Low-protein diet with yellow rice wine lees and application thereof in improving egg laying rate and egg quality of laying ducks

Abstract

The application discloses a low-protein daily ration of yellow rice lees and application of the daily ration in improving egg laying rate and egg quality of laying ducks. The daily ration comprises corn, soybean meal, brown rice, wheat bran, yellow rice lees and limiting amino acids, etc. Yellow rice lees is used to replace part of the soybean meal, so that the crude protein level of the daily ration is reduced by not more than 1 percent point compared with a conventional daily ration, and essential amino acids are kept in balance. The application proves that, by adding yellow rice lees in a specific proportion in the daily ration of laying ducks and combining with crystal amino acids, the crude protein can be reduced from 16.5% to 15.5%, which not only maintains the stability of average daily feed intake, egg laying rate, feed egg ratio and egg quality, but also significantly reduces the serum uric acid level, improves the intestinal metabolic profile, and maintains the stability of the cecal flora structure. The application provides a feasible scheme for high-value utilization of yellow rice lees and preparation of a low-protein daily ration of laying ducks, and has the dual advantages of reducing feed cost and reducing nitrogen excretion.

Description

Technical Field

[0001] This invention relates to the field of feed and aquaculture technology, specifically to a low-protein diet made from rice wine lees and its application in improving the egg production rate and egg quality of laying ducks. Background Technology

[0002] Yellow wine lees, as a novel protein supplement, possesses a complete range of amino acids, with a balanced composition of essential amino acids, making it superior to soybean meal overall. As an agricultural processing byproduct, yellow wine lees has a certain protein content and nutritional potential. It can be incorporated into livestock and poultry diets in appropriate proportions to replace some conventional high-priced protein sources (such as soybean meal), without significantly affecting livestock and poultry growth performance within suitable addition ratios. This study addresses the industry problem of large price fluctuations and high costs associated with soybean meal, a conventional protein source, in duck farming, and focuses on the high-value utilization of yellow wine lees, an unconventional feed ingredient. In their review, Ge Songtao (Ge Songtao, Xu Huangen, Shou Quanhong. Research progress on the development and utilization of rice wine lees resources [J]. Zhejiang Animal Husbandry and Veterinary Medicine, 2023, 48(01): 11-3) et al. clearly pointed out that when rice wine lees are used in livestock and poultry feed, they can replace some conventional protein sources (such as soybean meal) through appropriate treatment (such as fermentation and enzymatic hydrolysis), and have no significant negative impact on the production performance of some livestock and poultry. They also mentioned the potential of rice wine lees in reducing feed costs, which provides important support for this study to construct a low-protein diet system with rice wine lees as the core.

[0003] Currently, research on low-protein diets for poultry mainly focuses on laying hens and broilers, with balanced amino acids as the core technology. However, research on low-protein diets for laying ducks, especially those using rice wine lees as the main protein source, is still lacking. Studies by Han Qiang (Han Qiang, Li Kui, Chen Jie, et al. Effects of low-phosphorus and low-protein diets with added compound enzymes on the production performance and nitrogen and phosphorus metabolism of growing pigs [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2022, (20): 104-7) on growing pigs have shown that, under appropriate protein reduction, low-protein diets combined with enzymes can maintain normal production performance of growing pigs, while reducing nitrogen excretion and improving nitrogen metabolism efficiency. Although the research subjects are different, the mechanisms by which low-protein diets affect the production performance and nitrogen metabolism of livestock and poultry have certain commonalities, providing a reference framework for this study on low-protein diets for laying ducks. In addition, the intestinal metabolome and microbiome structure are important windows reflecting the nutritional metabolism and health status of animals, and their response mechanisms under low-protein diet conditions have not yet been systematically revealed in laying ducks.

[0004] Based on this, this application systematically evaluates the effects of replacing part of soybean meal with yellow wine lees in the diet on the production performance, egg quality, blood biochemical indicators, intestinal metabolome and cecal flora structure of laying ducks. The aim is to clarify the feasibility of applying yellow wine lees in the low-protein diet of laying ducks and the appropriate reduction in crude protein (CP), so as to provide experimental basis for the feed utilization of yellow wine lees and the formulation of low-protein diets for laying ducks.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a low-protein diet formulated by replacing part of the soybean meal with rice wine lees, which achieves multiple objectives of maintaining production performance, improving metabolic indicators and reducing feed costs, based on a clear and appropriate reduction in crude protein.

[0007] To achieve the above objectives, this invention provides a low-protein diet made from rice wine lees, comprising the following ingredients in parts by weight: 50-52 parts corn, 18-20 parts soybean meal, 6-7 parts brown rice, 7-8 parts wheat bran, 1.5-2.5 parts rice wine lees, 0.05-0.15 parts soybean oil, 1.0-1.2 parts dicalcium phosphate, 8.5-9.0 parts limestone powder, 0.15-0.25 parts sodium chloride, and 0.15-0.20 parts DL-methionine. 0.15-0.25 parts L-lysine sulfate, 0.03-0.08 parts L-threonine, 0.03-0.05 parts tryptophan, 0.04-0.08 parts isoleucine, 0.10-0.15 parts L-arginine, 1.0-1.5 parts potassium magnesium sulfate, 0.05-0.15 parts choline chloride, 0.02-0.05 parts phytase, 0.05-0.15 parts broiler multivitamins, and 0.05-0.15 parts organic minerals.

