Application of ocean-derived enterococcus durans YQ-6 in improving growth performance, slaughter performance and muscle quality of Leizhou black ducks

Abstract

The invention discloses application of ocean-derived enterococcus durans YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks, and belongs to the technical field of poultry breeding. Experiments prove that the marine-derived enterococcus durans YQ-6 can improve the growth performance and muscle quality of the Leizhou black ducks, enhance the immune function and oxidation resistance of organisms, improve the intestinal flora structure of the Leizhou black ducks and improve the intestinal health, and the effect is optimal when 1 * 10 < 9 > CFU / kg of the marine-derived enterococcus durans YQ-6 is added into feed. The feed disclosed by the invention complies with the development trend of'feed resistance-free ', and has important significance on green, safe and sustainable development of the breeding industry.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to application of marine-derived durable enterococci YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks. Background Art

[0002] The Leizhou Black Duck, with its long history of breeding, thrives naturally in the relatively enclosed tropical to subtropical coastal mudflats and represents a valuable livestock and poultry genetic resource for the Leizhou Peninsula. As a distinctively local black-feathered breed, the Leizhou Black Duck offers both high-quality egg production and excellent meat quality, renowned for its tender meat, delicious flavor, high egg production, and excellent adaptability to roughage. Research has shown that the muscle of the Leizhou Black Duck is rich in crude protein and crude fat, exhibiting a unique flavor and superior taste in terms of tenderness, color, and fat content, demonstrating significant advantages over Muscovy, Cherry Valley, and Gaoyou Ducks.

[0003] Currently, Leizhou Black Ducks are raised in a combination of captive breeding and tidal flat farming. While this approach has its unique advantages, it also leads to long breeding cycles, low feed efficiency, unstable duck health, and the frequent use of antibiotics. With the growing market demand for healthy and safe duck products, the development of new probiotics and feed additives that can replace antibiotics is becoming increasingly important. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of marine-derived durable enterococci YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the application of marine-derived durable enterococci YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks.

[0007] The second technical solution of the present invention is the use of marine-derived Enterococcus YQ-6 in the preparation of products that improve the antioxidant capacity and enhance the immune function of Leizhou black ducks.

[0008] The third technical solution of the present invention is the use of marine-derived durable enterococci YQ-6 in the preparation of products that promote the intestinal health of Leizhou black ducks.

[0009] The fourth technical solution of the present invention is a feed additive for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, wherein the feed additive includes marine-derived durable enterococci YQ-6.

[0010] The fifth technical solution of the present invention is a feed for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, wherein the feed includes marine-derived durable enterococci YQ-6 or the feed additive.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The present invention has confirmed through experiments that marine-derived persistent enterococci YQ-6 promotes the body's serum immunoglobulin and complement content, regulates the body's cytokine gene expression level, thereby maintaining a dynamic balance of immune function, improving the immune capacity of Leizhou black ducks, and by enhancing the body's antioxidant function, thereby improving its muscle quality, muscle nutritional value and flavor level. At the same time, marine-derived persistent enterococci YQ-6 can regulate the composition and stability of intestinal flora, increase the relative abundance of beneficial flora, promote intestinal villi development, improve intestinal tissue structure, and thus maintain intestinal health and improve the growth performance of Leizhou black ducks. The addition amount of marine-derived persistent enterococci YQ-6 to feed is 1×10 9 The present invention conforms to the development trend of "antibiotic-free feed" and has important significance for the green, safe and sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effects of marine-derived Enterococcus duracil YQ-6 on the antioxidant function of 21-day-old Leizhou black ducks. A. CAT activity; B. SOD activity; C. GSH-PX activity; D. MDA activity. *P < 0.05, **P < 0.01 compared with the control group. Same as below.

[0014] Figure 2 The effect of marine-derived Enterococcus durabilis YQ-6 on the complement and immunoglobulin levels of Leizhou black ducks: A. C3 content; B. C4 content; C. IgG content.

[0015] Figure 3 The immune function of marine-derived Enterococcus YQ-6 on the serum of Leizhou black ducks. Among them, A. IL-2 content; B. IL-4 content; C. IL-6 content; D. IFN-γ content; E. TNF-α content.

[0016] Figure 4 The effects of marine-derived Enterococcus durophilus YQ-6 on cytokine expression in the immune organs of Leizhou black ducks: A. cytokine expression in the thymus; B. cytokine expression in the spleen; C. cytokine expression in the bursa of Fabricius.

[0017] Figure 5The expression levels of CAT, SOD, and GSH-Px mRNA in the immune organs of Leizhou black ducks induced by marine-derived Enterococcus duracil YQ-6 are shown in Table 1. A. Cytokine expression levels in the thymus; B. Cytokine expression levels in the spleen; C. Cytokine expression levels in the bursa of Fabricius.

[0018] Figure 6 This is the histomorphology of the duodenum, jejunum and ileum of Leizhou black duck (100×).

[0019] Figure 7 This is a Venn diagram of the cecal microbiome of Leizhou black ducks. Note: Each color block in the diagram represents a group. The overlapping areas between blocks indicate the ASVs shared by the corresponding groups. The number in each block indicates the number of ASVs contained in that block.

[0020] Figure 8 Alpha diversity analysis of cecal microorganisms in Leizhou black ducks. A is the Shannon index and B is the Chao1 index.

[0021] Figure 9 Beta diversity analysis of cecal microorganisms in Leizhou black ducks.

[0022] Figure 10 This is the taxonomic level bacterial community structure and richness of the cecum of Leizhou black duck.

[0023] Figure 11 This is the taxonomic level bacterial community structure and richness of the cecum of Leizhou black duck.

[0024] Figure 12 This is a multi-level species difference discriminant analysis of LEfSe in the cecum of Leizhou black duck. DETAILED DESCRIPTION

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0027] The embodiments of the present invention provide the use of marine-derived Enterococcus durabilis YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks.

