Application of Rhodotorula glutinosae ZTHY2 in improving carcass traits of Leizhou black duck and promoting intestinal health

Abstract

The present invention discloses the application of rhodotorula ZTHY2 in improving the carcass traits of Leizhou black duck and promoting intestinal health, and belongs to the technical field of poultry breeding. The present invention verifies through experiments that rhodotorula ZTHY2 can improve its muscle quality, enhance the nutritional value and flavor level of the muscle; rhodotorula ZTHY2 can also regulate the composition structure and stability of intestinal flora, increase the relative abundance of beneficial flora while inhibiting the proliferation of harmful flora, effectively reduce intestinal permeability, promote the development of intestinal villi, improve intestinal tissue structure, and thus maintain intestinal health. The addition amount of rhodotorula ZTHY2 in feed is 2×10 9 The effect is best when the content of CFU / kg. The invention conforms to the development trend of "antibiotic-free feed" and has important significance for the green, safe and sustainable development of the breeding industry.

Description

Technical Field

[0001] The invention relates to the technical field of poultry breeding, and in particular to application of red yeast ZTHY2 in improving carcass traits of Leizhou black ducks and promoting intestinal health. Background Art

[0002] Leizhou Black Duck, also known as Leizhou Black Duck, has a long history of breeding. It has long lived and reproduced in a relatively isolated tropical and subtropical coastal mudflat environment without any systematic breeding. It is a high-quality livestock and poultry germplasm resource of the Leizhou Peninsula. Leizhou Black Duck is a black-feathered egg-and-meat dual-purpose duck with local characteristics. It has the characteristics of delicious meat, high egg production, and tolerance to roughage. The meat performance of Leizhou Black Duck has natural advantages. Studies have found that the muscles of Leizhou Black Duck contain relatively rich crude fat and crude protein content. Its muscle tenderness, meat color, crude fat rate and other aspects show a unique flavor and taste, which are significantly better than Muscovy Duck, Cherry Valley Duck and Gaoyou Duck.

[0003] At present, the breeding method of Leizhou black duck is a combination of pen breeding and tidal flat grazing, which leads to problems such as long investment cycle and uneven uniformity of Leizhou black duck population. As consumers' demand for healthy, safe and diversified duck products continues to increase, the development of new antibiotic-free feed additives has important economic value and research significance for promoting the slaughter performance and intestinal health of Leizhou black ducks. Summary of the invention

[0004] The purpose of the present invention is to provide an application of Rhodotorula glutinosus ZTHY2 in improving the carcass traits of Leizhou black ducks and promoting intestinal health, 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 Rhodotorula glutinosus ZTHY2 in improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks.

[0007] The second technical solution of the present invention is a feed additive for improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks, wherein the feed additive includes red yeast ZTHY2.

[0008] The third technical solution of the present invention is a feed for improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks, wherein the feed comprises the additive or red yeast ZTHY2.

[0009] Based on the above technical solution, the present invention has the following technical effects:

[0010] The present invention provides an application of rhodotorula ZTHY2 in improving the carcass traits of Leizhou black ducks and promoting intestinal health. Experiments have shown that rhodotorula ZTHY2 can improve the muscle quality, nutritional value and flavor level of the ducks. Rhodotorula ZTHY2 can also regulate the composition structure and stability of intestinal flora, increase the relative abundance of beneficial flora, and inhibit the proliferation of harmful flora, effectively reduce intestinal permeability, promote intestinal villus development, improve intestinal tissue structure, and thus maintain intestinal health. The addition amount of rhodotorula ZTHY2 in feed is 2×10 9 The effect is best when the content of CFU / kg. The invention conforms to the development trend of "antibiotic-free feed" and has important significance for the green, safe and sustainable development of the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 Effects of Rhodotorula glutinosae ZTHY2 on serum DAO activity (A) and D-LA content (B) of Leizhou black ducks;

[0013] Figure 2 The histomorphology of duodenum, jejunum and ileum of 21-day-old Leizhou black duck (100×);

[0014] Figure 3 This is the histomorphology of duodenum, jejunum and ileum of 42-day-old Leizhou black duck (100×);

[0015] Figure 4 The Venn diagram (A) and dilution curve (B) of the cecal flora of Leizhou black duck;

[0016] Figure 5 This is the Alpha diversity analysis of cecal microorganisms in Leizhou black ducks, where A is the Chao1 index and B is the Shannon index;

[0017] Figure 6 Beta diversity analysis of cecal microorganisms in Leizhou black ducks;