[0008] Preferably, the low-protein diet made from rice wine lees consists of the following ingredients in parts by weight: 51.66 parts corn, 19.54 parts soybean meal, 6.64 parts brown rice, 7.69 parts wheat bran, 1.99 parts rice wine lees, 0.1 parts soybean oil, 1.12 parts dicalcium phosphate, 8.87 parts limestone powder, 0.2 parts sodium chloride, 0.18 parts DL-methionine, 0.21 parts L-lysine sulfate, 0.05 parts L-threonine, 0.04 parts tryptophan, 0.06 parts isoleucine, 0.12 parts L-arginine, 1.2 parts potassium magnesium sulfate, 0.1 parts choline chloride, 0.03 parts phytase, 0.1 parts broiler multivitamins, and 0.1 parts organic minerals; the crude protein content of the diet is 15.5%.

[0009] Furthermore, the crude protein content of the low-protein diet made from rice wine lees provided by this invention is 15.5%.

[0010] The low-protein diet made from rice wine lees provided by this invention can be applied to improve the egg production rate and / or egg quality of laying ducks.

[0011] Preferably, the application is to use the diet to feed laying ducks of the Qingke No. 4 variety.

[0012] Preferably, improving egg quality includes increasing at least one of Haugh units, eggshell strength, and yolk color, or reducing the egg breakage rate.

[0013] Preferably, the application also includes reducing serum uric acid levels in laying ducks, improving intestinal amino acid metabolism, and maintaining the stability of the cecal flora structure.

[0014] The low-protein diet made from rice wine lees provided by this invention can also be used to reduce nitrogen excretion and / or reduce serum uric acid levels in laying ducks.

[0015] The present invention has the following advantages: This invention is the first to systematically apply rice wine lees as a major low-protein source in duck feed, providing a scientific basis and a practical technical solution for the high-value utilization of this agricultural by-product, which helps to alleviate the conflict between humans and livestock for grain and reduce dependence on imported soybean meal.

[0016] This invention successfully and safely reduced the crude protein level in laying duck diets from the conventional 16.5% to 15.5% while maintaining amino acid balance. Verification showed that this diet had no significant negative impact on the average daily feed intake, laying rate, feed conversion ratio, and key egg quality indicators such as egg shape index, eggshell strength, and Haugh units, thus ensuring farming efficiency while reducing feed formulation costs.

[0017] The low-protein diet provided by this invention can significantly reduce serum uric acid levels in laying ducks, indicating that it effectively reduces the catabolism of protein and the generation of nitrogenous waste in the body, thereby alleviating the metabolic burden on the liver and kidneys and having the environmental protection potential to reduce nitrogen emissions.

[0018] This invention, by precisely controlling the reduction rate (CP to 15.5%), minimizes the disturbance to the intestinal metabolic profile and cecal microbiota structure of laying ducks compared to excessive protein reduction (CP below 14.5%). It can positively regulate pathways related to amino acid metabolism, substance transport, and energy metabolism, and maintain the richness and structural stability of the cecal microbiota, providing a physiological basis for the healthy and efficient breeding of laying ducks. Attached Figure Description

[0019] Figure 1 OPLS-DA score plots and displacement test results for different dietary treatment groups under positive ion mode; among them, Figure 1 In the diagram, A, B, and C are the OPLS-DA score plots for CK vs LH, CK vs MH, and CK vs HH, respectively, while a, b, and c are the permutation test plots for CK vs LH, CK vs MH, and CK vs HH, respectively.

[0020] Figure 2 OPLS-DA score plots and displacement test results for different dietary treatment groups under negative ion mode; among them, Figure 2 In the diagram, A, B, and C are the OPLS-DA score plots for CK vs LH, CK vs MH, and CK vs HH, respectively, while a, b, and c are the permutation test plots for CK vs LH, CK vs MH, and CK vs HH, respectively.

[0021] Figure 3 The results of quantitative analysis of differential metabolites in different dietary treatment groups under positive and negative ion modes are shown. Figure 3 In this context, A represents positive ion mode and B represents negative ion mode.

[0022] Figure 4 The results of differential metabolite enrichment analysis in positive ion mode are shown for different dietary treatment groups; among them, Figure 4 A, B, and C in the table are the KEGG enrichment analysis results for CK vs LH, CK vs MH, and CK vs HH, respectively.

[0023] Figure 5 The results of the differential enrichment analysis of metabolites in different dietary treatment groups under negative ion mode are shown; among them, Figure 5 A, B, and C in the table are the KEGG enrichment analysis results for CK vs LH, CK vs MH, and CK vs HH, respectively.