[0028] In some specific embodiments, the slaughter performance is: improving carcass rate, half-eviscerated rate, full-eviscerated rate, breast muscle rate and leg muscle rate.

[0029] In some specific embodiments, the growth performance is: increasing average body weight and average daily gain, and reducing feed-to-gain ratio.

[0030] In some specific embodiments, the muscle quality is: reducing shear force and b-value of breast muscle, optimizing the amino acid and fatty acid composition in muscle, and increasing the content of aspartic acid, alanine and phenylalanine in muscle.

[0031] The embodiment of the present invention also provides the use of marine-derived Enterococcus durophilus YQ-6 in preparing a product for improving the antioxidant capacity and enhancing the immune function of Leizhou black ducks.

[0032] In some specific embodiments, the improvement of the antioxidant capacity and enhancement of the immune function of Leizhou black ducks is as follows: increasing the activities of SOD, CAT and GSH-Px in the serum of Leizhou black ducks, reducing the MDA content, increasing the levels of complement C3, C4 and immunoglobulin IgG in the serum of Leizhou black ducks, and regulating the expression levels of IFN-γ, IL-2, IL-4, IL-6 and TNF-α mRNA in the immune organ tissues of Leizhou black ducks.

[0033] The embodiments of the present invention also provide the use of marine-derived durable enterococci YQ-6 in the preparation of products that promote the intestinal health of Leizhou black ducks.

[0034] In some specific embodiments, the promoting of intestinal health of Leizhou black ducks is: increasing the height of villi, reducing the depth of crypts, reducing the villus-crypt ratio of duodenum and ileum, improving intestinal digestion and absorption capacity, maintaining the stability of intestinal flora of Leizhou black ducks and regulating the composition structure of flora;

[0035] At the phylum level, the relative abundance of Cyanobacteria increased, and the relative abundance of Ferrobacteria decreased; at the genus level, the relative abundance of Parabacteroides increased.

[0036] The embodiment of the present invention also provides a feed additive for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, wherein the feed additive includes marine-derived durable enterococci YQ-6.

[0037] The embodiment of the present invention also provides a feed for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, wherein the feed comprises marine-derived durable enterococci YQ-6 or the feed additive.

[0038] The present invention has confirmed through experiments that marine-derived Enterococcus YQ-6 can improve the growth performance and muscle quality of Leizhou black ducks, enhance the body's immune function and antioxidant capacity, improve the intestinal flora structure of Leizhou black ducks, and improve intestinal health. 9 The best effect is achieved when the CFU / kg

[0039] Example 1

[0040] Effects of marine-derived Enterococcus YQ-6 on growth performance, slaughter performance and meat quality of Leizhou black ducks

[0041] 1.1 Test strains

[0042] The marine-derived Enterococcus durans strain YQ-6 used in this study was isolated from small yellow croaker (Pseudosciaena spp.) in the South China Sea. This strain was deposited with the Guangdong Provincial Microbial Culture Collection on May 7, 2025, at Building 59, 5th Floor, 100 Xianlie Middle Road, Guangzhou, with the GDMCC No. 66265.

[0043] Lactobacillus acidophilus (BNCC185342) was purchased from Beina Biotechnology Co., Ltd.

[0044] Marine-derived Enterococcus duracil YQ-6 was inoculated into a culture medium (1% yeast extract, 2% glucose, 2% peptone, and 2% sodium chloride) and incubated at 37°C at 120 rpm for 48 hours. The culture was collected and centrifuged (conditions: 4°C, 10,000 rpm, 10 minutes), the supernatant discarded, and the suspension washed twice with sterile saline to prepare a bacterial suspension. The viable count of the suspension was calculated using the dilution plate method and adjusted to M1:1×10 based on experimental needs. 7 CFU / kg, M2: 1×10 8 CFU / kg, M3: 1×10 9 CFU / kg.

[0045] Lactobacillus acidophilus was inoculated into MRS culture medium and cultured anaerobically at 37°C, 180 rpm, and shaker for 24 hours. The culture medium was collected and centrifuged (conditions: 4°C, 8000 rpm, 10 minutes). The supernatant was discarded and the suspension was resuspended and washed twice with sterile saline to prepare a bacterial suspension. The viable count was calculated by the dilution plate method and finally adjusted to La: 1×10 according to the experimental needs. 9 CFU / kg.

[0046] 1.2 Test methods

[0047] 1.2.1 Experimental animals and group design

[0048] The experiment involved 180 one-day-old male Leizhou black ducks of similar weight and good health. They were randomly divided into five groups, each with six replicates, and six Leizhou black ducks per replicate. The experimental period was 42 days. The experimental groups are shown in Table 1:

[0049] Table 1 Experimental groups

[0050]

[0051] 1.2.2 Feeding management and basic feed

[0052] Before the experiment, the duck house and related equipment were thoroughly cleaned and disinfected, followed by ventilation and air purification to ensure the suitability of the experimental environment. During the experiment, the Leizhou black ducks were housed indoors on a gridded flat surface, fed with mixed feed, and had free access to food and water. Temperature, humidity, and lighting conditions were strictly controlled within the duck house. To ensure environmental hygiene and air quality, regular cleaning and good ventilation were maintained. Furthermore, the health of the Leizhou black ducks was closely monitored, with drinking water and feed quality regularly checked and detailed records kept. Feeding and vaccination procedures were carried out according to standard husbandry procedures. The basic feed was prepared according to the "Standard for Meat Duck Feeding" (NY / T 2122-2012). Its composition and nutrient levels are shown in Table 2.

[0053] Table 2 Feed composition and nutrient levels (air-dried basis) %

[0054]

[0055] Note: The premix provides per kilogram of feed: Vitamin A 8000IU, Vitamin B 12 mg, Vitamin D 2000IU, Vitamin E 20IU, Vitamin K 32 mg, Niacin 40 mg, D-pantothenic acid 15 mg, Folic acid 1 mg, Iron 60 mg, Copper 8 mg, Zinc 60 mg, Manganese 100 mg, Selenium 0.35 mg.