[0018] Figure 7 The structure and richness of the cecal flora at the taxonomic level of Leizhou black ducks, where A is the species composition at the phylum level, B is the relative abundance of Proteobacteria, and C is the relative abundance of Actinobacteria;

[0019] Figure 8The structure and richness of the flora at the taxonomic level of the cecum of Leizhou black ducks, where A is the species composition at the class level, B is the relative abundance of α-Proteobacteria, and C is the relative abundance of γ-Proteobacteria;

[0020] Fig. 9 The structure and richness of the cecal flora at the order, family and genus taxonomic levels of Leizhou black ducks, among which A is the species composition at the order taxonomic level, B is the species composition at the family taxonomic level, C is the species composition at the genus taxonomic level, A-1 is the relative abundance of Clostridium-UCG-014 order, B-1 is the relative abundance of the family of the coprostearate Eubacteriaceae group, B-2 is the relative abundance of the family of Oscillospiraceae, B-3 is the relative abundance of the family of Rikenellaceae, and C-1 is the relative abundance of the genus of the coprostearate Eubacteriaceae group;

[0021] Fig.10 This is the multi-level species difference discriminant analysis of LEfSe in the cecum of Leizhou black duck;

[0022] Fig.11 This is the Spearman correlation analysis of the antioxidant and immune indexes of the blood of Leizhou black ducks and the antioxidant and immune indexes of immune organs under the action of yeast ZTHY2, where A is blood and bursa of Fabricius, B is blood spleen, C is blood and thymus, and D is thymus and bursa of Fabricius;

[0023] Fig.12 This is the Spearman correlation analysis of the blood, immune organ antioxidant, immune indicators and intestinal health indicators (DAO, D-LA, i.e. DD) of Leizhou black ducks under the action of yeast ZTHY2, where A is blood and DD, B is bursa of Fabricius and DD, C is spleen and DD, and D is thymus and DD. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0030] The embodiment of the present invention provides the use of Rhodotorula glutinosus ZTHY2 in improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks.

[0031] In some specific embodiments, the carcass traits are dressing rate and evisceration rate.

[0032] In some specific embodiments, promoting the intestinal health of Leizhou black ducks includes: promoting intestinal development, maintaining the integrity of intestinal morphology, increasing the relative abundance of Actinobacteria, and reducing the relative abundance of Proteobacteria and its subordinate α-Proteobacteria and γ-Proteobacteria.

[0033] The embodiment of the present invention further provides a feed additive for improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks, wherein the feed additive comprises red yeast ZTHY2.

[0034] The embodiment of the present invention also provides a feed for improving the carcass traits of Leizhou black ducks and promoting the intestinal health of Leizhou black ducks. The feed includes the additive or red yeast ZTHY2.

[0035] The Rhodotorula glutinosus ZTHY2 used in the present invention is deposited in China Center for Type Culture Collection with the number M2015296. Lactobacillus acidophilus (BNCC185342) was purchased from Beina Biotechnology Co., Ltd.

[0036] Red yeast ZTHY2 was inoculated into a culture medium (1% yeast extract, 2% glucose, 2% peptone, 2% sodium chloride), and cultured at 30°C, 180r / min, and shaken for 72h. The bacterial solution was collected and centrifuged (conditions: 4°C, 4000r / min, 10min), the supernatant was discarded, and the solution was washed with sterile saline, and the process was repeated twice to prepare a bacterial suspension. The number of live bacteria in the bacterial suspension was calculated by the dilution plate method, and the concentration was adjusted to the required concentration according to the test needs.

[0037] Lactobacillus acidophilus was inoculated in MRS culture medium and cultured anaerobically at 37℃180r / min in a shaking incubator for 24h. After collecting the bacterial solution, centrifuge it (conditions: 4℃, 8000r / min, 10min), discard the supernatant, resuspend and wash twice with sterile saline to prepare a bacterial suspension. The number of viable bacteria was calculated by the dilution plate method, and finally adjusted to the required concentration according to the test needs.

[0038] Example 1

[0039] Effects of Rhodotorula on slaughter performance and meat quality of Leizhou black duck

[0040] 1 Test method

[0041] 150 one-day-old male Leizhou black ducks with good health and similar body weight were selected and randomly divided into 5 groups, with 3 replicates in each group and 10 Leizhou black ducks in each replicate. The control group was fed a basal diet, and the RM1, RM2, and RM3 groups were fed a basal diet with 2×10 7 CFU / kg, 2×10 8 CFU / kg, 2×10 9 CFU / kg Rhodotorula ZTHY2, LA group added 2×10 9 CFU / kg Lactobacillus acidophilus, the test period was 42 days.