[0024] Figure 6 The results of Alpha diversity analysis of cecal microorganisms in laying ducks under different dietary treatments are as follows: Figure 6 A, B, and C in the table represent the results of CK vs LH, CK vs MH, and CK vs HH, respectively.

[0025] Figure 7 The results of PCoA analysis of cecal microbiota in laying ducks under different dietary treatments are shown below. Figure 7 A, B, and C in the table represent the results of CK vs LH, CK vs MH, and CK vs HH, respectively.

[0026] Figure 8 The results of LEfSe LDA analysis of cecal microbiota in laying ducks under different dietary treatments are shown below. Figure 8A, B, and C in the table represent the results of CK vs LH, CK vs MH, and CK vs HH, respectively. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0028] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] This study constructed a low-protein diet system based on rice wine lees as the primary protein source and systematically evaluated the effects of different crude protein levels (16.5%, 15.5%, 14.5%, and 13.5%) on the production performance, egg quality, blood biochemical indicators, intestinal metabolome, and cecal microbiota structure of laying ducks. The aim was to explore the feasibility of replacing soybean meal with this diet and the appropriate reduction rate. Details are as follows: The experimental animals, their husbandry and management, and some materials involved in this embodiment are as follows: Experimental animal: Qingke No. 4 egg duck.

[0030] Experimental diets: Basal diets were formulated according to NRC (1994) and Chinese laying duck feeding standards. In the low-protein treatment groups, a portion of soybean meal was replaced with distillers' grains (DGD). By adjusting the proportions of corn, soybean meal, and DGD, the crude protein levels were achieved to 16.5% (control group, CK), 15.5% (LH), 14.5% (MH), and 13.5% (HH), respectively. The diets of all groups maintained consistent levels of metabolizable energy, calcium, and phosphorus, and synthetic amino acids were supplemented according to the ideal amino acid pattern to ensure a balanced supply of essential amino acids. The composition and nutrient levels of the diets are detailed in Table 1.

[0031] Table 1 Composition of the basic diet corn 50 51.66 53.31 55.68 soybean meal 24.33 19.54 14.72 10 brown rice 6.98 6.64 6.3 5.32 wheat bran 6.98 7.69 8.19 8.86 Yellow wine lees 0 1.99 4.14 6.01 soybean oil 0.1 0.1 0.1 0.1 Calcium hydrogen phosphate 1.11 1.12 1.15 1.17 stone powder 8.86 8.87 8.88 8.9 Sodium chloride 0.2 0.2 0.19 0.19 DL-methionine 0.15 0.18 0.21 0.24 L-Lysine sulfate 0.04 0.21 0.4 0.57 L-threonine 0 0.05 0.09 0.14 Tryptophan 0.02 0.04 0.06 0.08 Isoleucine 0 0.06 0.11 0.17 L-arginine 0 0.12 0.24 0.36 Potassium magnesium sulfate 0.9 1.2 1.58 1.88 choline chloride 0.1 0.1 0.1 0.1 Phytase 0.03 0.03 0.03 0.03 Broiler multivitamins 0.1 0.1 0.1 0.1 Organic polymineral 0.1 0.1 0.1 0.1 total 100 100 100 100 288 Qingke No. 4 laying ducks of similar weight and egg production rate at 180 days old were selected and randomly divided into 4 groups, with 6 replicates in each group and 12 ducks in each replicate. The pre-trial period was 30 days and the formal trial period was 12 weeks.

[0032] Experimental groups: A single-factor completely randomized design was adopted, with four dietary crude protein level gradients: 16.5% (control group, CK), 15.5% (LH group), 14.5% (MH group) and 13.5% (HH group).

[0033] Feeding and Management: The experimental ducks were raised in a semi-open duck house using a floor-raising method. The temperature inside the house was controlled at 13-25℃, the relative humidity at 55%-65%, and the light exposure at 16 hours per day. During the experiment, the ducks were guaranteed free access to feed and water, fed twice daily, with leftover feed and feces promptly removed to maintain environmental hygiene, and the duck house was disinfected regularly.

[0034] Experiment Example 1: Verification of the Effects of Low-Protein Diets Based on Rice Wine Fermentation on the Production Performance of Laying Ducks Based on the above grouping and feeding conditions, production performance was measured, as follows: During the formal trial, using replicates as the statistical unit, feed intake, egg production, total egg weight, and duck population were recorded weekly for each replicate. Average daily feed intake, egg production rate, average egg weight, and feed conversion ratio were calculated. The calculation formulas are as follows:

[0035] Average daily feed intake = feed intake / number of ducks; Egg production rate = (Number of eggs produced / Number of ducks) × 100%; Average egg weight = total egg weight / number of eggs laid; Feed intake to egg ratio = feed intake / total egg weight.

