Use of L-theanine in the preparation of a feed additive for improving intestinal health and / or alleviating weaning stress in weaned lambs

Abstract

The present application relates to the technical field of feed additive, and discloses application of L-theanine in preparation of a feed additive for improving intestinal health of weaned lambs and / or relieving weaning stress, and discloses a feed additive for improving intestinal health of weaned lambs and / or relieving weaning stress; the feed additive is L-theanine, the L-theanine is used as an active ingredient in the feed, and accounts for 1.0% or 1.5% of the total mass of dry matter of the feed; and a feed for improving intestinal health of weaned lambs and / or relieving weaning stress and a preparation method thereof are also disclosed.In the present application, the feed for weaned lambs is fed by adding L-theanine, the L-theanine accounts for 1.0% or 1.5% of the total mass of dry matter of the feed, can significantly reduce the diarrhea rate of weaned lambs, improve the serum immunoglobulin level and antioxidant capacity, and improve the intestinal mucosa morphology, thereby effectively relieving weaning stress of lambs.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to the application of L-theanine in the preparation of feed additives for improving the intestinal health of weaned lambs and / or alleviating weaning stress. Background Technology

[0002] Weaned lambs are typically goat or sheep lambs aged 35 to 60 days. Lambs at this age are in a special window of opportunity where maternal antibodies have faded and their own immune systems are not yet fully developed, making them particularly sensitive to weaning stress. In weaned lamb farming, antibiotic additives are often used to address problems such as diarrhea and growth retardation caused by weaning stress. While antibiotics can quickly inhibit pathogens, long-term use can easily lead to intestinal microecological imbalance and bacterial resistance, and poses a risk of drug residues, which is inconsistent with the development trend of green and healthy farming.

[0003] To address the aforementioned issues, existing technologies have further provided microecological preparations and functional amino acids or plant extracts. The efficacy of microecological preparations is highly dependent on strain activity, animal age, and rearing environment. In the variable microecological environment of the rumen and hindgut of ruminants, their colonization effect and functional stability are uncertain. For weaned lambs whose rumen function is not yet fully developed, exogenously added amino acids (such as glutamine) are easily degraded by rumen microorganisms, resulting in insufficient doses reaching the hindgut and acting on the intestinal mucosa, leading to poor targeting. Meanwhile, some plant extracts have palatability issues and may interfere with normal rumen fermentation function at higher doses. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for L-theanine, specifically in the preparation of feed additives for improving the gut health of weaned lambs and / or alleviating weaning stress, in order to address the lack of effective intervention programs specifically for improving the gut health of weaned lambs in the prior art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Application of L-theanine in the preparation of feed additives for improving gut health and / or alleviating weaning stress in weaned lambs.

[0006] In the feed, the effective dose of L-theanine accounts for 1.0% or 1.5% of the basal feed mass.

[0007] The present invention also provides a feed additive for improving the intestinal health of weaned lambs and / or alleviating weaning stress, wherein the feed additive is L-theanine; The L-theanine, as the active ingredient in the feed, accounts for 1.0% or 1.5% of the total dry matter mass of the diet.

[0008] The present invention also provides a feed for improving the intestinal health of weaned lambs and / or alleviating weaning stress, comprising L-theanine and a basal feed; The L-theanine accounts for 1.0% or 1.5% of the total dry matter mass of the feed; The basic feed consists of corn flour, soybean meal, peanut vines, and stone powder.

[0009] Preferably, the purity of the L-theanine is greater than 98%.

[0010] Preferably, the basic feed further includes lamb premix; The lamb premix provides the following per kilogram of feed: 8,000 IU of vitamin A, 2,000 IU of vitamin D3, 50 IU of vitamin E, 10 mg of copper, 80 mg of iron, 60 mg of manganese, 40 mg of zinc, 0.3 mg of selenium, and 0.4 mg of iodine.

[0011] The present invention also provides a method for preparing the feed, comprising mixing the L-theanine with the base feed evenly.

[0012] The beneficial effects of this invention are as follows: This invention is the first to propose the application of L-theanine in the preparation of feed additives for improving the intestinal health of weaned lambs and / or alleviating weaning stress, and clarifies two effective addition dosages of 1.0% and 1.5%. L-theanine is a naturally occurring amino acid component in tea, with high safety and no risk of drug residues or bacterial resistance. As a crystalline powder, it is stable and requires only an additional mixing step in existing feed formulation processes, making it easy to promote and apply.