[0056] 1.2.3 Growth performance

[0057] On the mornings of days 1, 7, 14, 21, 28, 35, and 42 of the formal experiment, the empty-stomach Leizhou black ducks were weighed and recorded, and their feed consumption was weighed daily. Average body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (FCR) were calculated.

[0058] BW (g) = total weight / number of ducks;

[0059] ADFI (g / d) = material consumption during the test period / number of test days;

[0060] ADG (g / d) = (average weight at the end of the test period - average weight at the beginning of the test period) / number of test days;

[0061] FCR=ADFI / ADG.

[0062] 1.2.4 Slaughter performance

[0063] After fasting for 12 hours in advance, two Leizhou black ducks of similar weight and health were selected from each replicate group on day 42 of the experiment and weighed before slaughter. The ducks were then bled from the jugular vein, carcass weight was measured, and the breast and leg muscles were dissected and collected from both sides. Dressing percentage, evisceration percentage, evisceration percentage, breast muscle percentage, leg muscle percentage, and lean meat percentage were measured and calculated according to the agricultural industry standard (NY / T 823-202).

[0064] Slaughter rate (%) = carcass weight / weight before slaughter × 100;

[0065] Eviscerated rate (%) = eviscerated weight / body weight before slaughter × 100;

[0066] Eviscerated rate (%) = eviscerated weight / body weight before slaughter × 100;

[0067] Leg muscle rate (%) = weight of leg muscles on both sides / total eviscerated weight × 100;

[0068] Chest muscle rate (%) = weight of chest muscles on both sides / total eviscerated weight × 100;

[0069] Lean meat rate (%) = (weight of chest muscles on both sides + weight of leg muscles on both sides) / total eviscerated weight × 100.

[0070] 1.2.5 Determination of meat quality indicators

[0071] The breast and leg muscles on the left side of 42-day-old Leizhou black ducks were selected and the pH, meat color, water loss rate, cooking loss rate, and shear force were measured. The test indicators are as follows:

[0072] (1) Meat color: Place the colorimeter probe vertically on the cross section of the muscle to measure the brightness value (L*), redness value (a*), and yellowness value (b*). Record the values ​​when they are stable and repeat three times.

[0073] (2) pH 45min : 45-60 minutes after slaughter, three different parts of the breast muscle and leg muscle were taken, and the pH value was measured by inserting the pH meter probe into the meat sample. After the value stabilized, it was recorded. The average value of the three parts was taken as the final pH value. 45min value.

[0074] (3) pH 24h : Store the meat sample in a 4℃ refrigerator and take it out 24 hours after slaughter to measure its pH 24h Numerical value, determination method is the same as pH 45min .

[0075] (4) Water loss rate: Use a sharp circular sampler to cut two pieces of meat samples (thickness 1 cm, diameter about 2.5 cm) from the chest muscle and leg muscle, weigh them and record them as m1, place the meat samples on a pressure table and cover them with 16 layers of qualitative filter paper on the top and bottom, apply 35 kg of pressure for 5 minutes, and weigh the weight after the pressure is completed and record it as m2.

[0076] (5) Water loss rate (%) = (m 1- m2) / m1×100%.

[0077] (6) Cooking loss: The breast and leg muscles were stored at 4°C until 24 h after slaughter, and the cooking loss was measured. The meat samples were trimmed into rectangular shapes along the muscle fiber direction and weighed as m3. The meat samples were placed in a ziplock bag and heated in an 80°C water bath until the core temperature of the meat sample reached 75°C. After the meat samples cooled to room temperature, the surface moisture was absorbed with filter paper and weighed as m4.

[0078] Cooking loss (%) = (m3-m4) / m3×100%

[0079] (7) Shear force: The shear force of the cooked meat sample was measured using a meat tenderizer. Each meat sample was measured twice and the average value was calculated.

[0080] 1.2.6 Determination of conventional nutrient content in muscle

[0081] After removing the surface fat and fascia of the breast and leg muscles, the nutritional components of the breast and leg muscles of 42-day-old Leizhou black ducks were determined: water, crude ash, crude fat, and crude protein.

[0082] (1) The moisture content was determined by the direct drying method in accordance with the national standard GB / 5009.3-2016;

[0083] (2) Crude fat content was determined by Soxhlet extraction method according to national standard GB / 5009.6-2016;

[0084] (3) Crude protein was determined by the Kjeldahl method in accordance with the national standard GB / 5009.5-2016;

[0085] (4) The crude ash content is determined according to the national standard GB / 5009.4-2016.

[0086] 1.2.7 Determination of fatty acid and amino acid content in muscle

[0087] Muscle tissue from 42-day-old Leizhou black ducks was minced and then analyzed for fatty acid and amino acid content. The amino acid determination method for this experiment was based on the national standard GB 5009.124-2016. An automatic amino acid analyzer was used to measure muscle amino acid content. The retention time and peak area of ​​amino acid standards were compared to determine the content of each amino acid in the muscle.

[0088] Fatty acid determination was performed in accordance with the national standard GB 5009.168-2016. Meat samples were hydrolyzed and fat extracted, followed by saponification and methylation to produce fatty acid methyl esters (FAMs). The percentage of each fatty acid was calculated using peak area normalization, using gas chromatography and the relative retention times of FAME standards.

[0089] 1.2.8 Data Analysis

[0090] SPSS 26.0 statistical software was used for one-way analysis of variance, and Duncan's multiple comparisons were performed. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. The results were expressed as mean ± standard deviation, and GraphPad Prism 8 software was used for graphics.