[0042] 1.1 Slaughter performance

[0043] Fasting for 12 hours in advance, at 42 days of the experimental period, two Leizhou black ducks with similar weight and health conditions were selected from each replicate group to weigh their pre-slaughter weight. Subsequently, the experimental ducks were bled from the jugular vein, the carcass weight was weighed, and the breast and leg muscles on both sides were dissected and separated and collected. The slaughter rate, half-eviscerated rate, full-eviscerated rate, breast muscle rate, leg muscle rate and lean meat rate were measured and calculated according to the agricultural industry standard (NY / T 823-202).

[0044] Slaughter rate (%) = carcass weight / pre-slaughter weight × 100;

[0045] Half-evisceration rate (%) = half-evisceration weight / body weight before slaughter × 100;

[0046] Eviscerated rate (%) = eviscerated weight / weight before slaughter × 100;

[0047] Leg muscle rate (%) = weight of leg muscles on both sides / total eviscerated weight × 100;

[0048] Chest muscle rate (%) = weight of chest muscles on both sides / total eviscerated weight × 100;

[0049] Lean meat rate (%) = (weight of chest muscles on both sides + weight of leg muscles on both sides) / total carcass weight × 100.

[0050] 1.2 Determination of meat quality indicators

[0051] The breast and leg muscles on the left side of the 42-day-old Leizhou black duck were selected to measure pH, meat color, water loss rate, cooking loss rate, and shear force. The test indicators are as follows:

[0052] 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.

[0053] pH 45min : 45-60 minutes after slaughter, take the breast muscle and leg muscle from three different parts, insert the pH meter probe into the meat sample to measure the pH value, record it after the value stabilizes, and take the average of the values ​​of the three parts as the final pH 45min value.

[0054] pH 24h : Store the meat samples in a 4℃ refrigerator and take them out 24 hours after slaughter to measure their pH 24h Numerical value, determination method is the same as pH 45min .

[0055] Water loss rate: Use a sharp round sampler to cut two pieces of meat samples (1 cm thick and about 2.5 cm in diameter) from the chest muscle and leg muscle, weigh them and record the weight as m1. Place the meat samples on a pressurized table and cover them with 16 layers of qualitative filter paper on the top and bottom. Apply a pressure of 35 kg for 5 minutes. After the pressurization ends, weigh the weight and record it as m2.

[0056] Water loss rate (%) = (m1-m2) / m1×100%.

[0057] Cook loss: Store breast and leg muscles at 4°C until 24 hours after slaughter to measure cooking loss. Cut the meat sample into a rectangular shape along the muscle fiber direction and weigh it as m3. Put the meat sample into a ziplock bag and heat it in a water bath at 80°C until the center temperature of the meat sample reaches 75°C. After the meat sample cools to room temperature, use filter paper to absorb the surface moisture and weigh it as m4.

[0058] Cooking loss (%) = (m3-m4) / m3×100%.

[0059] Shear force: Use a meat tenderizer to measure the shear force of the cooked meat sample. Measure each meat sample twice and calculate the average value.

[0060] 1.3 Determination of conventional nutrient content in muscle

[0061] After removing the surface fat and fascia of the pectoral muscle and leg muscle, the nutritional components of the pectoral muscle and leg muscle of 42d Leizhou black duck were determined: moisture, crude ash, crude fat and crude protein.

[0062] The moisture content is determined by the direct drying method in accordance with the national standard GB / 5009.3-2016; the crude fat content is determined by the Soxhlet extraction method in accordance with the national standard GB / 5009.6-2016; the crude protein content is determined by the Kjeldahl nitrogen determination method in accordance with the national standard GB / 5009.5-2016; and the crude ash content is determined by referring to the national standard GB / 5009.4-2016.

[0063] 1.4 Determination of fatty acid and amino acid content in muscle

[0064] The muscle tissue of 42-day-old Leizhou black duck was minced into meat paste and the fatty acid and amino acid content in the muscle of Leizhou black duck was detected. The determination method of amino acids in this test was carried out in accordance with the national standard GB 5009.124-2016. The amino acid content in the muscle was detected using a fully automatic amino acid analyzer, and the amino acid content in the muscle was analyzed by comparing the retention time and peak area of ​​the amino acid standard.

[0065] The determination method of fatty acids refers to the national standard GB5009.168-2016. After the meat sample is hydrolyzed and the fat is extracted, the fat is saponified and methylated to generate fatty acid methyl esters, which are analyzed by gas chromatography and combined with the relative retention time of fatty acid methyl ester standards, and the percentage of each fatty acid is calculated according to the peak area normalization method.