[0013] As a feed additive, L-theanine can activate the tryptophan metabolic pathway and / or branched-chain amino acid metabolic pathway in lambs, thereby improving the intestinal health of weaned lambs, reducing the incidence of diarrhea, reducing weight loss, increasing serum immunoglobulin A and / or immunoglobulin G levels, enhancing serum total antioxidant capacity, increasing the activity of superoxide dismutase and / or glutathione peroxidase and / or catalase, and improving jejunal villus height and / or colonic mucosal thickness to alleviate stress symptoms. Attached Figure Description

[0014] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0015] Figure 1Histological morphology (HE staining, 100×) of jejunum, ileum and colon of weaned lambs in different treatment groups in this application example. Figure 2 This invention provides a heatmap of the correlation between the top 20 upregulated differential metabolites in jejunal contents and jejunal morphological indicators. Red indicates a positive correlation, blue indicates a negative correlation, and the color intensity represents the magnitude of the correlation coefficient. Significance levels: *P<0.05, **P<0.01, ***P<0.001; Figure 3 This invention provides a heatmap showing the correlation between the top 20 downregulated differential metabolites in jejunal contents and jejunal morphological indices. Figure 4 This invention provides a heatmap showing the correlation between the top 20 upregulated differential metabolites in jejunal contents and serum antioxidant markers. Figure 5 This invention provides a heatmap showing the correlation between the top 20 downregulated differential metabolites in jejunal contents and serum antioxidant markers. Figure 6 This invention provides a heatmap showing the correlation between the top 20 upregulated differential metabolites in jejunal contents and serum immune indicators. Figure 7 This invention provides a heatmap showing the correlation between the top 20 downregulated differential metabolites in jejunal contents and serum immune indicators; Figure 8 This invention provides a heatmap showing the correlation between the top 20 upregulated differentially expressed metabolites in serum and jejunal morphological parameters. Red indicates a positive correlation, blue indicates a negative correlation, and the intensity of the color represents the magnitude of the correlation coefficient. Significance levels: *P<0.05, **P<0.01, ***P<0.001; Figure 9 This invention provides a heatmap showing the correlation between the top 20 downregulated differentially regulated metabolites in serum and jejunal morphological parameters; Figure 10 This invention provides a heatmap showing the correlation between the top 20 upregulated differential metabolites in serum and serum antioxidant indicators; Figure 11 This invention provides a heatmap showing the correlation between the top 20 downregulated differential metabolites in serum and serum antioxidant indicators; Figure 12 This invention provides a heatmap showing the correlation between the top 20 upregulated differential metabolites in serum and serum immune indicators; Figure 13 This invention provides a heatmap showing the correlation between the top 20 downregulated differential metabolites in serum and serum immune indicators. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] This invention provides a feed for improving the intestinal health of weaned lambs and / or alleviating weaning stress, comprising L-theanine and a basal feed; wherein the L-theanine accounts for 1.0% or 1.5% of the total dry matter mass of the feed (this invention relates only to these two embodiments, but it is known from common sense that intermediate values ​​are also within the scope of protection).

[0018] Preferably, the ratio of L-theanine to the basal feed is 1.5:100 by mass.

[0019] The L-theanine mentioned above is derived from chemical synthesis, microbial fermentation, or extraction from natural plants such as tea. The purity of L-theanine needs to meet the relevant hygiene standards for food-grade or feed-grade additives.

[0020] In this invention, the purity of the L-theanine is >98%.

[0021] In particular, the L-theanine improves the intestinal immune function of weaned lambs by activating the tryptophan metabolic pathway and / or the branched-chain amino acid metabolic pathway.

[0022] The basic feed can be designed for weaned lambs weighing about 10kg according to the "Standards for Meat Sheep Feeding" (NY / T816-2021). The composition and ratio of raw materials can be adjusted according to the breed, age, weight of the lambs and local feed resources, as long as the basic nutritional needs of the weaned lambs are met.

[0023] Preferably, it includes dry powder feed composed of corn flour, soybean meal, peanut vines, stone powder, and lamb premix.

[0024] The lamb premix provides the following per kilogram of complete diet: Vitamin A 8,000 IU; Vitamin D3 2,000 IU; Vitamin E 50 IU; Copper (copper sulfate) 10 mg; Iron (ferrous sulfate) 80 mg; Manganese (manganese sulfate) 60 mg; Zinc (zinc sulfate) 40 mg; Selenium (sodium selenite) 0.3 mg; Iodine (potassium iodide) 0.4 mg.

[0025] In this invention, cornmeal can be partially or completely replaced with other energy feeds such as wheat and sorghum; soybean meal can be replaced with other protein feeds such as rapeseed meal and cottonseed meal; and peanut vines can be replaced with roughage such as alfalfa meal and oat hay. These routine adjustments to the basic feed formulation do not affect the core function of L-theanine in improving the intestinal health of weaned lambs.