[0091] 1.2 Experimental Results

[0092] 1.2.1 Effects of marine-derived Enterococcus YQ-6 on the growth performance of Leizhou black ducks

[0093] As shown in Table 3, at different developmental time points (14, 21, 28, 35, and 42 days), the average body weight of each treatment group (M1, M2, M3, and LA) was significantly higher than that of the control group. In particular, at 35 and 42 days, the average body weight of the M3 group was significantly higher than that of the other groups (P < 0.05). The average daily feed intake of each treatment group did not differ significantly from that of the control group (P > 0.05). The average daily weight gain of each treatment group was generally higher than that of the control group, especially at 21-28 days and 28-35 days, when it was significantly higher than that of the other groups (P < 0.05). At 7-14 days and 21-28 days, the feed-to-gain ratio of the M3 group was significantly lower than that of the control group and the other treatment groups (P < 0.05). At other time points, the feed-to-gain ratio of each treatment group did not differ significantly from that of the control group (P > 0.05).

[0094] Table 3 Effects of marine-derived persistent enterococci YQ-6 on the growth performance of Leizhou black ducks

[0095]

[0096]

[0097] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), data with different uppercase letters indicate extremely significant differences (P<0.01), and data with the same or no letters indicate no significant differences (P>0.05).

[0098] 1.2.2 Effects of marine-derived persistent enterococci YQ-6 on the slaughter performance of 42-day-old Leizhou black ducks

[0099] As shown in Table 4, compared with the control group, the M3 group could significantly improve the carcass rate (P<0.05), half-eviscerated rate, full-eviscerated rate, breast muscle rate and leg muscle rate of Leizhou black duck (P<0.05).

[0100] Table 4 Effect of marine-derived persistent enterococci YQ-6 on the slaughter performance of Leizhou black ducks (%)

[0101]

[0102] 1.2.3 Effects of marine-derived Enterococcus YQ-6 on the quality and nutritional composition of 42-day-old Leizhou black duck breast muscle

[0103] As shown in Table 5, compared with the control group, the b values ​​of the other four groups were significantly lower (P < 0.05), and the M3 group significantly reduced the shear force and fat content of the breast muscle of Leizhou black ducks (P < 0.05). As the amount of Enterococcus durabilis YQ-6 added increased, the water loss rate tended to decrease, but no significant difference was observed.

[0104] Table 5 Effects of marine-derived persistent enterococci YQ-6 on the quality and nutritional components of Leizhou black duck breast muscle

[0105]

[0106]

[0107] 1.2.4 Effects of marine-derived Enterococcus YQ-6 on muscle quality and nutritional composition of 42-day-old Leizhou black duck leg meat

[0108] As shown in Table 6, compared with the control group, the M3 group can significantly reduce the shear force and fat content of the leg muscle of Leizhou black duck (P<0.05). With the increase of the amount of Enterococcus durabilis YQ-6 added, the water loss rate tended to decrease, but no significant difference was found.

[0109] Table 6 Effects of marine-derived Enterococcus YQ-6 on the muscle quality and nutritional components of Leizhou black duck leg

[0110]

[0111] 1.2.5 Effects of marine-derived Enterococcus durabilis YQ-6 on amino acid content in muscle of 42-day-old Leizhou black ducks

[0112] As shown in Table 7, compared with the control group, the contents of aspartic acid, alanine, phenylalanine and total amino acids in the muscles of Leizhou black ducks in the M3 group were significantly increased (P<0.05), and the contents of UAA and EAA in the M group were higher than those in the control group, but the differences were not significant.

[0113] Table 7 Effects of marine-derived Enterococcus YQ-6 on amino acids in Leizhou black duck muscle (%)

[0114]

[0115] Note: #. Essential amino acid; *. Umami amino acid.

[0116] 1.3 Conclusion

[0117] 1.3.1 Effects of marine-derived Enterococcus YQ-6 on the growth performance of Leizhou black ducks

[0118] In this experiment, adding different doses of YQ-6 to the feed could significantly increase the ADG of Leizhou black ducks and reduce the F / G. 9 The CFU / kgYQ-6 effect is extremely significant. This shows that YQ-6 can improve intestinal function and promote animal growth.

[0119] 1.3.2 Effects of marine-derived Enterococcus YQ-6 on the slaughter performance of Leizhou black ducks

[0120] The present study found that the slaughter rate of Leizhou black ducks ranged from 80.18% to 86.18%, and the evisceration rate ranged from 70.60% to 72.30%, further demonstrating their excellent meat production. During their growth cycle, Enterococcus durabilis, in addition to being rich in various nutrients, may also secrete a series of highly effective digestive enzymes, such as cellulase, amylase, protease, and lipase. These enzymes can optimize the balance of nutrients and promote the efficient absorption and utilization of various nutrients in the feed by the animal, thereby positively impacting slaughter performance.

[0121] 1.3.3 Effects of marine-derived Enterococcus YQ-6 on muscle quality of Leizhou black ducks

[0122] In the present invention, the b-values ​​of the breast muscles in the M1, M2, M3, and La groups were significantly lower than those in the control group, indicating better muscle quality. In the present invention, it was found that the shear force of the leg muscles of the breast muscles in the M3 group was significantly reduced, and the tenderness of the muscles was higher, indicating that the addition of YQ-6 maintained the freshness of the muscles and the stability of the meat color.

[0123] 1.3.4 Effects of marine-derived Enterococcus YQ-6 on general nutritional components of Leizhou black duck muscle

[0124] The nutritional composition of meat, including water, protein, fat, and collagen, largely influences consumer purchasing decisions. The discrepancies in these findings may be due to a variety of variables, including the type of strain fed, the duration of feeding, the animal species, and growth conditions.

[0125] 1.3.5 Effects of marine-derived Enterococcus durabilis YQ-6 on amino acids in Leizhou black duck muscle

[0126] The results of this example show that the content of non-essential amino acids (UAA) in the M group is higher than that in the control group, and the content of aspartic acid, alanine, phenylalanine and total amino acids in the M3 group are significantly higher than those in the control group, indicating that the addition of marine-derived Enterococcus YQ-6 can further enhance the nutritional level and flavor value of high-quality protein in muscle.