[0036] The effects of different crude protein levels in rice wine lees diets on the production performance of laying ducks are shown in Table 2. There were no significant differences in average daily feed intake, egg production rate, and feed conversion ratio among the treatment groups (P>0.05). Average egg weight did not differ significantly between the groups at week 0 (w) and week 2 (P>0.05), but significant differences appeared at weeks 4, 8, 10, and 12 (P<0.05). Specifically, at week 4, the HH group was significantly lower than the CK group; at week 8, the MH group was significantly lower than the CK group; at week 10, the LH and HH groups were significantly lower than the CK group; and at week 12, the MH and HH groups were significantly lower than the CK group. Overall, low-protein diets had a relatively small impact on feed intake and egg production rate in laying ducks, but had a certain impact on average egg weight, with the MH and HH groups showing the most significant effects.

[0037] Table 2. Effects of different crude protein levels in rice wine lees diets on the production performance of laying ducks. 0w Average egg weight / g 67.29±2.15 66.66±1.99 65.79±2.16 64.97±1.11 0.196 Egg production rate 0.93±0.06 0.90±0.06 0.93±0.04 0.90±0.08 0.386 Average daily feed intake 144.92±2.28 145.22±1.49 144.12±4.18 145.21±1.53 0.872 Egg ratio 2.33±0.19 2.43±0.19 2.30±0.09 2.50±0.24 0.249 2w Average egg weight / g 68.42±2.56 67.53±1.24 66.09±1.25 66.32±1.47 0.099 Egg production rate 0.93±0.10 0.90±0.06 0.93±0.10 0.93±0.06 0.915 Average daily feed intake 145.72±0.27 145.83±0.00 145.83±0.00 145.64±0.47 0.555 Egg ratio 2.31±0.28 2.40±0.16 2.40±0.33 2.37±0.12 0.905 4w Average egg weight / g <![CDATA[68.20±0.90 a ]]> <![CDATA[66.86±1.62 ab ]]> <![CDATA[66.33±1.66 ab ]]> <![CDATA[65.09±1.12 b ]]> 0.007 Egg production rate 0.95±0.07 0.86±0.16 0.92±0.09 0.95±0.07 0.457 Average daily feed intake 143.64±2.56 140.19±5.79 136.35±9.40 137.68±11.97 0.455 Egg ratio 2.24±0.17 2.53±0.62 2.26±0.22 2.24±0.13 0.417 6w Average egg weight / g 67.49±2.26 67.22±2.44 63.90±9.05 63.08±2.53 0.880 Egg production rate 0.90±0.10 0.86±0.17 0.90±0.11 0.92±0.13 0.894 Average daily feed intake 145.83±0.00 141.08±11.64 140.91±12.04 142.13±3.72 0.735 Egg ratio 2.42±0.34 2.66±0.67 2.50±0.42 2.50±0.38 0.857 8w Average egg weight / g <![CDATA[68.14±3.47 a ]]> <![CDATA[66.69±1.54 ab ]]> <![CDATA[63.78±2.50 b ]]> <![CDATA[64.84±2.23 ab ]]> 0.034 Egg production rate 0.95±0.07 0.88±0.13 0.88±0.14 0.82±0.10 0.306 Average daily feed intake 141.89±6.12 145.83±0.00 140.57±7.31 140.78±6.42 0.378 Egg ratio 2.22±0.22 2.55±0.43 2.56±0.31 2.70±0.45 0.171 10w Average egg weight / g <![CDATA[68.29±1.46 a ]]> <![CDATA[64.06±3.31 b ]]> <![CDATA[66.12±0.74 ab ]]> <![CDATA[63.83±2.09 b ]]> 0.005 Egg production rate 0.92±0.08 0.85±0.15 0.83±0.08 0.86±0.09 0.514 Average daily feed intake 140.29±7.66 145.72±7.42 141.50±5.51 140.90±8.79 0.587 Egg ratio 2.26±0.29 2.74±0.43 2.58±0.15 2.59±0.30 0.084 12w Average egg weight / g <![CDATA[69.76±1.28 a ]]> <![CDATA[67.74±1.58 a ]]> <![CDATA[64.74±0.85 b ]]> <![CDATA[64.07±2.95 b ]]> 0.0001 Egg production rate 0.85±0.10 0.82±0.08 0.83±0.14 0.75±0.16 0.486 Average daily feed intake 129.29±10.89 124.40±6.47 125.45±14.14 121.04±10.94 0.636 Egg ratio 2.20±0.18 2.27±0.25 2.34±0.16 2.64±0.59 0.168 Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05).

[0038] Experiment Example 2: Verification of the Effects of Low-Protein Diets Based on Rice Wine Fermentation on Egg Quality in Ducks Based on the above grouping and feeding conditions, egg quality was determined as follows: At the end of the 12th week of the formal experiment, 10 fresh duck eggs were randomly selected from each replicate for egg quality determination. The determination parameters included egg shape index, shell thickness, shell strength, yolk color, and Haugh units. The egg shape index was calculated based on the egg length and width; shell thickness was measured at three sites (blunt end, middle, and acute end) and the average value was taken; yolk color was determined using a Roche colorimetric fan; and shell strength and Haugh units were determined using an egg quality analyzer.

