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

Abstract

This invention discloses the application of marine-derived durable enterococci YQ-6 in improving the growth performance, slaughter performance, and muscle quality of Leizhou black ducks, belonging to the field of poultry farming technology. Experiments have demonstrated that marine-derived durable enterococci 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. Adding 1×10 [units of a specific amount] to the feed... 9 The optimal concentration of CFU / kg yields the best results. This invention aligns with the trend towards antibiotic-free feed and is of great significance for the green, safe, and sustainable development of the livestock industry.

Description

Technical Field

[0001] This invention relates to the field of poultry farming technology, and in particular to the application of marine-derived durable enterococcus YQ-6 in improving the growth performance, slaughter performance and muscle quality of Leizhou black duck. Background Technology

[0002] Leizhou Black Duck, with a long history of breeding, naturally thrives in the relatively enclosed tropical to subtropical coastal mudflats, making it a valuable livestock and poultry genetic resource of the Leizhou Peninsula. As a distinctive local black-feathered breed, Leizhou Black Duck is valued for both its egg and meat, and is renowned for its tender meat, delicious flavor, high egg production, and good adaptability to roughage. Studies have shown that Leizhou Black Duck muscle is rich in crude protein and crude fat, exhibiting a unique flavor and superior taste in terms of muscle tenderness, color, and fat ratio, showing significant advantages over Muscovy ducks, Cherry Valley ducks, and Gaoyou ducks.

[0003] Currently, Leizhou black ducks are raised using a combination of pen-raising and free-range grazing on mudflats. While this method has its unique advantages, it also leads to problems such as long breeding cycles, low feed utilization efficiency, unstable duck flock health, and frequent use of antibiotics. With the increasing market demand for healthy and safe duck products, the development of new probiotics and antibiotic alternatives in feed additives is particularly important. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention provides the following solution:

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

[0007] The second technical solution of this invention is the application of marine-derived durable enterococci YQ-6 in the preparation of products that enhance the antioxidant capacity and immune function of Leizhou black duck.

[0008] The third technical solution of this invention is the application 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 that improves the growth performance, slaughter performance, muscle quality, immune function and promotes the 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 that improves the growth performance, slaughter performance, muscle quality, immune function and promotes the 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] This invention experimentally demonstrates that marine-derived durable enterococci YQ-6 promotes serum immunoglobulin and complement levels, regulates cytokine gene expression levels, thereby maintaining the dynamic balance of immune function, enhancing the immunity of Leizhou black ducks, and improving muscle quality, nutritional value, and flavor by enhancing the body's antioxidant function. Simultaneously, marine-derived durable enterococci YQ-6 can regulate the composition and stability of the intestinal flora, increase the relative abundance of beneficial bacteria, promote intestinal villus development, and improve intestinal tissue structure, thereby maintaining intestinal health and improving the growth performance of Leizhou black ducks. The dosage of marine-derived durable enterococci YQ-6 in feed is 1×10⁻⁶. 9 The optimal concentration of CFU / kg yields the best results. This invention aligns with the trend towards antibiotic-free feed and is of great significance for the green, safe, and sustainable development of the livestock industry. Attached Figure Description

[0013] Figure 1 The effects of marine-derived durable enterococcus YQ-6 on the antioxidant function of 21-day-old Leizhou black ducks were investigated. The study included: A. CAT activity; B. SOD activity; C. GSH-PX activity; and D. MDA activity. *P<0.05, **P<0.01 compared to the control group (see figure below).

[0014] Figure 2 The effects of marine-derived durable enterococcus YQ-6 on complement and immunoglobulin levels in Leizhou black ducks were investigated. Specifically, the levels of A. C3; B. C4; and C. IgG were measured.

[0015] Figure 3 The study investigated the immune function of marine-derived durable enterococcus YQ-6 against the serum of Leizhou black duck. The study included: A. IL-2 content; B. IL-4 content; C. IL-6 content; D. IFN-γ content; and E. TNF-α content.

[0016] Figure 4 The study investigated the effects of marine-derived durable enterococcus YQ-6 on cytokine expression levels in the immune organs of Leizhou black duck. Specifically, the study measured: A. cytokine expression levels in the thymus; B. cytokine expression levels in the spleen; and C. cytokine expression levels in the bursa of Fabricius.

[0017] Figure 5This study investigated the expression levels of CAT, SOD, and GSH-Px mRNA in the immune organs of Leizhou black ducks by marine-derived durable enterococcus YQ-6. Specifically, the study included: A. Cytokine expression levels in the thymus; B. Cytokine expression levels in the spleen; and C. Cytokine expression levels in the bursa of Fabricius.