[0026] This feed is not limited to dry powder; it can also be milk replacer or liquid feed. The dosage should ensure that weaned lambs receive an effective daily dose of L-theanine. The specific application medium and method can be adjusted according to the actual management conditions of the farm.

[0027] This feed can be widely used in weaned calves and other ruminant young animals. Applicable animals include weaned lambs of the Huanghuai goat, as well as weaned lambs of goats (such as Boer goats and Nanjiang goats) and weaned lambs of sheep (such as Hu sheep and Xiaowei Han sheep).

[0028] The preferred choice is weaned lambs from the Huanghuai goat breed.

[0029] Due to the decline of maternal antibodies and the immaturity of their own immune system, weaning stress in lambs can easily trigger an over-activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to hypercortisolism, which in turn suppresses immunity and damages the intestines.

[0030] Specifically, L-theanine improves the intestinal immune function of weaned lambs through the following specific metabolic pathways: (1) Activation of the tryptophan metabolic pathway can promote the synthesis of 5-hydroxytryptamine and melatonin, inhibit the overactivation of the hypothalamus-pituitary-adrenal axis, and reduce serum cortisol levels.

[0031] (2) Activate branched-chain amino acid metabolic pathways, upregulate the level of peptide metabolites containing branched-chain amino acids, and enhance the body's metabolic adaptability under stress.

[0032] This feed can improve the intestinal immune function of weaned lambs, specifically by reducing the incidence of diarrhea, reducing weight loss, increasing serum immunoglobulin A and / or immunoglobulin G levels, enhancing serum total antioxidant capacity, increasing the activity of superoxide dismutase and / or glutathione peroxidase and / or catalase, and improving jejunal villus height and / or colonic mucosal thickness.

[0033] The main reason why existing technologies do not allow the direct use of sheep feed containing L-theanine is that ruminants have a unique rumen digestive system. The degradation degree of amino acid additives in the rumen is significantly different from that of monogastric animals. Although the rumen of weaned lambs is still in the development stage, it already has a certain microbial degradation function. Ordinary amino acid additives cannot reach the hindgut to exert their effects. If the dosage is directly increased, it will not only increase the cost of feed, but also have an adverse effect on the rumen fermentation function.

[0034] In particular, the intestinal immune system of weaned lambs is in a special window period of "maternal antibody regression and immature autoimmunity". Their metabolic regulatory network is completely different from that of adult sheep or monogastric animals. Without clear guidance on metabolic mechanisms, the effective dose and effect of L-theanine cannot be predicted.

[0035] This invention breaks through the technical prejudice that amino acid additives are difficult to apply to ruminants. It provides a special feed for lambs during the weaning period, which can effectively improve intestinal immunity, reduce the incidence of diarrhea during the weaning period, increase serum immunoglobulin levels, enhance antioxidant capacity, and improve intestinal mucosal morphology, thereby effectively alleviating the negative effects of weaning stress on lambs.

[0036] In summary, this invention provides a method for applying L-theanine. By feeding weaned lambs a diet containing a specific dose of L-theanine, the regulatory effect of L-theanine on the tryptophan metabolic pathway and branched-chain amino acid metabolic pathway can inhibit the overactivation of the hypothalamus-pituitary-adrenal axis, enhance the body's antioxidant capacity and humoral immunity, and repair the intestinal mucosal barrier, thereby achieving the goals of reducing diarrhea rate, alleviating weight loss, and improving intestinal health.

[0037] The following specific examples provide further details: Example 1:

[0038] 1. Preparation of test materials and additives L-Theanine source: crystalline powder, purchased from Zhejiang Yicun Biotechnology Co., Ltd. (Zhejiang, China), with a nominal purity of 99.8% as tested.

[0039] Basic feed: corn flour, soybean meal, peanut vines, lamb premix, limestone powder; the lamb premix provides the following per kilogram of complete diet: vitamin A 8,000 IU; vitamin D3 2,000 IU; vitamin E 50 IU; copper (copper sulfate) 10 mg; iron (ferrous sulfate) 80 mg; manganese (manganese sulfate) 60 mg; zinc (zinc sulfate) 40 mg; selenium (sodium selenite) 0.3 mg; iodine (potassium iodide) 0.4 mg.

[0040] 2. Preparation: 1 kg of L-theanine was mixed with 42 kg of corn flour, 15 kg of soybean meal, 40 kg of peanut vines, 2 kg of lamb premix, and 1 kg of limestone powder. The mixture was stirred evenly to ensure that L-theanine was evenly distributed in the feed, thus obtaining Example 1.