[0066] 1.5 Data Analysis

[0067] SPSS26.0 statistical software was used for one-way analysis of variance and Duncan's multiple comparisons. P < 0.05 was considered significant, and P < 0.01 was considered extremely significant. The results were expressed as mean ± standard deviation and plotted using GraphPad Prism8 software.

[0068] 2 Test results

[0069] 2.1 Effect of Rhodotorula glutinosae ZTHY2 on the slaughter performance of Leizhou black duck

[0070] As shown in Table 1, compared with the control group, the RM3 group can significantly improve the carcass rate of Leizhou black duck (P<0.05); with the increase of the addition amount of red yeast ZTHY2, the whole carcass rate, half carcass rate, breast muscle rate and leg muscle rate increased to varying degrees, but no significant difference was found (P>0.05).

[0071] Table 1 Effect of red yeast ZTHY2 on the slaughter performance of Leizhou black duck (%)

[0072]

[0073] 2.2 Effect of Rhodotorula glutinosae ZTHY2 on the quality of Leizhou black duck meat

[0074] As shown in Table 2, compared with the control group, the L* and b* values ​​of the flesh color in the pectoral muscle and leg muscle of the RM3 group were significantly reduced (P<0.05), and the a* value of the pectoral muscle of the RM3 group was significantly increased (P<0.05); the RM3 group could significantly increase the pH value of the pectoral muscle of Leizhou black duck. 24h (P<0.05) and significantly promoted leg muscle pH 24h The water loss rate of the pectoral and leg muscles in the RM3 group was significantly reduced (P<0.05).

[0075] Table 2 Effect of red yeast ZTHY2 on the quality of Leizhou black duck meat

[0076]

[0077]

[0078] 2.3 Effects of Rhodotorula glutinosae ZTHY2 on the content of conventional nutrients in Leizhou black duck

[0079] As shown in Table 3, compared with the control group, the RM3 group and the LA group could significantly increase the crude protein content of the breast muscle and leg muscle of Leizhou black duck (P<0.05), and the RM1, RM3 and LA groups significantly reduced the crude fat content of the breast muscle (P<0.05); Rhodotorula ZTHY2 had no significant effect on the moisture and crude ash content in the muscle of Leizhou black duck (P>0.05).

[0080] Table 3 Effect of red yeast ZTHY2 on the content of conventional nutrients in Leizhou black duck muscle (%)

[0081]

[0082] 2.4 Effect of Rhodotorula ZTHY2 on Amino Acids in Leizhou Black Duck Muscle

[0083] As shown in Table 4, compared with the control group, the threonine and tyrosine contents in the leg muscle of Leizhou black duck in the RM3 group were significantly increased (P<0.05), the methionine content in the LA group was significantly decreased (P<0.05), and the contents of TFAA, EAA, NEAA and DAA in each RM group were higher than those in the control group, but the differences were not significant (P>0.05).

[0084] Table 4 Effect of red yeast ZTHY2 on amino acids in Leizhou black duck muscle (%)

[0085]

[0086]

[0087] Note: Umami amino acids include: glycine, alanine, aspartic acid, glutamic acid, phenylalanine, and tyrosine.

[0088] 2.5 Effect of Rhodotorula ZTHY2 on the fatty acid composition of Leizhou black duck muscle

[0089] As shown in Table 5, compared with the control group, the RM groups significantly reduced the proportion of neuraminic acid in the muscles of Leizhou black duck (P<0.05), and the proportion of myristic acid, neuraminic acid and linoleic acid in the muscles of the LA group was significantly reduced (P<0.05); the proportion of SFA and PUFA in the muscles of the RM groups was lower than that of the control group, and the proportion of MUFA was higher than that of the control group (P>0.05).

[0090] Table 5 Effect of red yeast ZTHY2 on the fatty acid composition of Leizhou black duck muscle (%)

[0091]

[0092]

[0093] Slaughter performance can be used to evaluate the growth performance and nutrient deposition efficiency of livestock and poultry, and directly reflect the economic benefits of livestock and poultry farming. Meat ducks with good meat performance usually have a slaughter rate of more than 80% and a evisceration rate of more than 60%. In this example, the slaughter rate of Leizhou black ducks was 85.22-88.62%, and the evisceration rate was more than 72.80-76.13%, showing its superior meat production performance. Adding 2×10 9 CFU / kg red yeast ZTHY2 can significantly improve the slaughter rate of Leizhou black duck.