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

[0019] Figure 7 This is a Venn diagram of the cecal flora of Leizhou black ducks. Note: Each colored block in the diagram represents a group, the overlapping areas between colored blocks indicate the ASVs shared by the corresponding groups, and 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. Where A represents the Shannon index and B represents the Chao1 index.

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

[0022] Figure 10 To determine the cecal microbiota structure and richness of Leizhou black ducks at the taxonomic level.

[0023] Figure 11 Taxonomic structure and richness of the cecum genus in Leizhou black ducks.

[0024] Figure 12 Multi-level species difference discrimination analysis of LEfSe in the cecum of Leizhou black duck. Detailed Implementation

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

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

[0027] This invention provides the application of marine-derived durable enterococcus YQ-6 in improving the growth performance, slaughter performance, and muscle quality of Leizhou black duck.

[0028] In some specific implementations, the slaughter performance is: increased carcass yield, semi-eviscerated yield, fully eviscerated yield, breast muscle yield, and leg muscle yield.

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

[0030] In some specific implementations, the muscle quality is defined as: reducing shear stress and the b-value of the pectoral muscles, optimizing the amino acid and fatty acid composition of the muscles, and increasing the content of aspartic acid, alanine, and phenylalanine in the muscles.

[0031] This invention also provides the application of marine-derived durable enterococcus YQ-6 in the preparation of products that enhance the antioxidant capacity and immune function of Leizhou black duck.

[0032] In some specific implementation schemes, the enhancement of the antioxidant capacity and immune function of Leizhou black ducks is achieved by: increasing the activity of SOD, CAT and GSH-Px in the serum of Leizhou black ducks, reducing the content of MDA, increasing the content 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 organs and tissues of Leizhou black ducks.

[0033] This invention also provides the application of marine-derived durable enterococcus YQ-6 in the preparation of products that promote the intestinal health of Leizhou black duck.

[0034] In some specific implementation schemes, promoting the intestinal health of Leizhou black ducks means: increasing the height of intestinal villi, reducing crypt depth, decreasing the villi-crypt ratio of the duodenum and ileum, improving intestinal digestive and absorptive capacity, maintaining the stability of the intestinal flora of Leizhou black ducks, and regulating the composition and structure of the flora.

[0035] At the phylum level, the relative abundance of Cyanobacteria and Bacteria was increased, while the relative abundance of Iron-deficient Bacteria was decreased; at the genus level, the relative abundance of Parabacterium was increased.

[0036] This invention also provides a feed additive that improves the growth performance, slaughter performance, muscle quality, immune function, and promotes the intestinal health of Leizhou black ducks. The feed additive includes marine-derived durable enterococci YQ-6.

[0037] This invention also provides a feed that improves the growth performance, slaughter performance, muscle quality, immune function, and promotes the intestinal health of Leizhou black ducks. The feed includes marine-derived durable enterococci YQ-6 or the feed additives mentioned above.

[0038] This invention has demonstrated through experiments that marine-derived durable enterococci 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. Adding 1×10 to the feed... 9 The best results are achieved at CFU / kg.

[0039] Example 1

[0040] 1. Effects of marine-derived durable enterococci YQ-6 on growth performance, slaughter performance and meat quality of Leizhou black duck

[0041] 1.1 Test strains

[0042] In this experiment, the marine-derived durable enterococcus durans YQ-6 was isolated from small yellow croaker in the South China Sea. This strain was deposited on May 7, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66265.

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

[0044] Marine-derived durable enterococci YQ-6 were inoculated into a culture medium (1% yeast extract, 2% glucose, 2% peptone, 2% sodium chloride) and cultured at 37℃ and 120 rpm for 48 h on a shaker. The bacterial suspension was collected and centrifuged (4℃, 10000 rpm, 10 min), the supernatant was discarded, and the suspension was washed twice with sterile physiological saline to prepare a bacterial suspension. The viable count of the suspension was calculated using the dilution plating method, and the concentration was adjusted to M1: 1×10⁻⁶ as needed for the experiment. 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 anaerobically cultured at 37℃ and 180 rpm for 24 h on a shaker. The bacterial suspension was collected and centrifuged (4℃, 8000 rpm, 10 min), the supernatant was discarded, and the suspension was resuspended twice in sterile physiological saline to prepare a bacterial suspension. The viable count was calculated using the dilution plating method, and the final concentration was adjusted to La: 1 × 10⁻⁶ as needed for the experiment. 9 CFU / kg.