[0127] In summary, the addition of marine-derived Enterococcus YQ-6 to feed can increase the slaughter rate of Leizhou black ducks at 42 days, improve the average daily weight gain of Leizhou black ducks at different stages, and reduce the feed-to-weight ratio. The addition of marine-derived Enterococcus YQ-6 to feed can reduce the shear force and b value in the muscle, thereby maintaining the tenderness and muscle quality of the muscle and reducing the crude fat content in the muscle. Marine-derived Enterococcus YQ-6 can improve the nutritional level and flavor value of the muscle by increasing the content of aspartic acid, alanine, and phenylalanine. The addition amount is 1×10 9 The effect is better when the CFU / kg

[0128] Effects of marine-derived Enterococcus YQ-6 on antioxidant and immune functions of Leizhou black ducks

[0129] 2.1 Experimental methods

[0130] 2.1.1 Sample collection

[0131] On the 42nd day of the experiment, after fasting for 12 hours, two Leizhou black ducks with similar weight were selected from each replicate group for blood collection. 10 mL of blood was collected from the subwing vein. The blood was allowed to stand at room temperature and centrifuged at 3000 r / min at 4°C for 15 minutes, then serum was collected and stored at -20°C for later use. The Leizhou black ducks were then slaughtered, and the thymus, spleen and bursa of Fabricius were collected. After washing with sterile PBS, they were quickly frozen in liquid nitrogen and finally stored at -80°C. The intestinal segments were separated, the tissues were gently washed, and then fixed in 4% paraformaldehyde solution.

[0132] 2.1.2 Serum antioxidant index

[0133] According to the kit instructions, the activities of CAT, SOD, GSH-Px and MDA content in the serum of 42-day-old Leizhou black ducks were detected.

[0134] 2.1.3 Complement and immunoglobulin content determination

[0135] Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of C3, C4 and IgG in the serum at 42 days. The specific operation was carried out according to the instructions.

[0136] 2.1.4 Determination of serum cytokine levels

[0137] The levels of TNF-α, IFN-γ, IL-2, IL-4 and IL-6 in serum were detected according to the ELISA procedure.

[0138] 2.1.5 Analysis of immune organ-related gene expression

[0139] Total RNA was extracted from thymus, spleen, and bursa of Fabricius tissues and reverse transcribed into cDNA using a reverse transcription kit. Real-time fluorescence quantitative PCR was performed using the reverse-transcribed cDNA as template and the ChamQ SYBR qPCR Master Mix kit. β-actin was used as an internal reference gene to analyze the relative quantification of TNF-α, IFN-γ, IL-2, IL-4, SOD, GSH-Px, and CAT.

[0140] 2.2 Experimental Results

[0141] 2.2.1 Effects of marine-derived Enterococcus YQ-6 on the antioxidant function of Leizhou black ducks

[0142] like Figure 1 As shown, compared with the control group, the activities of serum CAT and SOD in group M3 were significantly increased; the levels of GSH-Px in group M1 and group La were significantly increased, and those in group M2 and group M3 were extremely significantly increased; the levels of MDA in group M2, group M3 and group La were extremely significantly decreased.

[0143] 2.2.2 Effects of marine-derived Enterococcus YQ-6 on complement and immunoglobulin levels in Leizhou black ducks

[0144] like Figure 2 As shown, compared with the control group, the C3 level in the M3 group increased significantly (P<0.05), and the levels of C4 and IgG increased very significantly (P<0.01).

[0145] 2.2.3 Immune function of marine-derived Enterococcus durabilis YQ-6 against serum from Leizhou black ducks

[0146] like Figure 3 As shown in the data, compared with the control group, the IL-2 level in the M2 group was significantly increased (p<0.05), and the IL-6 level was significantly decreased (p<0.05); the IL-2 and IL-4 levels in the M3 group were extremely significantly increased (p<0.01), and the IL-6, IFN-γ and TNF-α levels were significantly decreased (p<0.05); the IL-6, FN-γ and TNF-α levels in the La group were also significantly decreased (p<0.05).

[0147] 2.2.4 Effects of marine-derived Enterococcus durabilis YQ-6 on the expression levels of genes related to the immune organs of Leizhou black ducks

[0148] like Figure 4As shown in Figure A, compared with the control group, the expressions of TNF-α and IFN-γ mRNA in the thymus of the M2, M3 and La groups were significantly reduced (p<0.05). The expression of TNF-α mRNA in the thymus of the M3 group was extremely significantly reduced (p<0.01), and the expression of IL-2 and IL-4 mRNA was extremely significantly increased (p<0.01). In the La group, the expressions of TNF-α and IFN-γ mRNA in the thymus were both reduced (p<0.05), and the expressions of IL-2 and IL-4 mRNA were both increased (p<0.05), and the effects were significant.

[0149] like Figure 4 As shown in Figure B, compared with the control group, the expression of TNF-α mRNA in the spleen of the M3 group was decreased, and the expression of IL-2 and IL-4 mRNA was increased, and the effect was extremely significant; the expression of TNF-α mRNA in the M2 group was significantly decreased (p<0.05), and the expression of IL-2 and IL-4 mRNA was significantly increased (p<0.05).

[0150] like Figure 4 As shown in center C, the M3 group reduced TNF-α mRNA expression in the bursa of Fabricius (p<0.05) and IFN-γ mRNA expression in the experimental area (p<0.05 or p<0.01), with the M2 and M3 groups showing extremely significant reductions (p<0.01). The M2, M3, and La groups all increased IL-2 and IL-4 mRNA expression (p<0.05 or p<0.01), with the M3 group showing the most significant effect.