[0039] The effects of different crude protein levels in rice wine lees diets on the quality of duck eggs are shown in Table 3. It can be seen that there were no significant differences among the groups in egg shape index, shell thickness, shell strength, yolk color, and Haugh units (P>0.05), indicating that different crude protein levels in rice wine lees diets did not have a significant impact on the quality of duck eggs, suggesting that constructing a low-protein diet using rice wine lees is feasible for maintaining the quality of duck eggs.

[0040] Table 3. Effects of different crude protein levels in rice wine lees diets on egg quality in laying ducks. Egg-shaped index 1.36±0.01 1.38±0.03 1.36±0.02 1.37±0.02 0.353 Eggshell thickness 0.46±0.04 0.46±0.05 0.46±0.04 0.45±0.05 0.644 Eggshell strength 4.35±0.96 4.06±1.02 4.16±1.12 4.02±1.02 0.604 Egg yolk color 5.77±1.50 5.07±1.28 5.10±0.92 5.13±1.33 0.110 Huff unit 69.14±10.60 70.82±12.80 62.22±15.71 64.50±16.10 0.067 Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05).

[0041] Experiment Example 3: Verification of the Effects of Low-Protein Diet Based on Rice Wine Fermentation Grains on Blood Biochemical Indicators of Laying Ducks Based on the above grouping and feeding conditions, the blood biochemical indicators of laying ducks were tested, as follows: At the end of week 12 of the formal experiment, two laying ducks close to the average weight of each replicate were selected. After fasting for 12 hours, blood was collected via the subwing vein. Blood samples were allowed to stand at room temperature, then centrifuged at 3000 r / min for 15 min to separate the serum, which was then stored at -80℃ for later analysis. The measured parameters included total protein, albumin, globulin, uric acid, blood urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase activity. Each parameter was measured according to the instructions of the corresponding kit.

[0042] The effects of different crude protein levels in a rice wine lees diet on the blood biochemical indicators of laying ducks are shown in Table 4. It can be seen that, in terms of protein metabolism, there were no significant differences in serum total protein, albumin, globulin, and urea nitrogen content among the groups (P>0.05), indicating that the low-protein rice wine lees diet did not have a significant adverse effect on protein metabolic homeostasis and liver function in laying ducks. Uric acid (Ua) levels were significantly higher in the CK group than in the HH group (P<0.05), while the LH and MH groups were in the middle range and showed no significant difference from the CK and HH groups. Liver function indicators (AST, ALT, ALP) remained stable among the groups without significant changes, indicating that the amount of rice wine lees added was controlled within a low range and did not significantly interfere with liver metabolic function. Meanwhile, serum uric acid is the main end product of avian protein catabolism, and its level can indirectly reflect the amount of nitrogenous waste generated and the overall nitrogen excretion level. Therefore, the significantly lower serum uric acid levels in the HH and LH groups indicate that the low-protein rice wine lees diet of this invention has the potential to reduce nitrogen excretion.

[0043] Table 4. Effects of different crude protein levels in rice wine lees diets on blood biochemical parameters of laying ducks. TP 66.44±7.35 64.57±9.12 63.17±8.76 63.81±13.14 0.849 ALB 16.71±2.19 17.29±2.18 16.73±2.37 15.04±1.37 0.059 GLB 49.74±5.43 47.27±7.14 46.44±6.65 48.77±11.60 0.751 UREA 0.74±0.50 1.05±1.26 0.56±1.21 0.28±0.16 0.232 Ua <![CDATA[356.02±139.68 a ]]> <![CDATA[279.62±92.98 ab ]]> <![CDATA[292.71±41.72 ab ]]> <![CDATA[237.64±99.87 b ]]> 0.046 AST 82.79±21.51 70.93±19.53 70.65±16.05 61.47±19.30 0.073 ALT 90.15±32.08 67.31±33.87 88.13±38.52 78.52±24.94 0.311 ALP 304.46±257.12 238.97±121.06 251.51±98.13 226.72±106.76 0.649 Note: TP: Total protein (g / L); ALB: Albumin (g / L); GLB: Globulin (g / L); UREA: Blood urea nitrogen (mmol / L); Ua: Uric acid (μmol / L); AST: Aspartate aminotransferase (U / L); ALT: Alanine aminotransferase (U / L); ALP: Alkaline phosphatase (U / L). Different letters in the superscript of the same data indicate significant differences (P<0.05).

[0044] Experiment Example 4: Effects of a low-protein diet made from rice wine lees on the intestinal metabolome of laying ducks. Based on the above grouping and feeding conditions, the effects of different crude protein levels in rice wine lees diets on the intestinal metabolome of laying ducks were investigated, including the following: 1. OPLS-DA Analysis To analyze the effects of a low-protein diet based on rice wine lees on the intestinal metabolic characteristics of laying ducks, a non-targeted metabolomics analysis was performed on jejunal contents. Specifically, at the end of week 12 of the formal experiment, one laying duck close to the average weight of that replicate was randomly selected from each replicate, and jejunal contents were collected. Immediately after collection, the samples were flash-frozen in liquid nitrogen and then stored at -80 °C. A suitable amount of jejunal contents was taken and metabolites were extracted using a methanol-acetonitrile-water mixture. After vortexing, low-temperature sonication, and centrifugation, the supernatant was collected for subsequent UPLC-MS / MS analysis.