[0041] Example 2:

[0042] The experimental materials and additives were prepared and the preparation method was the same as in Example 1, except that the content of L-theanine was 1.5 kg.

[0043] Comparative Example 1: No L-theanine added.

[0044] Application example: 1. Experimental animals Eighteen 45-day-old, healthy, and well-adjusted weaned lambs of the Huanghuai goat breed (purchased from Muteng Agricultural Technology Development Co., Ltd., Anhui, China) with similar initial weights (10.16±0.18 kg) were selected. The lambs were randomly divided into 3 groups with 6 replicates per group and 1 lamb per replicate, and were housed in individual pens.

[0045] 2. Grouping Control group (CON group): fed with control group 1.

[0046] Experimental group 1 (LT1.0 group): Feeding Example 1.

[0047] Experimental Group 2 (LT1.5 group): Feeding Example 2.

[0048] 3. Feeding and Management Procedures A three-day pre-trial period was set up to allow the lambs to acclimatize to the experimental diet and feeding environment. After the pre-trial period, a six-day formal trial period began. During the trial, the lambs were fed twice daily, at 08:00 and 17:00, ensuring that there was sufficient feed left in the troughs and allowing them free access to feed. Clean and sufficient drinking water was also provided for the lambs to drink freely.

[0049] The detection methods and results are as follows: 1. Nutritional level testing: Detection method: The crude ash content (Ash) was determined according to GB / T6438-2007, which uses a muffle furnace.

[0050] Test results: The nutritional levels of the feed prepared in Example 1 were tested and found to be as follows: dry matter (DM) 89.99%, crude protein (CP) 8.13%, neutral detergent fiber (NDF) 24.30%, acid detergent fiber (ADF) 7.27%, crude ash (Ash) 5.74%, and crude fat (EE) 2.45%.

[0051] The contents of dry matter (DM), crude protein (CP), crude fat (EE), crude ash (Ash), neutral detergent fiber (NDF) and acid detergent fiber (ADF) in the basal diet were determined by the corresponding standard methods. Specifically, dry matter was determined by GB / T6435-2014, crude protein by GB / T 6432-2018, crude fat by GB / T 6433-2006, and crude ash (Ash) by GB / T6438-2007; the concentrations of ADF and NDF were quantified according to the method of Van Soest et al.

[21] .

[0052] 2. Detect diarrhea and weight changes in weaned lambs. Detection method: On day 0 (initial) and day 6 (before slaughter), all lambs were weighed on an empty stomach before morning feeding, and the initial and final weights were recorded, respectively. Weight changes during the experiment were also calculated. The fecal characteristics of each lamb were observed and recorded daily before morning feeding. The fecal scoring criteria were as follows: 1 point for strip-shaped or granular feces; 2 points for soft but formed feces; 3 points for feces without separation of feces and water, and difficult-to-form feces; 4 points for feces with separation of feces and water, and mucus and blood streaks. A score ≥3 points was considered diarrhea. The number of days with diarrhea refers to the total number of days each lamb scored ≥3 points during the trial period. The diarrhea rate was calculated using the formula: Diarrhea rate (%) = [Number of lambs with diarrhea / (Number of lambs in the experiment × Number of days in the experiment)] × 100%.

[0053] Test results: The effects of L-theanine content in the diet on fecal score, number of days with diarrhea, and diarrhea rate in weaned lambs are shown in Table 1. Table 1

[0054] Note: Different lowercase letters in the superscript of data from the same line indicate significant differences (P<0.05). SEM values ​​are standard errors.

[0055] The comparison results of the initial weight and weight on day 6 of the lambs in each group are shown in Table 2: Results Analysis: Table 1 shows that, compared with the control group, the addition of L-theanine to the diet effectively improved the diarrhea status of weaned lambs. Specifically, the diarrhea rate in the 1.5% L-theanine group (LT1.5) significantly decreased from 63.9% in the control group to 30.6% (P=0.023), and the fecal score on day 6 decreased from 2.83 to 1.50 (see Table 1). Regarding body weight, the control group lambs lost 0.25 kg during the experiment, while the LT1.5 group lambs lost only 0.09 kg (see Table 2), indicating that L-theanine can effectively alleviate weight loss caused by weaning stress.