[0094] In this embodiment, feeding red yeast ZTHY2 can significantly reduce the water loss rate and pH value of the breast and leg muscles of Leizhou black ducks. 24 and L* and b* values ​​in flesh color, and as the addition amount increases to 2×10 9CFU / kg, leg muscle a* value significantly increased, and Rhodotorula ZTHY2 had a tendency to improve the tenderness of the muscle. The results of this example also showed that the addition of Rhodotorula ZTHY2 could increase the crude protein content in the breast and leg muscles of Leizhou black duck and reduce the crude fat content in the breast muscle.

[0095] In this example, Rhodotorula glutinosae ZTHY2 promoted the accumulation of total amino acids (TEAA), essential amino acids (EAA), non-essential amino acids (NEAA) and umami amino acids (DAA) in the muscle tissue of Leizhou black duck, and when the addition amount of Rhodotorula glutinosae ZTHY2 was 2×10 9 CFU / kg significantly increased the content of flavor amino acids: threonine and tyrosine. Tyrosine is an aromatic amino acid, and its increased content can improve the quality and flavor of meat. According to the test results, Leizhou black duck is a relatively high-quality protein source, and adding rhodotorula ZTHY2 to the diet can further improve the nutritional level and flavor value of high-quality protein in muscle.

[0096] In summary, Rhodotorula ZTHY2 significantly reduced the proportion of neuraminic acid in the muscle of Leizhou black duck, while increasing the proportion of monounsaturated fatty acids (MUFA). Although the proportion of neuraminic acid decreased, the proportion of high-quality fatty acids such as palmitoleic acid and oleic acid increased. Overall, Rhodotorula ZTHY2 can improve the fatty acid composition of muscle by reducing the proportion of saturated fatty acids (PUFA) and polyunsaturated fatty acids (SFA) and increasing the proportion of MUFA to a certain extent, thereby improving the nutrition and flavor of meat, and has no adverse effects on the fatty acid composition of Leizhou black duck muscle.

[0097] Example 2

[0098] Effects of Rhodotorula glutinosae ZTHY2 on intestinal health of Leizhou black ducks

[0099] 1. Serum barrier factor determination and intestinal tissue morphology analysis

[0100] 1.1 Serum barrier factor determination

[0101] The 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.

[0102] 1.2 Analysis of intestinal tissue morphology

[0103] (1) Sampling: The intestinal tissues of 21d and 42d were fixed in 4% paraformaldehyde solution for more than 72h. A scalpel blade was used to cut out intestinal segments of about 2-3 mm in size, with the two ends of the cross section parallel and flat.

[0104] (2) Dehydration and transparency: Place the tissue in an embedding box and rinse it slowly with running water for 5 hours overnight, then perform gradient alcohol dehydration, which are: 70% alcohol for 12 hours, 75% alcohol for 2 hours, 80% alcohol for 30 minutes, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes, and anhydrous ethanol for 45 minutes. After dehydration, perform transparency, which are: xylene alcohol mixture for 15 minutes, xylene I for 10 minutes, and xylene II for 10-15 minutes.

[0105] (3) Wax immersion embedding: embedding in wax I (xylene: paraffin 1:1) for 45 min, wax II for 50 min, and wax III for 50 min.

[0106] (4) Sectioning: Use a microtome to slice at a constant speed with a thickness of 5 μm. Spread the slices at 48°C, carefully remove them with a glass slide, mark them, and finally place them in a constant temperature box at 36°C to dry overnight.

[0107] (5) HE staining: First, the sections were dewaxed and hydrated with xylene I and II for 10 min each, anhydrous ethanol I and II for 2 min each, 95% alcohol for 2 min, 85% alcohol for 2 min, and 75% alcohol for 2 min, and then washed with water. Then, staining was started with hematoxylin staining for 8 min, washing with water, differentiation solution for 10 s, washing with water for blueing, eosin staining for 6 min, and washing with water.

[0108] (6) Dehydration, transparency and sealing: 70% alcohol for 10 seconds, 80% alcohol for 30 seconds, 90% alcohol for 30 seconds, anhydrous ethanol I and II for 1 minute each, xylene I and II for 5 minutes each, and finally use neutral resin to seal the slides. The sealing process should be gentle to avoid section displacement and bubble generation.

[0109] (7) Image collection and measurement: After the sample is completely dried and solidified, it is observed under a microscope to measure the intestinal villus height (VH) and crypt depth (CD), and the villus-crypt ratio (V / C) is calculated.

[0110] 1.3 Analysis of intestinal flora structure

[0111] On the 42nd day of the experimental period, two Leizhou black ducks with similar body weights were selected from each replicate group for slaughter, and the cecal contents were aseptically collected in a sterile centrifuge tube, immediately quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator for testing.