[0046] 1.2 Test Methods

[0047] 1.2.1 Experimental Animals and Group Design

[0048] One hundred and eighty healthy, one-day-old male Leizhou black ducks of similar weight were selected for the experiment. They were randomly divided into five groups, with six replicates per group and six Leizhou black ducks per replicate. The experiment lasted for 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] Prior to the experiment, the duck house and related equipment underwent a thorough cleaning and disinfection process, followed by ventilation and air purification to ensure the suitability of the experimental environment. During the experiment, Leizhou Black Ducks were raised in an indoor wire mesh floor system, fed with mixed feed, and the ducks had free access to food and water. Temperature, humidity, and lighting conditions within the duck house were strictly controlled. To ensure hygiene and air quality in the duck house, regular cleaning was performed and good ventilation was maintained. Furthermore, the health status of the Leizhou Black Ducks was closely monitored, and the quality of drinking water and feed was regularly checked and detailed records were kept. Feeding management and immunization procedures were carried out according to standard feeding procedures. The basic feed was formulated with reference to the "Standards for Meat Duck Feeding" (NY / T 2122-2012), and its composition and nutritional levels are shown in Table 2.

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

[0054]

[0055] Note: The premix provides the following per kilogram of feed: Vitamin A 8000 IU, Vitamin B 12 mg, Vitamin D 2000 IU, Vitamin E 20 IU, Vitamin K 32 mg, Niacin 40 mg, Dextrorotatory 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, fasted Leizhou black ducks were weighed and their feed consumption was recorded daily. Average body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR) were calculated.

[0058] BW(g) = Total weight / Number of ducks;

[0059] ADFI(g / d) = Amount of material consumed during the test / 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, at day 42 of the experiment, two Leizhou black ducks of similar weight and health status were selected from each replicate group and weighed before slaughter. Subsequently, the experimental ducks were bled from the jugular vein, weighed, and the pectoral and leg muscles were dissected and collected. The dressing percentage, semi-eviscerated percentage, fully eviscerated percentage, pectoral muscle percentage, leg muscle percentage, and lean meat percentage were determined and calculated according to the agricultural industry standard (NY / T 823-202).

[0064] Slaughter rate (%) = Slaughter weight / Pre-slaughter weight × 100;

[0065] Semi-eviscerated yield (%) = Semi-eviscerated weight / Pre-slaughter body weight × 100;

[0066] Full evisceration rate (%) = Full eviscerated weight / Pre-slaughter body weight × 100;

[0067] Leg muscle percentage (%) = (Weight of both legs' muscles / Total net body weight) × 100;

[0068] Pectoral muscle percentage (%) = Weight of both pectoral muscles / Total net chest weight × 100;

[0069] Lean meat percentage (%) = (Weight of both pectoral muscles + Weight of both leg muscles) / Total eviscerated weight × 100.

[0070] 1.2.5 Determination of meat quality indicators

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

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

[0073] (2) pH 45min 45-60 minutes after slaughter, three different parts of the meat—breast muscle, leg muscle, and ham muscle—are sampled. A pH meter probe is inserted into the meat sample to measure the pH value. Once the value stabilizes, it is recorded, and the average value of the three parts is taken as the final pH. 45min value.

[0074] (3) pH 24h Meat samples were stored at 4°C and removed 24 hours after slaughter to determine their pH. 24h The numerical value and measurement method are the same as pH. 45min .

[0075] (4) Water loss rate: Two meat samples (1 cm thick and about 2.5 cm in diameter) were cut from the pectoral muscle and leg muscle using a sharp round sampler. The weight was recorded as m1. The meat samples were placed on the pressure table and covered with 16 layers of qualitative filter paper on the top and bottom. A pressure of 35 kg was applied for 5 min. After the pressure was applied, the weight was recorded as m2.

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

[0077] (6) Cooking loss: The cooking loss of pectoral and leg muscles was measured after being stored at 4°C for 24 hours post-slaughter. The meat samples were trimmed into cuboids along the muscle fiber direction and weighed, recorded as m3. The meat samples were placed in a self-sealing bag and heated in an 80°C water bath until the center temperature of the meat sample reached 75°C. After the meat samples were cooled to room temperature, the surface moisture was absorbed with filter paper and weighed, recorded 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 tenderness meter. Each meat sample was measured twice and the average value was calculated.