[0056] Table 2

[0057] 3. Detection of serum antioxidant capacity Detection method: On day 6 of the experiment, patients were fasted for 12 hours prior to slaughter. 10 mL of blood was collected via the jugular vein, allowed to stand at room temperature for 30 min, and then centrifuged at 3000×g for 15 min at 4°C to separate serum. The serum was aliquoted into 1.5 mL sterile centrifuge tubes and stored at -20°C for the determination of antioxidant and immunomodulatory indicators. Serum antioxidant indicators were detected using the corresponding kits manufactured by Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Shanghai, China), strictly following the instructions. Total antioxidant capacity (T-AOC) was determined using the FRAP method kit (catalog number: ml092646); catalase (CAT) activity was determined using the UV absorption method (catalog number: ml092621); superoxide dismutase (SOD) activity was determined using the WST-8 method (catalog number: ml076328); glutathione peroxidase (GSH-Px) activity was determined using the micro-method kit (catalog number: ml076447); and malondialdehyde (MDA) content was determined using the micro-method (catalog number: ml022446).

[0058] Test results: The effects of dietary L-theanine supplementation on serum antioxidant levels in weaned lambs are shown in Table 3. Table 3

[0059] Note: Different lowercase letters in the superscript of data from the same line indicate significant differences (P<0.05). SEM values ​​are standard errors.

[0060] Results Analysis: Compared with the control group, the addition of 1.0% and 1.5% L-theanine to the diet significantly improved the serum antioxidant capacity of weaned lambs. On day 6, the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities in the LT1.0 and LT1.5 groups were significantly increased (P<0.05) (see Table 3). Among them, the improvement effect in the LT1.5 group was more obvious, with T-AOC, SOD, GSH-Px, and CAT activities increasing by 25.3%, 7.2%, 13.2%, and 12.4% respectively compared with the control group.

[0061] 4. Detect serum immune markers. Detection method: On day 6 of the experiment, after a 12-hour fast prior to slaughter, 10 mL of blood was collected via the jugular vein. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000×g for 15 minutes at 4°C to separate the serum. The serum was aliquoted into 1.5 mL sterile centrifuge tubes and stored at -20°C for the determination of antioxidant and immunomodulatory indicators. Serum immunomodulatory indicators were detected using the corresponding kits manufactured by Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Shanghai, China), strictly following the instructions. The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were determined using enzyme-linked immunosorbent assay (ELISA), kit numbers YJ025601, YJ025602, and YJ025607, respectively.

[0062] Test results: The effects of dietary L-theanine supplementation on serum immune markers in weaned lambs are shown in Table 4. Table 4

[0063] Different lowercase letters in the superscript of data from the same peer indicate significant differences (P<0.05). SEM values ​​are standard errors.

[0064] Results analysis: On day 6, compared with the control group, the addition of L-theanine to the diet significantly increased the levels of immunoglobulin A (IgA) and immunoglobulin G (IgG) in the serum of weaned lambs (P<0.05), and this increase was dose-dependent (see Table 4). The LT1.5 group showed the best effect, with IgA and IgG levels increasing by 7.5% and 4.9%, respectively, compared to the control group. There was no significant difference in immunoglobulin M (IgM) levels among the groups (P>0.05).

[0065] 5. Detection of intestinal tissue morphology Detection method: After blood collection, the animals were slaughtered, and the jejunum, ileum, and mid-colon were quickly harvested, with approximately 2 cm from each section. These sections were fixed in 4% paraformaldehyde for histological observation. The fixed jejunum, ileum, and colon tissues were embedded in paraffin and sectioned, then stained with hematoxylin and eosin (H&E) for observation of intestinal morphology under a light microscope. For the jejunum and ileum, villus height (VH) and crypt depth (CD) were measured, and the villus height / crypt depth ratio (VH / CD) was calculated. For the colon, crypt depth (CD), mucosal thickness (MT), and muscularis propria thickness (MLT) were measured. Quantitative analysis was performed using Image-Pro Plus 6.0 image analysis software. Five non-overlapping fields of view were randomly selected from each section, and three sites were measured in each field. The average of all measurements was taken as the final data for that section.

[0066] Test results: Adding L-theanine to the diet can effectively improve the intestinal mucosal morphology of weaned lambs. Figure 1 As shown.

[0067] Table 5 shows the effects of dietary L-theanine supplementation on the morphological parameters of intestinal tissue in weaned lambs. Table 5

[0068] Results Analysis: Compared with the control group, the villus height (VH) of the jejunum was significantly increased in both the LT1.0 and LT1.5 groups (P=0.013), with the VH in the LT1.5 group increasing from 586.89 μm to 867.79 μm, an increase of 47.8% (see Table 5). Simultaneously, the villus height / crypt depth ratio (VH / CD) in the LT1.5 group was significantly higher than that in the control group (3.15 vs 2.10, P=0.078). In the colon, the mucosal thickness (MT) was significantly increased in the L-theanine-added groups (P=0.013), increasing from 569.36 μm to 765.66 μm in the LT1.5 group.