[0151] 2.2.5 Effects of marine-derived Enterococcus durabilis YQ-6 on CAT, SOD, and GSH-Px mRNA expression levels in immune organs of Leizhou black ducks

[0152] like Figure 5 As shown in Figure A, compared with the control group, the expression of CAT mRNA in the thymus of the M3 group was significantly increased (p<0.05), and the expression of SOD and GSH-Px mRNA in the M2 and La groups was significantly increased (p<0.05), and the increase in the M3 group was extremely significant (p<0.01). Figure 5 As shown in Figure B, compared with the control group, the expression of CAT, SOD, and GSH-Px mRNA in the M3 group increased (p<0.05 or p<0.01), and the increase of CAT and SOD was extremely significant (p<0.01); the expression of CAT mRNA in the La group increased extremely significantly (p<0.01). Figure 5 As shown in middle C, compared with the control group, the expression of CAT and SOD mRNA in the M3 group was extremely significantly increased (p<0.01), the expression level of GSH-Px mRNA was significantly increased (p<0.05), and the expression of CAT and SOD mRNA in the La group was significantly increased (p<0.05).

[0153] 2.3 Conclusion

[0154] 2.3.1 Effect of marine-derived Enterococcus YQ-6 on the antioxidant capacity of Leizhou black ducks

[0155] Adding YQ-6 to the diet can improve the activity of SOD, GSH-Px and CAT in the serum of Leizhou black ducks, eliminate free radicals in the body and maintain the balance of the internal environment. Fluorescence quantitative PCR analysis showed that feeding Enterococcus durophilus could upregulate the expression levels of CAT, SOD and GSH-Px mRNA in the spleen, thymus and bursa of Leizhou black ducks. 9 The effect was most significant when the CFU / kg was 0.5, indicating that the addition of YQ-6 to feed can inhibit the production of MDA by increasing the activities of CAT, SOD and GSH-Px and upregulating the mRNA expression levels of antioxidant enzyme-related genes, thereby enhancing the body's antioxidant capacity and maintaining redox balance.

[0156] 2.3.2 Effects of marine-derived Enterococcus YQ-6 on the immune function of Leizhou black ducks

[0157] In this embodiment, 1×10 9 CFU / kgYQ-6 can promote the increase of serum C3 and C4 levels in Leizhou black ducks, while 1×10 9 CFU / kgYQ-6 can increase the serum IgG content of Leizhou black ducks. This shows that YQ-6 can promote the production of immunoglobulins and complement in the serum of Leizhou black ducks, mediate the body's humoral immunity, and thus improve the disease resistance of livestock and poultry. 9 CFU / kgYQ-6 had the best effect.

[0158] The present invention discovered that YQ-6 has different effects on the expression of immune factors in different immune organs. The results showed that compared with the control group, high-concentration YQ-6 significantly decreased the expression of TNF-α and IFN-γ in the thymus, while increasing the expression levels of IL-2 and IL-4. The results for the bursa of Fabricius were similar to those for the thymus, but in the spleen, the expression of TNF-α and IL-4 decreased, while the expression levels of IL-2 and IL-4 increased, while the expression of IFN-γ did not change significantly. This indicates that YQ-6 has a significant effect on improving the immune function of Leizhou black ducks. Throughout the immune response, various immune cells stimulate each other by secreting different cytokines to balance pro-inflammatory and anti-inflammatory responses, thereby regulating the overall immune response.

[0159] In summary, the addition of marine-derived Enterococcus durabilis YQ-6 to the feed increased serum CAT, GSH-Px, and SOD activities, reduced MDA levels, and upregulated the expression of antioxidant enzyme genes in the thymus, spleen, and bursa of Fabricius. By producing multiple antioxidant enzymes, it inhibited the formation of free radicals in the body, thereby inhibiting lipid peroxidation and enhancing the body's antioxidant capacity. Marine-derived Enterococcus durabilis YQ-6 maintained immune homeostasis and improved the body's immune function by increasing serum immunoglobulin IgG and complement C3 and C4 levels in Leizhou black ducks and regulating the levels and mRNA expression of IL-2, IL-4, IL-6, TNF-α, and IFN-γ in serum and immune organs.

[0160] Effects of marine-derived Enterococcus YQ-6 on intestinal health of Leizhou black ducks

[0161] 3.1 Test method

[0162] 3.1.1 Serum barrier factor determination and intestinal tissue morphology analysis. The DAO activity and D-LA content in the serum of 42-day-old Leizhou black ducks were detected using kits according to the kit instructions.

[0163] 3.1.2 HE staining was used for intestinal tissue morphology analysis.

[0164] 3.1.3 Analysis of intestinal flora structure

[0165] On the 42nd day of the experimental period, two Leizhou black ducks with similar body weight were selected from each replicate group for slaughter, and the cecal contents were aseptically collected into sterile centrifuge tubes, immediately frozen in liquid nitrogen, and stored in a -80℃ refrigerator for testing.

[0166] After extracting total DNA from the microbiome, performing PCR amplification of the target fragment, recovering and purifying the amplified product, and performing fluorescence quantification, sequencing was performed using a PacBioSequel sequencer. Finally, the sequence was denoised using the QIIME2dada2 analysis pipeline. Sequences with abundances less than 5 were filtered out to obtain the final amplicon sequence variants (SVs) and signature table, which were then used to analyze the microbial flora of the cecal contents of Leizhou black ducks. All of the above experimental procedures were assisted by Nanjing Paisonno Gene Technology Co., Ltd.

[0167] 3.2 Experimental Results

[0168] 3.2.1 Effects of marine-derived Enterococcus YQ-6 on intestinal morphology of Leizhou black ducks

[0169] As shown in Table 8, compared with the control group, the addition of marine-derived Enterococcus YQ-6 to the feed can promote the growth of intestinal villi length in the duodenum and ileum of Leizhou black ducks and increase the villus-crypt ratio of each intestinal segment, and effectively reduce the crypt depth. 9CFU / kg, the effect was most significant (P<0.01).