[0045] The OPLS-DA score plots and displacement test plots for each group under positive and negative ion modes are shown in the following figures. Figure 1 and Figure 2As shown in the figure, the CK group showed varying degrees of separation from the LH, MH, and HH groups, indicating that different crude protein levels in the distillers' grains diet can alter the intestinal metabolic profile of laying ducks. With decreasing crude protein levels, the separation between the treatment groups and the CK group gradually increased, with the MH and HH groups showing more significant differences from the CK group. The permutation test results showed that the model was stable and reliable, suggesting that this model can be used for subsequent differential metabolite screening.

[0046] 2. Differential metabolite screening analysis Differential metabolite analysis was performed on each group under both positive and negative ion modes. Non-targeted metabolomics analysis was conducted using a UPLC-MS / MS platform with a Waters ACQUITY UPLC BEH C18 column and an electrospray ionization source for mass spectrometry. Data were acquired in both positive and negative ion modes. Quality control samples were included during the analysis to evaluate instrument stability and data repeatability.

[0047] The quantitative analysis results of differential metabolites in each group under positive and negative ion modes are shown in the figure. Figure 3 As shown in the figure, a certain number of differentially expressed metabolites were screened in each comparison group, with both up-regulated and down-regulated metabolites present. Under positive ion mode, 76, 196, and 310 differentially expressed metabolites were screened in the CK vs LH, CK vs MH, and CK vs HH groups, respectively. Among these, 48, 68, and 54 metabolites were up-regulated, and 28, 128, and 256 metabolites were down-regulated, respectively. Under negative ion mode, 81, 283, and 479 differentially expressed metabolites were screened in the same three comparison groups, respectively. Among these, 42, 32, and 33 metabolites were up-regulated, and 39, 251, and 446 metabolites were down-regulated, respectively. Overall, the number of differentially expressed metabolites gradually increased with increasing levels of rice wine lees, with down-regulated metabolites being the predominant type, especially in the HH group where the changes were most pronounced. This suggests that a high-level rice wine lees-based, low-protein diet has a stronger impact on the intestinal metabolic network.

[0048] 3. Enrichment analysis of differentially metabolized metabolites via the KEGG pathway Differential metabolite enrichment analysis was performed on each group. After peak identification, peak alignment, peak area normalization, and quality control of the raw data, metabolite annotation was performed using databases such as HMDB and METLIN. PCA was used to analyze the overall sample distribution and QC sample aggregation, and OPLS-DA was used to analyze the differences in metabolomics profiles between different treatment groups. VIP > 1 and P < 0.05 were used as the screening criteria for differential metabolites, and KEGG pathway enrichment analysis was performed on the differential metabolites.

[0049] The results of differential enrichment analysis of substances in each group under positive and negative ion modes are shown below. Figure 4 , Figure 5As shown, under positive ion mode, the differential metabolites in the CK vs LH group were mainly enriched in pathways such as riboflavin metabolism, primary bile acid biosynthesis, fatty acid biosynthesis, unsaturated fatty acid biosynthesis, and ABC transporters; the CK vs MH group was mainly enriched in pathways such as biotin metabolism, primary bile acid biosynthesis, tyrosine metabolism, α-linolenic acid metabolism, histidine metabolism, glycine / serine / threonine metabolism, and arginine and proline metabolism; the CK vs HH group was further enriched in pathways such as α-linolenic acid metabolism, primary bile acid biosynthesis, cofactor biosynthesis, riboflavin metabolism, biotin metabolism, mTOR signaling pathway, FoxO signaling pathway, ABC transporters, programmed necrosis, arginine and proline metabolism, purine metabolism, sphingolipid metabolism, and glutathione metabolism. In negative ion mode, the differential metabolites of the CK VS LH and CK VS MH groups were mainly enriched in ABC transporters and various sugar, amino acid and nucleotide metabolism pathways, including starch and sucrose metabolism, fructose and mannose metabolism, pyrimidine metabolism, purine metabolism, arginine and proline metabolism, arachidonic acid metabolism and pantothenic acid and coenzyme A biosynthesis, etc.; while in addition to ABC transporters, the CK VS HH group was also significantly enriched in terpene skeleton biosynthesis, pentose phosphate pathway, oxidative phosphorylation, glycolysis / gluconeogenesis, carbon metabolism, porphyrin metabolism and 2-oxocarboxylic acid metabolism pathways.