[0080] 1.2.6 Determination of routine nutrient content in muscle

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

[0082] (1) Moisture content was determined by direct drying method according to national standard GB / 5009.3-2016;

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

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

[0085] (4) The crude ash content was determined in accordance with the national standard GB / 5009.4-2016.

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

[0087] The fatty acid and amino acid content of 42-day-old Leizhou black duck muscle tissue was determined after mincing into a paste. The amino acid determination method in this experiment followed the national standard GB 5009.124-2016. A fully automated amino acid analyzer was used to detect the amino acid content in the muscle. The content of each amino acid in the muscle was analyzed by comparing the retention time and peak area of ​​amino acid standards.

[0088] The determination method for fatty acids was based on the national standard GB 5009.168-2016. After hydrolysis and fat extraction of meat samples, the fat was saponified and methylated to generate fatty acid methyl esters. The fatty acid methyl esters were analyzed by gas chromatography and the percentage content of each fatty acid was calculated by peak area normalization in conjunction with the relative retention time of fatty acid methyl ester standards.

[0089] 1.2.8 Data Analysis

[0090] One-way ANOVA was performed using SPSS 26.0 statistical software, and multiple comparisons were performed using Duncan's software. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. The results are expressed as mean ± standard deviation, and graphs were generated using GraphPad Prism 8 software.

[0091] 1.2 Experimental Results

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

[0093] Table 3 shows that 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. Particularly 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). There was no significant difference in average daily feed intake between the treatment groups and 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, significantly higher than that of the other groups (P<0.05). At 7-14 days and 21-28 days, the feed conversion ratio of the M3 group was significantly lower than that of the control group and other treatment groups (P<0.05). At other time points, there was no significant difference in feed conversion ratio between the treatment groups and the control group (P>0.05).

[0094] Table 3 Effects of marine-derived durable enterococcus YQ-6 on the growth performance of Leizhou black duck

[0095]

[0096]

[0097] Note: Different lowercase letters in the same row headings indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and identical or no letters indicate no significant differences (P>0.05). The same applies to the following table.

[0098] 1.2.2 Effects of marine-derived durable 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 can significantly improve the carcass rate (P<0.05), semi-eviscerated rate, fully eviscerated rate, breast muscle rate and leg muscle rate of Leizhou black duck (P<0.05).

[0100] Table 4. Effects of marine-derived durable enterococcus YQ-6 on the slaughter performance of Leizhou black duck (%)

[0101]

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

[0103] Table 5 shows that, compared with the control group, the b-values ​​of the other four groups were significantly reduced (P<0.05), and the M3 group significantly reduced the shear force and fat content of the breast muscle of Leizhou black duck (P<0.05). With the increase of the amount of durable enterococcus YQ-6 added, the water loss rate tended to decrease, but no significant difference was observed.

[0104] Table 5. Effects of marine-derived durable enterococcus YQ-6 on the quality and nutritional composition of breast muscle in Leizhou black duck.

[0105]

[0106]

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

[0108] As shown in Table 6, compared with the control group, the M3 group significantly reduced the shear force and fat content of the leg muscles of Leizhou black ducks (P<0.05). With the increase of the amount of durable enterococcus YQ-6 added, the water loss rate tended to decrease, but no significant difference was observed.

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

[0110]

[0111] 1.2.5 Effects of marine-derived durable enterococcus YQ-6 on amino acid content in the muscle of 42-day-old Leizhou black duck

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

[0113] Table 7. Effects of marine-derived durable enterococcus YQ-6 on amino acid content in the muscle of Leizhou black duck (%)

[0114]

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

[0116] 1.3 Conclusion

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

[0118] In this experiment, adding different doses of YQ-6 to the diet significantly increased the ADG (adverse glucose tolerance) and decreased the F / G (feed to glucose ratio) of Leizhou black ducks, and 1×10 9 The effect of CFU / kg YQ-6 is extremely significant. This indicates that YQ-6 can improve intestinal function and promote animal growth.

[0119] 1.3.2 Effects of marine-derived durable enterococci YQ-6 on the slaughter performance of Leizhou black duck

[0120] This invention reveals that the slaughter rate of Leizhou Black Duck ranges from 80.18% to 86.18%, while the eviscerated yield ranges from 70.60% to 72.30%, further highlighting its excellent meat production characteristics. Durable Enterococcus, during its growth cycle, in addition to being rich in various nutrients, may also possess the ability to secrete a series of highly efficient digestive enzymes, such as cellulase, amylase, protease, and lipase. These enzymes can optimize the balance of nutrients, promote the efficient absorption and utilization of various nutrients in feed by the animal, thereby positively impacting slaughter performance.