[0170] Table 8 Effects of marine-derived persistent enterococci YQ-6 on the intestinal morphology of Leizhou black ducks

[0171]

[0172] Note: Data in the same industry with different lowercase letters indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and the same data or no letters indicate no significant differences (P>0.05).

[0173] like Figure 6 As shown in the results, from the morphological observation of intestinal structure, it can be seen that the intestinal villi structure of the duodenum, jejunum and ileum of each group are complete, with clear layers and obvious crypts, and no obvious lesions were observed; compared with the control group, after feeding the marine-derived persistent enterococcus YQ-6, the intestinal villi of the Leizhou black ducks were more closely arranged, with more complete edges, which can better maintain the integrity of the intestinal morphological structure of the Leizhou black ducks.

[0174] 3.2.2 Effects of marine-derived Enterococcus YQ-6 on the intestinal flora of Leizhou black ducks

[0175] The number of common and unique ASVs in the five groups of samples is as follows Figure 7 As shown, there were 128 ASVs in total among the five groups of samples, and the control group, M1 group, M2 group, M3 group, and La group had 252 ASVs, 478 ASVs, 500 ASVs, 634 ASVs, and 549 ASVs, respectively.

[0176] 3.2.3 Alpha Diversity Analysis

[0177] like Figure 8 As shown, there were no significant differences in the Shannon index among the five groups of samples, indicating that species diversity did not differ significantly among the groups. Group M3 had the highest Shannon index, indicating that this group had the highest species diversity. There were no significant differences in the Chao1 index among the five groups of samples, indicating that species richness did not differ significantly among the groups. Group M3 had the highest Chao1 index, indicating that this group had the highest species diversity.

[0178] 3.2.4 Beta Diversity Analysis

[0179] Principal coordinate analysis (PCoA) based on Bray-Curtis distance and scatter plots were used to show the β diversity of bacterial flora. Figure 9As shown, there were significant inter-group differences in the bacterial flora composition of the five groups (R2=0.1743, P=0.004). There was no overlap between the La group, the Control group, and the M2 and M3 groups, indicating that there were significant differences in the bacterial community composition between the La group, the Control group, and the M2 and M3 groups. There was partial overlap between the M1, M2, and M3 groups, indicating that there were similarities in the bacterial flora composition between the three groups. The Control group was farther away from the other groups, indicating that the bacterial flora composition of the Control group was quite different from that of the other groups. The scatter plot shows that there were significant differences in the bacterial communities between the Control group and the M2 and M3 groups (P<0.05). There were also significant differences in the bacterial communities between the La group and the M2 and M3 groups (P<0.05).

[0180] 3.2.5 Analysis of bacterial flora structure at the phylum level

[0181] 16S amplicon sequencing revealed 3549 ASVs representing 12 different phyla in the duck gut bacteria. The top 11 phyla by relative abundance were selected for visualization, with the remaining phyla grouped as "Other." The top ten phyla by abundance included: Firmicutes, Bacteroidota, Campilobacterota, Desulfobacterota, Proteobacteria, Deferribacterota, Actinobacteria, Spirochaetes, Cyanobacteria, Synergistota, and Elusimicrobiota. Compared with the control group, the relative abundance of Firmicutes increased in the M3 and La groups (P < 0.05). The relative abundance of Cyanobacteria increased in the M2 and M3 groups (P < 0.05). Compared with the control group, the relative abundance of Ferrobacteria was significantly decreased in the M3 group (P<0.05).In the five groups, Firmicutes was the dominant bacterial phylum in the duck intestine.

[0182] 3.2.6 Analysis of bacterial flora structure at the genus level

[0183] The top 11 genera by relative abundance were selected for visualization, with the remaining phyla classified as "Other." The top nine genera by relative abundance included Bacteroides, Faecalibacterium, Helicobacter, Desulfovibrio, Streptococcus, Prevotellaceae (UCG-001), Phascolarctobacterium, Clostridia (UCG-014), and Parabacteroides. The relative abundance of Parabacteroides increased in the M3 group compared to the control group (P < 0.05).

[0184] 3.2.7 Linear discriminant analysis of intestinal flora

[0185] In this example, 1 phylum, 2 classes, 3 orders, 7 families, 10 genera, and 4 species were found to be significantly different between the groups. These taxa will also be used as biomarkers to distinguish the groups. Figure 12As shown. Specifically, in the M3 group, 1 order, 3 families, 5 genera, and 1 species were identified, namely Monoglobales, Monoglobaceae, Eggerthellaceae, Oxalobacter, CHKCI001, NK4A214_group, Monoglobus, Eisenbergiella, Oxalobacter, and Oxalobacter_formigenes. In the M2 group, 1 phylum, 1 class, 1 order, 2 families, and 1 genus were identified, namely Deferribacterota, Deferribacteres, Deferribacterales, Deferribacteraceae, Marinifilaceae, and Mucispirillum. In the M1 group, 2 genera were identified, namely CHKCI002 and Subdoligranulum. In the control group, one genus and one species were identified, namely Enterococcus_columbae and Butyricimonas. In the La group, one phylum, one class, one order, two families, one genus, and one species were identified, namely Spirochaetota, Spirochaetia, Spirochaetales, Spirochaetaceae, Barnesiellaceae, Sphaerochaeta, and Bacteroides_sp.

[0186] Overall, the most biomarkers were identified in M3, while the Control group had only two biomarkers.

[0187] 3.3 Conclusion

[0188] 3.3.1 Effects of marine-derived Enterococcus YQ-6 on the intestinal villi of Leizhou black ducks

[0189] In this example, the addition of YQ-6 significantly increased VH and VH / CD in the duodenum and ileum of Leizhou black ducks and reduced CD. The increased intestinal villi indicates that enterococci can enhance intestinal absorptive capacity. This suggests that different strains of enterococci can improve intestinal morphology in poultry, promote nutrient absorption, accelerate metabolism, and enhance animal growth.

