (1) and the reference solution from step (2), inject them into the liquid chromatograph, and determine the chromatogram. Record the chromatogram for 135 min. Import the chromatogram results of the 10 batches of samples into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software for analysis. Use the "median method" with a time width of 0.2 min, perform multi-point correction and automatic matching of the chromatographic peaks of each fingerprint spectrum to generate a common fingerprint spectrum pattern. See the UPLC fingerprint spectra of the 10 batches of natural plant compositions. Figure 2 The generated comparison spectrum can be found in [the image / image]. Figure 3 The fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention time as the mixed reference standard, and the chromatogram of the test sample should be substantially consistent with the fingerprint chromatogram of the reference standard.

[0055] (5) Fingerprint analysis: The fingerprint spectrum generated 16 common peaks. Each common peak was numbered. Peak 3, paeoniflorin, had a moderate retention time and good peak shape in all batches of samples, so it was selected as the reference peak. The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 4 and 5. The similarity of the fingerprint spectra among the 10 batches of samples was calculated based on the common peaks, and the similarity evaluation was performed. The similarity should all be greater than 0.90, specifically 1, 0.994, 0.993, 0.983, 0.993, 0.994, 0.996, 0.988, 0.993, and 0.994. The relative retention time RSD was less than 1.0%, therefore the determined material reference preparation process is stable and feasible.

[0056] Table 4. Relative retention times of common peaks in fingerprint spectra of 10 batches of samples.

[0057]

[0058] Table 5. Relative peak areas of common peaks in fingerprint spectra of 10 batches of samples.

[0059]

[0060]

Claims

1. A method for quality testing of a functional feed-grade natural plant composition for preventing subclinical mastitis in dairy cows during pregnancy and lactation, characterized in that, The process includes the determination of chemical components and the establishment of fingerprint spectra, with the following specific steps: (1) Preparation of test solution: Accurately weigh 10-50 parts of Eucommia ulmoides leaves, 5-25 parts of Paeonia lactiflora, 10-40 parts of Cuscuta chinensis, 10-40 parts of Taraxacum mongolicum, 5-20 parts of Perilla frutescens leaves, 10-30 parts of Dolichos lablab and 5-20 parts of Glycyrrhiza uralensis, place them in a stoppered conical flask, add 25 mL of 80% methanol, weigh, let stand for 0.5 h, extract by ultrasonication for 1 h, cool and make up for the weight loss, shake well and take an appropriate amount, filter through a 0.45 μm microporous membrane to obtain the filtrate as the test solution; (2) Preparation of reference solutions: Accurately weigh chlorogenic acid, paeoniflorin, chicoric acid, perilla ketone and glycyrrhizic acid and add them to methanol to prepare mixed mother liquors with concentrations of 100.3 µg / mL, 180 µg / mL, 99.5 µg / mL, 100.2 µg / mL and 98.5 µg / mL, respectively. Then dilute stepwise to prepare 7 reference solutions with different concentrations. (3) Content determination method: Accurately pipette 2 μL of the test solution in step (1) and the five reference solutions of different concentrations in step (2) into the liquid chromatograph, plot the standard curve with peak area as the ordinate and concentration as the abscissa, and then calculate the content of the main chemical components in the test sample based on the standard curve. (4) Establishment of fingerprint spectrum: The test solution in step (1) and the reference solution in step (2) are precisely pipetted into the liquid chromatograph and measured. The chromatogram is recorded at an absorption wavelength of 232 nm. The chromatogram results are imported into the software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine 2012 Edition" for analysis. The "median method" is used and the time width is set to 0.2 min. Multi-point correction and automatic matching are performed on the chromatographic peaks of each fingerprint spectrum to generate a common pattern of fingerprint spectrum. The fingerprint spectrum is obtained and similarity analysis is performed. The liquid chromatography and chromatographic conditions for step (3) are as follows: Chromatographic system: Waters 2695 high performance liquid chromatography system, Waters 2998 PDA detector, Empower TM Chromatography workstation software; using ACQUITY TM UPLC BEH C 18 Chromatographic column, 100 mm × 2.1 mm, 1.7 µm; Column temperature: 30 ℃; injection volume: 2 μL; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution; flow rate: 0.4 mL / min; detection wavelength: 232 nm; The gradient elution conditions are as follows: 0 min, flow rate 0.4 mL / min, acetonitrile 5%, 0.1% phosphoric acid solution 95%; 10 min, flow rate 0.4 mL / min, acetonitrile 12%, 0.1% phosphoric acid solution 88%; 18 min, flow rate 0.4 mL / min, acetonitrile 16%, 0.1% phosphoric acid solution 84%; 22 min, flow rate 0.4 mL / min, acetonitrile 27%, 0.1% phosphoric acid solution 73%; 30 min, flow rate 0.4 mL / min, acetonitrile 60%, 0.1% phosphoric acid solution 40%; 31 min, flow rate 0.4 mL / min, acetonitrile 95%, 0.1% phosphoric acid solution 5%; 32 min, flow rate 0.4 mL / min, acetonitrile 5%, 0.1% phosphoric acid solution 95%.

2. The quality inspection method according to claim 1, characterized in that, Step (1) The ingredients consist of 25 parts Eucommia ulmoides leaves, 15 parts Paeonia lactiflora, 20 parts Cuscuta chinensis, 20 parts Taraxacum mongolicum, 15 parts Perilla frutescens leaves, 10 parts Dolichos lablab and 10 parts Glycyrrhiza uralensis.

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