[0121] 1.3.3 Effects of marine-derived durable enterococcus YQ-6 on the muscle quality of Leizhou black duck

[0122] In this invention, the b-values ​​of the pectoral muscles in groups M1, M2, M3, and La were significantly lower than those in the control group, indicating better muscle quality. This invention also revealed that the shear force of the pectoral muscles in group M3 was significantly reduced, resulting in higher muscle tenderness, demonstrating that the addition of YQ-6 maintains muscle freshness and color stability.

[0123] 1.3.4 Effects of marine-derived durable enterococci YQ-6 on the routine nutritional components of Leizhou black duck muscle

[0124] The nutritional composition of meat mainly includes water, protein, fat, and collagen, and these components largely influence consumer purchasing preferences. The differences in the above research results may stem from variations in various variables such as the type of bacterial strain fed, the duration of feeding, the animal species, and the growth conditions.

[0125] 1.3.5 Effects of marine-derived durable enterococcus YQ-6 on amino acid content in Leizhou black duck muscle

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

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

[0128] 2. Effects of marine-derived durable enterococci YQ-6 on the antioxidant and immune functions of Leizhou black ducks

[0129] 2.1 Experimental Methods

[0130] 2.1.1 Sample Collection

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

[0132] 2.1.2 Serum antioxidant indicators

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

[0134] 2.1.3 Determination of complement and immunoglobulin levels

[0135] The levels of C3, C4, and IgG in serum on day 42 were detected using enzyme-linked immunosorbent assay (ELISA). The specific procedure was performed in accordance with the instruction manual.

[0136] 2.1.4 Serum cytokine level measurement

[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 expression of immune organ-related genes

[0139] Total RNA was extracted from the thymus, spleen, and bursa of Fabricius, and reverse transcribed into cDNA using a reverse transcription kit. Using the reverse-transcribed cDNA as a template, real-time quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit. β-actin was used as an internal reference gene to perform relative quantitative analysis 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 durable enterococcus YQ-6 on the antioxidant function of Leizhou black duck

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

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

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

[0145] 2.2.3 Marine-derived durable enterococcus YQ-6's effect on the serum immune function of Leizhou black duck

[0146] like Figure 3 As shown, 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 significantly increased (p<0.01), and the levels of IL-6, IFN-γ and TNF-α were significantly decreased (p<0.05); the IL-6, IFN-γ and TNF-α levels in the La group were also significantly decreased (p<0.05).

[0147] 2.2.4 Effects of marine-derived durable enterococcus YQ-6 on the expression levels of immune organ-related genes in Leizhou black ducks

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

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

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

[0151] 2.2.5 Effects of marine-derived durable enterococcus YQ-6 on the expression levels of CAT, SOD, and GSH-Px mRNA in the 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 group M3 was significantly increased (p<0.05), the expression of SOD and GSH-Px mRNA in groups M2 and La was significantly increased (p<0.05), and the increase in group M3 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 was increased in the M3 group (p<0.05 or p<0.01), and the increase in CAT and SOD was extremely significant (p<0.01); the expression of CAT mRNA was extremely significant in the La group (p<0.01). Figure 5 As shown in Figure C, compared with the control group, the expression of CAT and SOD mRNA was significantly increased in the M3 group (p<0.01), the expression level of GSH-Px mRNA was significantly increased (p<0.05), and the expression of CAT and SOD mRNA was significantly increased in the La group (p<0.05).

[0153] 2.3 Conclusion

[0154] 2.3.1 Effects of marine-derived durable enterococci YQ-6 on the antioxidant capacity of Leizhou black duck

[0155] Adding YQ-6 to the diet can improve the activity of SOD, GSH-Px, and CAT in the serum of Leizhou black ducks, thereby scavenging free radicals and maintaining the balance of the internal environment. Quantitative real-time PCR analysis showed that feeding with *Enterococcus durableis* upregulated the expression levels of CAT, SOD, and GSH-Px mRNA in the spleen, thymus, and bursa of Fabricius of Leizhou black ducks, with an addition amount of 1×10⁻⁶. 9 The effect was most significant at CFU / kg. This indicates that adding YQ-6 to the feed can enhance the body's antioxidant capacity and maintain redox balance by increasing the activity of CAT, SOD and GSH-Px and upregulating the mRNA expression levels of antioxidant enzyme-related genes, inhibiting MDA production, and thus maintaining the redox balance.