[0112] After the steps of total DNA extraction of microbiome, PCR amplification of target fragments, recovery and purification of amplified products and fluorescence quantification, the sequence was sequenced using PacBioSequel sequencer, and finally the sequence was denoised using QIIME2dada2 analysis process. After filtering out sequences with abundance less than 5, the final amplicon sequence variation SVs and feature table were obtained to analyze the microbial flora of the cecal contents of Leizhou black ducks. The above experimental processes were all completed with the assistance of Nanjing Paisonno Gene Technology Co., Ltd.

[0113] 1.4 Data Analysis

[0114] Same as Example 1.

[0115] 2 Test results

[0116] 2.1 Effects of Rhodotorula glutinosae ZTHY2 on serum barrier factors and intestinal tissue morphology of Leizhou black ducks

[0117] 2.1.1 Effects of Rhodotorula glutinosae ZTHY2 on DAO activity and D-LA content in Leizhou black duck

[0118] like Figure 1 As shown in Figure A, compared with the control, the RM1 group significantly reduced the serum DAO activity of Leizhou black ducks (P<0.05), and the RM2 and RM3 groups extremely significantly reduced the DAO activity (P<0.01). Figure 1 As shown in B, the RM1 group significantly reduced the serum D-LA content of Leizhou black ducks (P<0.05), and the RM3 group extremely significantly reduced the D-LA content (P<0.01).

[0119] 2.1.2 Effects of Rhodotorula glutinosae ZTHY2 on intestinal morphology of Leizhou black ducks

[0120] As shown in Table 6, compared with the control group, the addition of red yeast ZTHY2 to the feed can promote the growth of intestinal villus length in the duodenum, jejunum and ileum of Leizhou black ducks at 21 days and 42 days, and increase the villus-to-crypt ratio of each intestinal segment, and effectively reduce the crypt depth of the jejunum at 21 days and 42 days. 9 CFU / kg, the effect was most significant (P<0.01). Figure 2 and Figure 3 As shown, from the intestinal structural morphological observation, it can be seen that the intestinal villi structure of the duodenum, jejunum and ileum of each group are complete, the layers are clear, the crypts are clear and obvious, and no obvious lesions were observed; compared with the control group, after feeding with red yeast ZTHY2, the intestinal villi of Leizhou black ducks were arranged more closely, the edges were more complete, and the integrity of the intestinal morphological structure of Leizhou black ducks was better maintained.

[0121] Table 6 Effects of red yeast ZTHY2 on the intestinal morphology of Leizhou black ducks

[0122]

[0123]

[0124] 2.2 Effect of Rhodotorula glutinosae ZTHY2 on the intestinal flora structure of Leizhou black ducks

[0125] 2.2.1 Analysis of sequencing sequence and OTU validity

[0126] In this study, high-throughput sequencing was used to sequence and analyze the cecal flora of Leizhou black ducks. Figure 4 As shown in A, there were 145 ASVs in the cecal content samples of the Control group, RM1 group, RM2 group, RM3 group and LA group, among which the Control group, RM1 group, RM2 group, RM3 group and LA group had 282 ASVs, 386 ASVs, 383 ASVs, 556 ASVs and 454 ASVs, respectively. The results showed that feeding Rhodotorula colloidinum ZTHY2 could change the structure of the cecal flora of Leizhou black ducks.

[0127] Rarefaction curves can be used to assess the number of samples sequenced and compare the species richness in each group of samples, such as Figure 4 As shown in Figure B, the dilution curve of each sample tends to be flat as the sequencing depth increases. The results show that the sample size of this sequencing is reasonable and the sequencing depth is sufficient to cover the species characteristics of the cecal flora of Leizhou black ducks in each group.

[0128] 2.2.2 Alpha Diversity Analysis

[0129] The Chao1 index can reflect the species richness in the sample. The species richness in the bacterial community increases as the value increases. Figure 5 As shown in A, there was no significant difference in Chao1 index among the samples in each group, indicating that there was no significant difference in species richness among the groups.

[0130] The Shannon index reflects the diversity of species in a sample. The larger the value, the higher the diversity of species in the sample. Figure 5 As shown in B, there was no significant difference in the Shannon index of the samples in each group, indicating that there was no significant difference in species diversity among the groups. However, compared with the Control group, the Shannon index of the RM1 group and the RM2 group increased (P﹥0.05).

[0131] 2.2.3 Beta diversity analysis

[0132] The principal coordinate analysis (PCoA) based on the Bray-Curtis distance was used to show the β diversity of the bacterial flora. Figure 6It can be seen that there are significant differences in the bacterial flora composition of each group (R 2 =0.1962, P=0.001). There was no overlap between the Control, RM1, and LA groups and the RM2 and RM3 groups, indicating that there were significant differences in the bacterial community composition between the Control, RM1, and LA groups and the RM2 and RM3 groups. The RM3 group was far away from the other groups, indicating that there were obvious inter-group differences in the intestinal flora composition of the RM3 group of meat ducks.