[0069] 6. Detection of the molecular mechanism of L-theanine This invention further reveals the mechanism by which L-theanine improves intestinal health in weaned lambs at the molecular level using LC-MS / MS non-targeted metabolomics technology.

[0070] Detection method: On day 6 of the experiment, patients were fasted for 12 hours prior to slaughter. 10 mL of blood was collected via the jugular vein, allowed to stand at room temperature for 30 min, and then centrifuged at 3000×g for 15 min at 4°C to separate the serum. The serum was aliquoted into 1.5 mL sterile centrifuge tubes and stored at -80°C for untargeted metabolomics analysis. After slaughter, approximately 2 g of the contents of the mid-jejunum was rapidly collected, placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for untargeted metabolomics analysis of the jejunal contents.

[0071] Jejunal contents samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for non-targeted metabolomics analysis. Sample pretreatment steps were as follows: Approximately 10 mg of jejunal contents was accurately weighed into a 2 mL centrifuge tube containing one 6 mm grinding bead, and 200 μL of extraction buffer (methanol:water = 4:1, v / v, containing 0.02 mg / mL L-2-chlorophenylalanine and four other internal standards) was added. The mixture was ground in a cryo-tissue homogenizer at -10℃ and 50 Hz for 6 min, followed by ultrasonic extraction at 5℃ and 40 kHz for 30 min, and then allowed to stand at -20℃ for 30 min. The mixture was centrifuged at 4℃ and 13,000 × g for 15 min, and the supernatant was directly transferred to a vial with an inner tube for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Quality control samples (QC) were prepared by mixing 20 μL of supernatant from each of the jejunal contents samples, and one QC sample was inserted every 5–15 analytical samples in the analytical sequence.

[0072] LC-MS / MS analysis was performed using a Vanquish Horizon UHPLC system and an Exploris 480 mass spectrometer (ThermoFisher Scientific). The chromatographic column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm; Waters), with a column temperature of 40 °C and an injection volume of 3 μL. Mobile phase A was water:acetonitrile (95:5, containing 0.1% formic acid), and mobile phase B was acetonitrile:isopropanol:water (47.5:47.5:5, containing 0.1% formic acid). Positive and negative ion modes were used for acquisition, with a scan range of m / z 70–1050. The primary full scan resolution was set to 60,000, and the secondary mass spectrometry (MS / MS) resolution was set to 15,000 in data-dependent acquisition (DDA) mode.

[0073] Raw data were processed using Progenesis QI v3.0 (Waters), and metabolite identification was performed based on accurate mass numbers (bias <10ppm) and secondary mass spectrometry information, compared with HMDB, Metlin, and a self-built database.

[0074] Serum samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for non-targeted metabolomics analysis. 100 μL of serum was taken and 400 μL of extraction buffer (methanol:acetonitrile = 1:1, v / v, containing 0.02 mg / mL L-2-chlorophenylalanine and four other internal standards) was added. The mixture was vortexed, sonicated at 5℃ and 40 kHz for 30 min, and then incubated at -20℃ for 30 min. The mixture was centrifuged at 4℃ and 13,000 × g for 15 min, and the supernatant was dried under nitrogen. 120 μL of acetonitrile:water (1:1) was added to reconstitute the supernatant, vortexed, and sonicated again for 5 min (5℃, 40 kHz). The mixture was then centrifuged (4℃, 13,000 × g, 10 min), and the supernatant was used for injection. Quality control samples (QC) were prepared by mixing the supernatants of each serum sample, with one QC sample inserted between every 5–15 samples.

[0075] The LC-MS / MS detection conditions, data processing, and metabolite identification methods are the same as in Section 2.3.5, except that the resolution of the secondary mass spectrometry (MS / MS) in DDA mode is set to 7,500 (the resolution of the primary full scan is 60,000).

[0076] Test results and analysis: (1) Activation of the metabolic pathway of branched-chain amino acids (BCAAs) Metabolomics analysis of jejunal contents (see) Figures 2 to 7 The results showed that the addition of L-theanine significantly activated the metabolic pathway of branched-chain amino acids (BCAAs).

[0077] In the LT1.0 group, the branched-chain amino acid-containing tripeptide Ile-Phe-Asp was significantly upregulated (Log2FC=1.379); in the LT1.5 group, another branched-chain amino acid-containing metabolite, Lys-Glu-Lys, was also significantly upregulated (Log2FC=1.441). Activation of BCAA catabolism can provide substrates for the tricarboxylic acid cycle and activate the mammalian target of rapamycin (mTOR) signaling pathway, promoting protein synthesis and tissue repair.