[0156] 2.3.2 Effects of marine-derived durable enterococcus YQ-6 on the immune function of Leizhou black duck

[0157] In this embodiment, 1×10 9 CFU / kg YQ-6 can promote the increase of serum C3 and C4 levels in Leizhou black ducks, while 1×10 9 CFU / kg YQ-6 can increase the serum IgG content of Leizhou black ducks. This indicates that YQ-6 can promote the production of immunoglobulins and complement in the serum of Leizhou black ducks, mediate humoral immunity, and thus improve the disease resistance of livestock and poultry. Adding 1×10 CFU / kg YQ-6 to the feed... 9 CFU / kgYQ-6 is most effective.

[0158] This invention discovered that YQ-6 affects the expression levels of immune factors in different immune organs. The results showed that, compared to the control group, in the thymus, high concentrations of YQ-6 significantly decreased the expression of TNF-α and IFN-γ, while increasing the expression levels of IL-2 and IL-4. The results in the bursa of Fabricius were similar to those in the thymus, but in the spleen, TNF-α expression decreased, while IL-2 and IL-4 expression increased, and IFN-γ expression showed no significant change. Therefore, YQ-6 has a significant effect on enhancing the immune function of Leizhou black duck. In the entire 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 durable enterococci YQ-6 to feed can increase the activities of CAT, GSH-Px, and SOD in the serum of Leizhou black ducks, reduce MDA content, and upregulate the expression levels of antioxidant enzyme genes in the thymus, spleen, and bursa of Fabricius. By producing various antioxidant enzymes, it inhibits the formation of free radicals in the body, thereby inhibiting lipid peroxidation and enhancing the body's antioxidant capacity. Marine-derived durable enterococci YQ-6 also increases the levels of immunoglobulin IgG and complement C3 and C4 in the serum of Leizhou black ducks, and regulates the levels and mRNA expression levels of IL-2, IL-4, IL-6, TNF-α, and IFN-γ in serum and immune organs, thereby maintaining immune homeostasis and improving the body's immune function.

[0160] 3. The effects of marine-derived durable enterococci YQ-6 on the gut health of Leizhou black duck

[0161] 3.1 Test Methods

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

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

[0164] 3.1.3 Analysis of Gut Microbiota Structure

[0165] On day 42 of the experiment, two Leizhou black ducks with similar weights were selected from each replicate group for slaughter. The contents of the cecum were aseptically collected into sterile centrifuge tubes, immediately flash-frozen in liquid nitrogen, and stored at -80°C for analysis.

[0166] After steps including total DNA extraction from the microbiome, PCR amplification of the target fragment, recovery and purification of the amplification product, and quantitative fluorescence analysis, sequencing was performed using a PacBioSequel sequencer. Finally, the sequences were denoised using the QIIME2dada2 analysis workflow, filtering out sequences with an abundance less than 5 to obtain the final amplicon sequence variants (SVs) and feature table, which were then used to analyze the microbial community of the cecal contents of Leizhou black ducks. All of the above experimental procedures were assisted by Nanjing Paisennong Gene Technology Co., Ltd.

[0167] 3.2 Experimental Results

[0168] 3.2.1 Effects of marine-derived durable enterococci YQ-6 on intestinal morphology of Leizhou black duck

[0169] As shown in Table 8, compared with the control group, the addition of marine-derived durable enterococci YQ-6 to the feed promoted the growth of intestinal villi length in the duodenum and ileum of Leizhou black ducks and increased the villus-crypt ratio in each intestinal segment, while also effectively reducing crypt depth. The addition amount was 1×10⁻⁶. 9The effect was most significant at CFU / kg (P<0.01).

[0170] Table 8. Effects of marine-derived durable enterococcus YQ-6 on intestinal morphology of Leizhou black duck.

[0171]

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

[0173] like Figure 6 As shown, morphological observation of the intestinal structure revealed that the villi of the duodenum, jejunum, and ileum in each group were intact, with clear layers and distinct crypts. No obvious lesions were observed. Compared with the control group, the villi of Leizhou black ducks fed with marine-derived durable enterococci YQ-6 were more tightly arranged and had more intact edges, thus better maintaining the integrity of the intestinal morphology of Leizhou black ducks.