[0133] 2.2.4 Analysis of bacterial community structure and richness at the phylum level

[0134] Based on the analysis of the bacterial flora structure of phyla, the results of 16S amplicon sequencing showed that the intestinal bacteria of Leizhou black ducks contained 3119 ASVs representing 14 different phyla. Figure 7 As shown in A, the top ten phyla in terms of abundance include: Bacteroidota, Desulfobacterota, Firmicutes, Proteobacteria, Verrucomicrobiota, Fusobacteriota, Elusimicrobiota, Deferribacteres, Spirochaetota, and Cyanobacteria. Based on the test results, it can be seen that Bacteroidetes is the dominant phylum in the intestine of Leizhou black duck. Figure 7 As shown in Figures 7B and 7C, compared with the Control group, the relative abundance of Proteobacteria in the RM3 group was significantly decreased (P<0.05), and the relative abundance of Actinobacteria in the RM1 group was extremely significantly increased (P<0.01).

[0135] 2.2.5 Analysis of bacterial community structure and richness at the class level

[0136] Based on the analysis of microbial flora structure at the class level, the top 10 classes with the highest relative abundance of species were selected for visualization. Figure 8 As shown in A, the top ten classes in abundance include: Bacteroidia, Clostridia, Desulfovibrionia, Negativicutes, Bacilli, Gammaproteobacteria, Alphaproteobacteria, Fusobacteria, Deferribacteres, and Vampirovibrionia. Figure 8 As shown in B and C, compared with the Control group, the relative abundance of α-Proteobacteria in the RM3 group was significantly reduced (P<0.05), and the relative abundance of γ-Proteobacteria in each RM group showed a downward trend (P﹥0.05).

[0137] 2.2.6 Analysis of bacterial community structure and richness at the order, family and genus levels

[0138] Analysis of bacterial flora structure based on order, family and genus levels, such as Fig. 9 As shown in Figures A-1 and B-3, compared with the control group, the addition of red yeast ZTHY2 to the feed can reduce Clostridia_UCG-014 and Rikenellaceae, but the difference is not significant (P﹥0.05); Fig. 9 As shown in B-1, B-2 and C-1, the relative abundance of the coprostanologenic Eubacterium group and its subordinate genera ([Eubacterium]_coprostanoligenes_group) in group RM1 was significantly decreased (P<0.05); the relative abundance of the Oscillospiraceae family (Oscillospiraceae) in groups RM2 and RM3 was increased (P﹥0.05).

[0139] 2.2.7 Linear discriminant analysis of intestinal flora

[0140] LEfSe analysis, or linear discriminant analysis, is an analytical tool for the discovery and interpretation of biological features of high-dimensional data. It enables comparisons between two or more different groups, and can also be used to analyze comparisons between different subgroups in the same group, so as to find biological features with significant differences between groups, i.e., biomarkers. In this experiment, based on the genus-level intestinal flora of the five groups, linear judgment was used to find biomarkers that can distinguish different groups. At the same time, a bar chart is used to display taxonomic units with LDA values ​​greater than 2. The LDA value is used to estimate the impact of the taxonomic unit on the differences in intestinal flora between groups. The larger the LDA value, the higher the contribution of the taxonomic unit to the differences in intestinal flora between groups.

[0141] like Fig.10As shown, in this example, 2 phyla, 2 classes, 5 orders, 5 families, 12 genera, and 5 species were found to be significantly different between the groups, and these taxa will also be used as biomarkers to distinguish the groups. In the Control group, 1 phylum, 1 class, 2 orders, 2 families, 2 genera, and 2 species were identified, and the genus and species levels were: Rikenellaceae-RC9 genus (Rikenellaceae_RC9_gut_group), Lactobacillus genus (Lactobacillus), Streptococcus alactolyticus (Streptococcus_alactolyticus), Lachnoclostridium_phocaeense. In the RM1 group, 1 phylum, 1 class, 1 order, 2 families, 4 genera, and 2 species were identified, and the genus and species levels were Prevotellaceae_Ga6A1_group genus, olobacterium genus, Olsenella genus (Olsenella), Bacteroides_gallinaceum species, and Lactobacillus gingivalis (Olsenella). In the RM2 group, one order, one family, and two genera were identified, of which Monoglobus and Peptococcus were identified at the genus level. In the RM3 group, one order, two families, and one genus were identified, of which Muribaculaceae was identified at the genus level. In the LA group, one genus was identified, which was Eubacterium_brachy_group.