[0190] 3.3.2 Effect of marine-derived Enterococcus YQ-6 on improving the intestinal flora of Leizhou black ducks

[0191] In this example, alpha diversity analysis revealed that although the Shannon index and Chao1 index of the cecum of Leizhou black ducks were not significantly different between the marine-derived persistent enterococci YQ-6 and the control group, the species diversity and richness of the M3 group were the highest, indicating that the marine-derived persistent enterococci YQ-6, as a feed additive, can affect the relative abundance of specific bacterial communities by regulating the composition of intestinal flora without affecting alpha diversity.

[0192] This example further explored the effects of YQ-6 on the intestinal microbiota by analyzing changes in bacterial composition and abundance at different taxonomic levels at the phylum level. The results showed that in the cecal environment of newborn ducks, the intestines are initially nearly sterile. Over time, Proteobacteria dominated during the first three days after birth; from the fourth day onward, Firmicutes gradually increased in number and replaced Proteobacteria as the dominant bacterial community. In subsequent developmental stages, in addition to Proteobacteria and Firmicutes, significant numbers of Bacteroidetes were also detected in the intestines of domestic ducks raised under greenhouse conditions. Firmicutes bacteria have the ability to degrade dietary fiber, producing metabolites such as acetate and butyrate, thereby improving intestinal permeability. Bacteroidetes bacteria are considered the primary decomposers of polysaccharides. In this example, the relative abundance of Firmicutes was higher in the M3 group, suggesting that YQ-6, by regulating the ratio of Firmicutes to Bacteroidetes in the intestine, influences lipid metabolism, thereby regulating weight and promoting health.

[0193] To further explore the specific effects of specific bacterial genera on the production performance of broiler ducks, this example analyzed the compositional changes of the intestinal microbiota of Leizhou black ducks at the genus level. The results showed that Bacteroides dominated the cecal contents, and the increase in its abundance was positively correlated with the host's weight gain. While degrading intestinal carbohydrates, Bacteroides secreted a variety of short-chain fatty acids, including acetate, propionate, and butyrate. These metabolites actively participated in and influenced the host's energy metabolism. Bacteroides fragilis has the ability to repair human immune deficiencies and correct acquired immune imbalances. By increasing white blood cell count and strengthening macrophage function, it effectively eliminates infections and reduces cancer risk, achieving a two-way regulation of immunity. In contrast, Parabacteroides is more adept at degrading complex proteins and has a relatively low tolerance to bile acids. Based on these studies, it is speculated that YQ-6 enriches the intestinal microbiota structure by increasing the number of dominant bacterial genera, maintaining intestinal health, enhancing immunity, and improving production performance.

[0194] Furthermore, this example employed the LEfSe analysis method to identify gut microbial biomarkers with significant differences between groups. The results showed that different doses of the marine-derived Enterococcus durconium YQ-6 had differential effects on the gut microbiota of Leizhou black ducks, with the relative abundance of key gut microbiota groups shifting accordingly. Combined with the results of α-diversity and β-diversity analyses, this further confirmed that YQ-6 significantly modulated the abundance and composition of the gut microbiota in Leizhou black ducks.

[0195] In summary, the addition of marine-derived Enterococcus durabilis YQ-6 to the feed can increase villus height, reduce crypt depth, and improve intestinal digestion and absorption capacity in Leizhou black ducks. Marine-derived Enterococcus durabilis YQ-6 can maintain the stability of the intestinal flora of Leizhou black ducks. By regulating the composition of the intestinal microbiome and increasing the relative abundance of beneficial bacterial genera, it facilitates the establishment of the intestinal microbial barrier, improves intestinal health, and promotes animal growth performance.

[0196] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of marine-derived persistent enterococci YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black ducks.

2. The use according to claim 1, characterized in that The slaughtering performance is: improving the carcass rate, half-eviscerated rate, full-eviscerated rate, breast muscle rate and leg muscle rate.

3. The use according to claim 1, characterized in that The growth performance is: increasing average body weight and average daily gain, and reducing feed-to-weight ratio.

4. The use according to claim 1, characterized in that The muscle quality is as follows: reducing shear force and b-value of pectoral muscle, optimizing amino acid and fatty acid composition in muscle, and increasing the content of aspartic acid, alanine and phenylalanine in muscle.

5. Application of marine-derived persistent enterococci YQ-6 in the preparation of products that improve the antioxidant capacity and enhance the immune function of Leizhou black ducks.

6. The use according to claim 5, characterized in that The method of improving the antioxidant capacity and enhancing the immune function of Leizhou black ducks comprises: improving the activities of SOD, CAT and GSH-Px in the serum of Leizhou black ducks, reducing the MDA content, increasing the contents of complement C3, C4 and immunoglobulin IgG in the serum of Leizhou black ducks, and regulating the expression levels of IFN-γ, IL-2, IL-4, IL-6 and TNF-α mRNA in the immune organ tissues of Leizhou black ducks.

7. Application of marine-derived durable enterococci YQ-6 in the preparation of products that promote intestinal health of Leizhou black ducks.

8. The use according to claim 7, characterized in that The said promotion of intestinal health of Leizhou black ducks is as follows: increasing the height of villi, reducing the depth of crypts, reducing the villus-crypt ratio of duodenum and ileum, improving the digestion and absorption capacity of the intestine, maintaining the stability of the intestinal flora of Leizhou black ducks and regulating the composition structure of the flora; At the phylum level, the relative abundance of Cyanobacteria increased, and the relative abundance of Ferrobacteria decreased; at the genus level, the relative abundance of Parabacteroides increased.

9. A feed additive for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, characterized in that: The feed additive includes marine-derived Enterococcus durabilis YQ-6.

10. A feed for improving the growth performance, slaughter performance, muscle quality, immune function and intestinal health of Leizhou black ducks, characterized in that: The feed includes marine-derived Enterococcus durabilis YQ-6 or the feed additive according to claim 9.

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