[0174] 3.2.2 Effects of marine-derived durable enterococci YQ-6 on the gut microbiota of Leizhou black duck

[0175] The number of common and unique ASVs in the five groups of samples are as follows: Figure 7 As shown, there are a total of 128 ASVs among the five groups of samples. The control group, M1 group, M2 group, M3 group and La group have 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, indicating that there were no significant differences in species diversity among the groups. Group M3 had the highest Shannon index, indicating that this group had the highest species diversity. Similarly, there were no significant differences in the Chao1 index among the five groups, indicating that there were no significant differences in species richness 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) and scatter plots based on Bray-Curtis distance are used to visualize the β-diversity of bacterial communities. For example... Figure 9As shown, there were significant differences in bacterial community composition among the five groups (R² = 0.1743, P = 0.004). There was no overlap between groups La and Control and groups M2 and M3, indicating significant differences in bacterial community composition. Groups M1, M2, and M3 showed partial overlap, indicating similarity in bacterial community composition among these three groups. Group Control was significantly distancing itself from the other groups, indicating a large difference in bacterial community composition. The scatter plot showed significant differences in bacterial community composition between groups Control and M2 and M3 (P < 0.05). Significant differences also existed between groups La and M2 and M3 (P < 0.05).

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

[0181] 16S amplicon sequencing results showed that the duck gut bacteria contained 3549 ASVs representing 12 different phyla. The top 11 phyla by relative abundance were selected for visualization, and the remaining phyla were grouped into the "Other" top ten phyla, including: Firmicutes, Bacteroidetes, Campylobacterota, Desulfobacterota, Proteobacteria, Deferribacterota, Actinobacteria, Spirochaetota, Cyanobacteria, Synergistota, and Elusimicrobiota. Compared with the control group, the relative abundance of Firmicutes was increased in groups M3 and La (P<0.05). The relative abundance of Cyanobacteria was increased in groups M2 and M3 (P<0.05). Compared with the Control group, the relative abundance of iron-deficient bacilli was significantly reduced in the M3 group (P<0.05). Firmicutes was the dominant phylum in the duck gut in all five groups.

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

[0183] The top 11 genera in terms of relative abundance were selected for visualization, with the remaining phyla categorized as "Other." The top nine most abundant genera included: *Bacteroides*, *Faecalibacterium*, *Helicobacter*, *Desulfovibrio*, *Streptococcus*, *Prevotellaceae_UCG-001*, *Phascolarctobacterium*, *Clostridia_UCG-014*, and *Parabacteroides*. Compared to the Control group, the relative abundance of *Parabacteroides* was increased in the M3 group (P<0.05).

[0184] 3.2.7 Linear discriminant analysis of gut microbiota

[0185] This embodiment found significant differences among the groups in 1 phylum, 2 classes, 3 orders, 7 families, 10 genera, and 4 species. These taxonomic units will also serve as biomarkers to distinguish between the groups, such as... Figure 12As shown. Specifically, in group M3, one order, three families, five genera, and one species were identified: Monoglobus, Monoglobusaceae, Eggerthellaceae, Oxalobacter, CHKCI001, NK4A214_group, Monoglobus, Eisenbergiella, Oxalobacter, and Oxalobacter_formigenes. In group M2, one phylum, one class, one order, two families, and one genus were identified: Deferribacterota, Deferribacteres, Deferribacterales, Deferribacteraceae, Marinifilaceae, and Mucispirillum. In group M1, two genera were identified: CHKCI002 and Subdoligranulum. One genus and one species were identified in the Control group: *Enterococcus columbae* and *Butyricimonas*. One phylum, one class, one order, two families, one genus, and one species were identified in the La group: *Spirochaetota*, *Spirochaetia*, *Spirochaetales*, *Spirochaetaceae*, *Barnesiellaceae*, *Sphaerochaeta*, and *Bacteroides* sp.

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

[0187] 3.3 Conclusion

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

[0189] In this embodiment, the addition of YQ-6 significantly increased the VH and VH / CD in the duodenum and ileum of Leizhou black ducks and decreased CD. The increase in intestinal villi indicates that Enterococcus can enhance the absorptive capacity of the intestine. Therefore, it is speculated that different strains of Enterococcus can improve the intestinal morphology of poultry, promote the absorption of nutrients, accelerate metabolism, and promote animal growth.

[0190] 3.3.2 Marine-derived durable enterococci YQ-6 can improve the gut microbiota of Leizhou black ducks.