[0078] This demonstrates the molecular basis for L-theanine's ability to enhance the metabolic adaptability of weaned lambs under stress and promote intestinal mucosal repair.

[0079] In addition, the pro-inflammatory remission factor Resolvin D1 was significantly upregulated in the L-theanine group, and the level of the glutathione derivative Glutathione Monoisopropyl Ester was increased. The weaning stress-related metabolite Tyr-Phe-His-Glu was significantly downregulated in the LT1.5 group (Log2FC=-3.292).

[0080] These changes collectively indicate that L-theanine can optimize the metabolic environment of the gut microbiota, promote the active resolution of inflammation, and restore redox balance.

[0081] (2) Activate the tryptophan-5-HT / melatonin metabolic axis Serum metabolomics analysis (see) Figures 8 to 13 The results showed that, compared with the control group, the serum melatonin level in lambs in the L-theanine group was significantly increased in a dose-dependent manner, with log2 fold differences (Log2FC) of 0.321 and 0.536 in the LT1.0 and LT1.5 groups, respectively. Simultaneously, the level of 2-methyl-5-hydroxytryptamine in the jejunal contents was also significantly upregulated (LT1.5 vs CON, Log2FC = 1.168). 2-methyl-5-hydroxytryptamine is a stable analogue of serotonin (5-HT), which plays a crucial role in regulating intestinal motility, secretion, and immune function.

[0082] Correspondingly, serum levels of the stress hormone cortisol were significantly downregulated in both the LT1.0 and LT1.5 groups (Log2FC -0.144 and -0.126, respectively). These results demonstrate for the first time that L-theanine can systematically alleviate weaning stress by activating the tryptophan-5-HT / melatonin metabolic axis. This is achieved by enhancing 5-HTergic signaling locally in the gut to regulate intestinal function and by increasing circulating melatonin levels to inhibit the overactivation of the hypothalamus-pituitary-adrenal (HPA) axis.

[0083] As can be seen from the above application examples, by adding 1.0% and 1.5% L-theanine by dry matter to the diet of weaned lambs, the present invention can significantly reduce the diarrhea rate of weaned lambs (from 63.9% to 30.6%), reduce weight loss, increase serum immunoglobulin (IgA, IgG) levels and antioxidant enzyme (T-AOC, SOD, GSH-Px, CAT) activity, and significantly improve jejunal villus height (increased by 47.8%) and colonic mucosal thickness.

[0084] Metabolomics analysis further revealed that L-theanine exerts a synergistic effect across multiple dimensions, including neuroendocrine regulation, energy metabolism adaptation, and intestinal barrier repair, by activating the tryptophan metabolic pathway (promoting 5-HT / melatonin synthesis and inhibiting cortisol release) and branched-chain amino acid metabolic pathways. Therefore, the technical solution provided by this invention can effectively improve the intestinal immune function of weaned lambs and alleviate weaning stress syndrome, with the overall effect of a 1.5% dose being superior to that of a 1.0% dose.

[0085] Through systematic animal feeding experiments, the functions of this invention are as follows: (1) Reduce the diarrhea rate: Adding 1.5% L-theanine to the basic diet can reduce the diarrhea rate of weaned lambs from 63.9% to 30.6%, a reduction of 52.1% (P=0.023).

[0086] (2) Reduced weight loss: During the experiment, the lambs in the control group lost 0.25 kg, while the lambs in the 1.5% L-theanine group lost only 0.09 kg, a reduction of 64.0% in weight loss.

[0087] (3) Increased serum immunoglobulin levels: Compared with the control group, the serum immunoglobulin A content of lambs in the 1.5% L-theanine group increased from 89.917 μg / mL to 96.660 μg / mL (P=0.021), an increase of 7.5%; the immunoglobulin G content increased from 170.767 μg / mL to 179.057 μg / mL (P=0.016), an increase of 4.9%.

[0088] (4) Enhanced serum antioxidant capacity: Compared with the control group, the total antioxidant capacity of lamb serum in the 1.5% L-theanine group increased from 0.297 U / mL to 0.372 U / mL (P=0.012), an increase of 25.3%; superoxide dismutase activity increased from 12.308 U / mL to 13.191 U / mL (P=0.046), an increase of 7.2%; glutathione peroxidase activity increased from 62.989 U / mL to 71.321 U / mL (P=0.010), an increase of 13.2%; catalase activity increased from 16.402 U / mL to 18.433 U / mL (P=0.001), an increase of 12.4%.