[0142] In this example, the addition of red yeast ZTHY2 can significantly reduce the serum DAO activity and D-LA content of Leizhou black ducks, indicating that red yeast ZTHY2 has a protective effect on the intestinal mucosa and maintains the integrity of the intestinal mucosa. Red yeast ZTHY2 significantly increases the VH and V / C ratios of the duodenum, jejunum and ileum of Leizhou black ducks, and reduces the CD values ​​of the jejunum at 21d and 42d, thereby promoting intestinal development and maintaining the integrity of intestinal morphology.

[0143] In this example, through Alpha diversity analysis, it was found that although Rhodotorula ZTHY2 could improve the Shannon index of the cecum of Leizhou black ducks, it had no significant effect on the richness of the flora; in β diversity analysis, Rhodotorula ZTHY2 could affect the composition of the intestinal flora, and there was a significant difference compared with the control group. This shows that Rhodotorula ZTHY2, as a feed additive, can affect the relative abundance of specific flora by regulating the composition of the intestinal flora without affecting Alpha diversity.

[0144] This example further explored the effect of Rhodotorula Glucans ZTHY2 on intestinal flora by analyzing the changes in flora structure and richness at different classification levels. The results showed that Bacteroidetes dominated the intestines of Leizhou black ducks, and feeding Rhodotorula Glucans ZTHY2 could significantly increase the relative abundance of Actinobacteria and reduce the relative abundance of Proteobacteria and their subordinate α-Proteobacteria and γ-Proteobacteria.

[0145] Example 3

[0146] Correlation analysis of ZTHY2 yeast on antioxidant and immune indicators in the blood of Leizhou black ducks, antioxidant and immune indicators in immune organs, and intestinal health indicators

[0147] After the Leizhou black ducks were fed with yeast ZTHY2, Spearman correlation analysis was performed on their blood antioxidant and immune indicators and the antioxidant and immune indicators of immune organs. The results showed that blood antioxidant enzymes CAT, GSH-Px, SOD were positively correlated with bursal antioxidant enzymes CAT, SOD, IL-6, TNF-α (p<0.05), while MDA was negatively correlated with these enzymes (p<0.05). Except for C4, various blood immune indicators were positively correlated with bursal antioxidant enzymes (CAT, SOD, GSH-Px) and cytokines (IL-4, IL-6, TNF-α) (p<0.05), such as Fig.11 As shown in A. In the spleen, blood antioxidant enzymes were positively correlated with spleen enzymes and cytokines (IL-6, TNF-α) (p<0.05), and MDA was negatively correlated with GSH-Px and SOD (p<0.05). Fig.11 As shown in B. In the thymus, blood antioxidant indexes were positively correlated with thymus index (GSH-Px, SOD, IL-6, IL-2) (p<0.05, IL-2 was significant at p<0.01), while MDA was negatively correlated with GSH-Px (p<0.05), as shown in Fig.11 As shown in C. Spearman correlation analysis of the antioxidant and immune indicators of the thymus and bursa showed that except for IL-2 and IFN-γ in the bursa (p<0.05, IL-2 was significantly higher than p<0.01), the other indicators were positively correlated with those in the thymus, such as Fig.11 As shown in D.

[0148] Spearman correlation analysis showed that the blood parameters, antioxidant and immune indicators of Leizhou black ducks were negatively correlated with intestinal health indicators after ZTHY2 treatment. DAO and D-LA were negatively correlated with GSH-Px, SOD, TNF-α, IL-2, IgG, C3, IFN-γ and IL-4 in the blood (p<0.05, Fig.12 In the bursa of Fabricius, DAO and D-LA were negatively correlated with GSH-Px, IL-4, IL-6 and TNF-α (p<0.05, Fig.12 In spleen, only DAO was negatively correlated with TNF-α and IL-6 (p<0.05, Fig.12 In the thymus, DAO and D-LA were generally negatively correlated with gut health markers, except for GSH-Px (p<0.05), and especially with IL-2 (p<0.01, Fig.12 (middle D).

[0149] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The application of red yeast ZTHY2 in preparing feed for improving the carcass traits of Leizhou black duck and promoting the intestinal health of Leizhou black duck, characterized in that: The carcass trait is slaughter rate; the promoting intestinal health of Leizhou black duck is to reduce α - Relative abundance of Proteobacteria; the deposit number of the red yeast ZTHY2 is CCTCC NO: M2015296; The amount of red yeast ZTHY2 added to the feed is 2×10 9 CFU / kg.

Images