[0191] In this embodiment, Alpha diversity analysis revealed that although marine-derived durable enterococci YQ-6 showed no significant difference in Shannon and Chao1 indices of Leizhou black duck cecum compared to the control group, the M3 group had the highest species diversity and richness. This indicates that marine-derived durable enterococci YQ-6, as a feed additive, can influence the relative abundance of specific bacterial groups by regulating the composition of the intestinal flora without affecting Alpha diversity.

[0192] This embodiment further explores the impact of YQ-6 on the gut microbiota by analyzing changes in the structure and abundance of bacteria at different taxonomic levels. Results showed that in a cecal environment, the intestines of newborn ducks were nearly sterile in the initial stages. Over time, Proteobacteria dominated for the first three days after birth; from the fourth day onwards, Firmicutes gradually increased in number and replaced Proteobacteria as the dominant phylum. In subsequent developmental stages, in addition to Proteobacteria and Firmicutes, a large number of Bacteroidetes were also detected in the intestines of domestic ducks raised in greenhouses. Firmicutes have the ability to degrade dietary fiber, producing metabolites such as acetic acid and butyric acid, thereby optimizing intestinal permeability. Bacteroidetes are considered major polysaccharide decomposers. In this embodiment, the M3 group had a relatively high abundance of Firmicutes, indicating that YQ-6 influences lipid metabolism by regulating the ratio of Firmicutes to Bacteroidetes in the gut, thereby regulating body weight and promoting health.

[0193] To delve into the specific impacts of particular bacterial genera on the production performance of broiler ducks, this study analyzed the compositional changes of the gut microbiota in Leizhou black ducks at the genus level. The results showed that *Bacteroides* spp. dominated the cecal contents, and the increase in the abundance of this genus was positively correlated with the increase in host weight. While degrading intestinal carbohydrates, *Bacteroides* spp. secreted various short-chain fatty acids, including acetic acid, propionic acid, and butyric acid. These metabolites actively participated in and influenced the host's energy metabolism processes. *Bacteroides fragilis* possesses the ability to repair human immune deficiencies and correct acquired immune imbalances. It effectively eliminates infections and reduces cancer risk by increasing white blood cell count and strengthening the function of macrophage immune cells, achieving bidirectional regulation of immunity. In contrast, *Parabacteroides* spp. are better at degrading structurally complex proteins and have relatively low tolerance to bile acids. Based on these findings, it is speculated that YQ-6 enriches the gut microbiota structure, maintains intestinal health, enhances immunity, and improves its production performance by increasing the dominant bacterial genera.

[0194] Furthermore, this embodiment adopted the LEfSe analysis method to identify gut microbiota biomarkers with significant differences among groups. The results showed that different doses of marine-derived durable enterococci YQ-6 had differential effects on the gut microbiota of Leizhou black ducks, and the relative abundance of key gut microbiota among groups also changed accordingly. Combined with the analysis results of α-diversity and β-diversity, this further confirmed that YQ-6 has a significant regulatory effect on the abundance and composition of the gut microbiota of Leizhou black ducks.

[0195] In summary, adding marine-derived durable enterococci YQ-6 to feed can increase intestinal villus height, reduce crypt depth, and improve intestinal digestive and absorptive capacity in Leizhou black ducks. Marine-derived durable enterococci YQ-6 can maintain the stability of the intestinal flora in Leizhou black ducks, and by regulating the composition of the intestinal microbiota, increase the relative abundance of beneficial bacteria, thereby facilitating the establishment of the intestinal microbial barrier, improving intestinal health, and promoting animal growth performance.

[0196] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of marine-derived durable enterococcus YQ-6 in improving the slaughter performance of Leizhou black duck, characterized in that, The slaughtering performance is as follows: improved carcass yield, semi-eviscerated yield, fully eviscerated yield, breast muscle yield, and leg muscle yield; the marine-derived durable enterococcus YQ-6 was deposited on May 7, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66265.

2. A feed additive for improving the slaughter performance of Leizhou black duck, characterized in that, The feed additive includes marine-derived durable enterococci YQ-6; the slaughter performance is: improved carcass yield, semi-eviscerated yield, fully eviscerated yield, breast muscle yield, and leg muscle yield; the marine-derived durable enterococci YQ-6 was deposited on May 7, 2025 at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66265.

3. A feed for improving the slaughter performance of Leizhou black ducks, characterized in that, The feed includes marine-derived durable enterococci YQ-6 or the feed additive described in claim 2; the slaughter performance is: improved carcass yield, semi-eviscerated yield, fully eviscerated yield, breast muscle yield, and leg muscle yield; the marine-derived durable enterococci YQ-6 was deposited on May 7, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66265.

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