[0089] (5) Improved intestinal mucosal morphology: Compared with the control group, the jejunal villus height of lambs in the 1.5% L-theanine group increased from 586.89 μm to 867.79 μm (P=0.013), an increase of 47.8%; the colonic mucosal thickness increased from 569.36 μm to 765.66 μm (P=0.013), an increase of 34.5%. The ratio of jejunal villus height to crypt depth increased from 2.10 to 3.15 (P=0.078).

[0090] (6) Optimization of metabolome: Metabolomics analysis confirmed that L-theanine can significantly upregulate serum melatonin levels (the logarithm of the difference in the 1.5% group compared with the control group was Log2FC=0.536) and 2-methyl-5-hydroxytryptamine levels in jejunal contents (Log2FC=1.168), while downregulating serum cortisol levels (Log2FC=-0.126) and upregulating the levels of branched-chain amino acid-related metabolites.

[0091] (7) Safe and non-toxic, with no risk of residue L-Theanine is a naturally occurring amino acid found in tea leaves. It has been approved for use as a food additive and dietary supplement and is recognized for its safety. Using it as a feed additive carries no risk of drug residues or bacterial resistance, aligning with the development direction of green farming.

[0092] (8) It is easy to apply and promotes industrialization. This feed only requires the addition and mixing of L-theanine to the existing feed formulation process, without changing existing breeding facilities or feeding management methods. L-theanine is a crystalline powder, which is stable, easy to store and transport, has a simple mixing process, is highly accepted by farmers, and is easy to promote and apply in large-scale sheep farms.

[0093] Existing technologies such as probiotics, functional amino acids, or plant extracts all have unresolved drawbacks. However, this feed contains L-theanine, a crystalline powder that is unaffected by fluctuations in the rumen and hindgut microecological environment. It also has a dual mechanism of action, acting not only locally in the intestine (increasing jejunal villus height by 47.8% and colonic mucosal thickness by 34.5%), but also acting as a signaling molecule to activate the body's own tryptophan and branched-chain amino acid metabolic pathways, amplifying endogenous repair capabilities. Therefore, the absolute dose requirement for reaching the hindgut is relatively low. Through systemic-local synergy, it compensates for the potential deficiency of the hindgut targeted dose, enabling its application in ruminant feeding without inhibiting rumen function. This systematically solves the weaning stress problem faced by weaned lambs (35-60 days old) during the special window period of maternal antibody decline and immature autoimmune system.

[0094] This invention primarily protects the application of L-theanine in the preparation of feed additives for improving the intestinal health of weaned lambs and / or alleviating weaning stress. Feeding lambs with feed containing L-theanine during the weaning period can activate the tryptophan metabolic pathway and / or branched-chain amino acid metabolic pathway in the lamb's body, thereby improving the intestinal health of weaned lambs, reducing the incidence of diarrhea, reducing weight loss, increasing serum immunoglobulin A and / or immunoglobulin G levels, enhancing serum total antioxidant capacity, increasing the activity of superoxide dismutase and / or glutathione peroxidase and / or catalase, and improving jejunal villus height and / or colonic mucosal thickness to alleviate stress symptoms.

[0095] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. Application of L-theanine in the preparation of feed additives for improving gut health and / or alleviating weaning stress in weaned lambs.

2. The application according to claim 1, characterized in that, In the feed, the effective dose of L-theanine accounts for 1.0% or 1.5% of the basal feed mass.

3. A feed additive for improving intestinal health and / or alleviating weaning stress in weaned lambs, characterized in that, The feed additive is L-theanine; The L-theanine, as the active ingredient in the feed, accounts for 1.0% or 1.5% of the total dry matter mass of the diet.

4. A feed for improving intestinal health and / or alleviating weaning stress in weaned lambs, characterized in that, Including L-theanine and basal feed; The L-theanine accounts for 1.0% or 1.5% of the total dry matter mass of the feed; The basic feed consists of corn flour, soybean meal, peanut vines, and stone powder.

5. The feed additive according to claim 3 or the feed according to claim 4, characterized in that, The purity of the L-theanine is greater than 98%.

6. The feed according to claim 4, characterized in that, The basic feed also includes lamb premix; The lamb premix provides the following per kilogram of feed: 8,000 IU of vitamin A, 2,000 IU of vitamin D3, 50 IU of vitamin E, 10 mg of copper, 80 mg of iron, 60 mg of manganese, 40 mg of zinc, 0.3 mg of selenium, and 0.4 mg of iodine.

7. A method for preparing the feed according to claim 4, characterized in that, This includes mixing the L-theanine with the basal feed evenly.

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