[0050] In summary, treatment with a low-protein diet containing fermented rice wine lees significantly altered the intestinal metabolic profile of laying ducks. The CK group and all low-protein groups showed varying degrees of separation in OPLS-DA analysis. Furthermore, the number of differentially expressed metabolites gradually increased with decreasing CP levels, primarily downregulated metabolites, with the most significant changes observed in the HH group. KEGG enrichment analysis revealed that differentially expressed metabolites were mainly enriched in pathways involving arginine and proline metabolism, histidine metabolism, glycine / serine / threonine metabolism, purine metabolism, ABC transporters, primary bile acid biosynthesis, α-linolenic acid metabolism, sphingolipid metabolism, and glutathione metabolism. This indicates that the low-protein diet not only affects amino acid and nitrogenous metabolism in the laying duck's intestines but also participates in regulating lipid metabolism, substance transport, and redox homeostasis. This may be because a decrease in dietary CP levels reduces the amount of protein and nitrogenous substrates entering the intestines, leading to a decrease in amino acid catabolism and purine metabolite formation. Simultaneously, the presence of dietary fiber, phenolic substances, and fermentation-active components in fermented rice wine lees may further influence bile acid metabolism, membrane lipid metabolism, and the formation of microbial metabolites. In summary, low-protein diets based on rice wine lees can reshape the intestinal metabolic network of laying ducks. The metabolic changes in the 15.5% CP group were relatively mild, while the metabolic disturbances in the 14.5% and 13.5% CP groups were more severe, suggesting that excessive reduction of dietary protein levels may affect intestinal metabolic homeostasis.

[0051] Experimental Example 5: Analysis of the Effects of Low-Protein Diet Based on Rice Wine Fermentation Grains on Cecal Microbiota in Laying Ducks 1. Alpha diversity analysis of cecal microbiota At the end of week 12 of the formal experiment, one laying duck with a weight close to the average weight of that replicate was randomly selected from each replicate, and cecal contents were collected. Immediately after collection, the samples were flash-frozen in liquid nitrogen and stored at -80°C. Total DNA was extracted from the cecal contents using a microbial genomic DNA extraction kit. After the DNA concentration and purity were verified to be within acceptable limits, it was used for subsequent PCR amplification.

[0052] The analysis results of Alpha diversity analysis of cecal microorganisms in each group of laying ducks are as follows: Figure 6 As shown in the figure, there were no significant differences in Chao1, Goods coverage, Simpson, and Shannon indices between the CK and LH groups. There were also no significant differences in any of the alpha diversity indices between the CK and MH groups. Compared with the HH group, the CK group showed a significantly lower Chao1 index (P=0.025) and a significantly higher Goods coverage (P=0.0094), while the Simpson and Shannon indices showed no significant differences (P>0.05). These results indicate that the low-protein diet of rice wine lees had a relatively small impact on cecal microbiota alpha diversity in the LH and MH groups, while the HH group had a more significant impact on microbiota richness and sequencing coverage.

[0053] 2. Analysis of Beta diversity of cecal microbiota PCR amplification was performed on the V3-V4 region of the 16S rRNA gene using primers 338F and 806R. After purification, quantification, and library construction, the amplified products were sequenced using the Illumina MiSeq platform. The sequencing data underwent quality filtering, noise reduction, and chimera removal to obtain representative ASV sequences, which were then annotated based on the Silva database.

[0054] The PCoA analysis results of the cecal flora of each group of laying ducks are shown below. Figure 7 As shown in the figure. PCoA results showed that the explanation rates for the first two principal axes of the CK and LH groups were 22.1% and 12.3%, respectively, indicating some overlap between the two groups; the explanation rates for the first two principal axes of the CK and MH groups were 22.4% and 10.8%, respectively, showing a certain trend of separation between the groups; and the explanation rates for the first two principal axes of the CK and HH groups were 22.5% and 16.3%, respectively, with further enhanced separation. These results indicate that a low-protein diet based on rice wine lees can alter the cecal microbiota structure of laying ducks, and the difference between the community structure and the control group gradually increases with increasing treatment levels.

[0055] 3. LEfSe analysis of cecal microbiota QIIME 2 was used to analyze cecal microbiota alpha diversity, including Chao1, Shannon, Simpson, and Goodscoverage indices; PCoA was used to analyze beta diversity; and LEfSe analysis was used to screen for differentially expressed microbiota among groups, with the LDA score threshold set at 4.0.

[0056] To screen for differentially significant marker bacteria between different treatment groups and the control group, LEfSe was used to analyze the CK group, LH group, MH group, and HH group. The LEfSe LDA analysis results of the cecal flora of laying ducks are as follows: Figure 8 As shown. In the comparison between the CK and LH groups, the differentially enriched bacterial communities in the CK group mainly included *Fimimorpha* and *Erysipelatoclostridium*, while the LH group mainly enriched *Spirochaetia*, *Treponemataceae*, *Sphaerochaetaceae*, *Christensenellales*, and *Ornithospirochaeta*. In the comparison between the CK and MH groups, the differentially enriched bacterial communities in the CK group were still mainly *Negativicutes*, *Firmicutes C*, *Acidaminococcales*, *Lachnospiraceae*, and *Phascolarctobacterium A*, while the MH group mainly enriched *Bacteroides H massiliensis*. In the comparison between the CK and HH groups, the CK group mainly enriched *Firmicutes C*, *Acidaminococcales*, *Phascolarctobacterium A*, and *Phocaeicola A*, while the HH group mainly enriched *Ornithinicoccus A*. The results showed that low-protein diets could significantly alter the composition of differentially expressed marker bacteria in the cecal flora of laying ducks, and different treatment levels formed enrichment patterns of their respective characteristic bacterial communities.

[0057] In summary, the low-protein diet based on rice wine lees did not alter the dominant phylum structure of the cecal microbiota in laying ducks, which was dominated by Bacteroidota and Firmicutes_A, but it significantly affected the relative abundance of different phyla and characteristic bacterial groups. Specifically, the Chao1 index was significantly reduced in the HH group, and PCoA analysis showed varying degrees of segregation between the low-protein treatment group and the CK group, indicating that a high-level low-protein diet based on rice wine lees may reduce cecal microbiota richness and cause community structure shift. LEfSe analysis showed that different treatment groups developed their own differential marker bacteria. This may be because the reduced dietary CP level decreased the amount of protein and nitrogenous substrates entering the cecum, altering the available nutrient substrates for microorganisms; simultaneously, rice wine lees contain a certain amount of dietary fiber and fermentation-active substances, which can serve as fermentation substrates for regulating cecal microbiota composition. In summary, the 15.5% CP group had a relatively small impact on the cecal microbiota, while the 14.5% and 13.5% CP groups showed more significant changes in microbiota structure. This suggests that moderately reducing the protein level in a low-protein diet based on rice wine lees is beneficial for maintaining the stability of the cecal microecology, while excessively reducing the CP level may lead to simplification of the microbiota structure and changes in intestinal microecological homeostasis.

[0058] In summary, under the condition of supplementing crystalline amino acids and maintaining a basically consistent level of major nutrients, replacing part of the soybean meal with rice wine lees to construct a low-protein diet, reducing the crude protein level of the laying duck diet from 16.5% to 15.5%, did not have significant adverse effects on average daily feed intake, laying rate, feed conversion ratio, major egg quality indicators, and serum liver function-related enzyme activity. Furthermore, the impact on intestinal metabolic profile and cecal microbiota structure was relatively small. At the same time, moderately reducing the dietary crude protein level helps reduce the formation of nitrogenous metabolites and has the potential to reduce nitrogen emissions.

[0059] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A low-protein diet made from rice wine lees, characterized in that, It is composed of the following raw materials in parts by weight: corn 50-52 parts, soybean meal 18-20 parts, brown rice 6-7 parts, wheat bran 7-8 parts, rice wine lees 1.5-2.5 parts, soybean oil 0.05-0.15 parts, dicalcium phosphate 1.0-1.2 parts, limestone powder 8.5-9.0 parts, sodium chloride 0.15-0.25 parts, DL-methionine 0.15-0.20 parts, L-lysine sulfate 0.15-0.20 parts. 0.25 parts, L-threonine 0.03~0.08 parts, tryptophan 0.03~0.05 parts, isoleucine 0.04~0.08 parts and L-arginine 0.10~0.15 parts, potassium magnesium sulfate 1.0~1.5 parts, choline chloride 0.05~0.15 parts, phytase 0.02~0.05 parts, broiler multivitamins 0.05~0.15 parts and organic multiminerals 0.05~0.15 parts.

2. The low-protein diet made from rice wine lees according to claim 1, characterized in that, The diet consists of the following ingredients in parts by weight: 51.66 parts corn, 19.54 parts soybean meal, 6.64 parts brown rice, 7.69 parts wheat bran, 1.99 parts rice wine lees, 0.1 parts soybean oil, 1.12 parts dicalcium phosphate, 8.87 parts limestone powder, 0.2 parts sodium chloride, 0.18 parts DL-methionine, 0.21 parts L-lysine sulfate, 0.05 parts L-threonine, 0.04 parts tryptophan, 0.06 parts isoleucine, 0.12 parts L-arginine, 1.2 parts potassium magnesium sulfate, 0.1 parts choline chloride, 0.03 parts phytase, 0.1 parts broiler multivitamins, and 0.1 parts organic minerals; the crude protein content of the diet is 15.5%.

3. The low-protein diet made from rice wine lees according to claim 1 or 2, characterized in that, The crude protein content of the diet is 15.5%.

4. The application of the low-protein diet made from rice wine lees as described in any one of claims 1 to 3 in improving the egg production rate and / or egg quality of laying ducks.

5. The application according to claim 4, characterized in that, The application is to use the diet to feed laying ducks of the Qingke No. 4 variety.

6. The application according to claim 4, characterized in that, Improving egg quality includes increasing at least one of the following: Haugh unit, eggshell strength, yolk color, or reducing egg breakage rate.

7. The application according to claim 4, characterized in that, The applications also include reducing serum uric acid levels in laying ducks, improving intestinal amino acid metabolism, and maintaining the stability of the cecal flora.

8. The use of the low-protein diet made from rice wine lees as described in any one of claims 1 to 3 in reducing nitrogen excretion and / or reducing serum uric acid levels in laying ducks.

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