Use of rumen microbial markers in identifying the meat quality grade of chinese black snowflake beef and in preparing products for identifying the meat quality grade of chinese black snowflake beef

Abstract

The application discloses application of a rumen microbial marker in identifying meat quality grades of Chinese black hair snow flower beef cattle and preparing products for identifying meat quality grades of Chinese black hair snow flower beef cattle, relates to the technical field of biological detection, and the rumen microbial marker comprises at least one of Fibrobacter , Sharpea , Bradymonadales , Mogibacterium The application first finds that the four kinds of rumen microorganisms can be used alone or jointly to identify the meat quality grades of Chinese black hair snow flower beef cattle, so that the meat quality grades of the Chinese black hair snow flower beef cattle to be detected can be quickly and accurately confirmed, and the rumen microbial marker has a wide application prospect in breeding and grade identification of Chinese black hair snow flower beef cattle.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to the application of a rumen microbial marker in identifying the beef quality grade of Chinese Black Marble beef and in preparing products for identifying the beef quality grade of Chinese Black Marble beef. Background Technology

[0002] Chinese Black Marble Beef Cattle is an internationally recognized high-grade and high-quality beef cattle breed, with its marbling grade being a core economic trait.

[0003] Existing methods for distinguishing the meat quality grade of Chinese Black Wagyu beef cattle are mostly based on meat composition testing or visual inspection. These methods are not very accurate and are labor-intensive. Summary of the Invention

[0004] The main objective of this invention is to propose the application of rumen microbial markers in identifying the beef quality grade of Chinese Black Marble beef and in preparing products for identifying the beef quality grade of Chinese Black Marble beef, aiming to provide a method that can more accurately identify the beef quality grade of Chinese Black Marble beef.

[0005] To achieve the above objectives, this invention proposes the application of a rumen microbial marker in the preparation of products used to identify the quality grade of Chinese Black Marble beef, wherein the rumen microbial marker includes... Fibrobacter , Sharpea , Bradymonadales , Mogibacterium At least one of them.

[0006] In one embodiment, the product includes at least one of test strips, reagent kits, and microfluidic chips.

[0007] In one embodiment, the product includes a kit, which includes at least one of a real-time quantitative PCR kit, a multiplex PCR / nucleic acid microarray kit, a digital PCR kit, a high-throughput sequencing kit, a colloidal gold immunochromatographic kit, and an enzyme-linked immunosorbent assay (ELISA) kit.

[0008] In one embodiment, the product includes a kit comprising primers and probes, the primers and probes being nucleic acids, and the primers and probes comprising targeting... Fibrobacter , Sharpea , Bradymonadales , Mogibacterium At least one of the primers and probes.

[0009] In one implementation, the following steps are included: S1. Provide Chinese Black Wagyu beef cattle to be tested and obtain the types and levels of their rumen microbial markers; S2. Compare the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested with the rumen microbial markers of known A1-2, A3, A4, and A5 grades of Chinese Black Wagyu beef cattle to determine the grade of the Chinese Black Wagyu beef cattle to be tested.

[0010] In one embodiment, step S2, the determination method includes: If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain... Fibrobacter If the absolute abundance is ≥106.19, it is determined to be a Chinese Black Sheep marbled beef cattle of grade A1-2; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain Sharpea If the absolute abundance is ≥111.54, it is determined to be A3 grade Chinese Black Wagyu beef cattle; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain Bradymonadales If the absolute abundance is ≥30.23, it is determined to be A4 grade Chinese Black Wagyu beef cattle; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain Mogibacterium If the absolute abundance is ≥105.38, it is determined to be A5 grade Chinese Black Wagyu beef cattle.

[0011] This invention also provides the application of rumen microbial markers in identifying the quality grade of Chinese Black Marble beef, wherein the rumen microbial markers include... Fibrobacter , Sharpea , Bradymonadales , Mogibacterium At least one of them.

[0012] In the technical solution of this invention, it is discovered for the first time that the above four rumen microorganisms can be used alone or in combination to identify the meat quality grade of Chinese Black Wagyu beef cattle, thereby quickly and accurately confirming the meat quality grade of the Chinese Black Wagyu beef cattle to be tested. This rumen microbial marker has broad application prospects in the breeding and grading of Chinese Black Wagyu beef cattle. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0014] Figure 1The figure shows the Alpha diversity analysis results of rumen microorganisms of Chinese Black-haired Snowflake beef cattle of grades A1-2 to A5 in Example 2 of the present invention. The left figure is the Chao1 analysis result figure and the right figure is the Shannon analysis result figure. Figure 2 This is an NMDS analysis diagram of the rumen microorganisms of Chinese Black-haired Snowflake beef cattle of grades A1-2 to A5 in Example 2 of the present invention; Figure 3 This is a diagram showing the phylum-level (left) and genus-level (right) community composition of rumen microorganisms in A1-2 to A5 grade Chinese Black-haired Snowflake beef cattle in Example 1 of the present invention. Figure 4 The image shows the LEfSe analysis results of rumen microorganisms of Chinese Black-haired Snowflake beef cattle of grades A1-2 to A5 in Example 3 of the present invention. The left image is an evolutionary branching diagram, and the right image is an LDA score diagram.

[0015] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Existing methods for distinguishing the meat quality grade of Chinese Black Wagyu beef cattle are mostly based on meat composition testing or visual inspection. These methods are not very accurate and are labor-intensive.

[0018] In view of this, the present invention proposes the application of a rumen microbial marker in the preparation of products used to identify the quality grade of Chinese Black Marble beef, wherein the rumen microbial marker includes Fibrobacter (Fibrobacterium spp.) Sharpea (Spp. Sharpel) Bradymonadales (Bradymonales) MogibacteriumAt least one of the genera (Mogibacterium).

[0019] In the technical solution of this invention, it is discovered for the first time that the above four rumen microorganisms can be used alone or in combination to identify the meat quality grade of Chinese Black Wagyu beef cattle, thereby quickly and accurately confirming the meat quality grade of the Chinese Black Wagyu beef cattle to be tested. This rumen microbial marker has broad application prospects in the breeding and grading of Chinese Black Wagyu beef cattle.

[0020] In some embodiments, the product includes at least one of test strips, reagent kits, and microfluidic chips. That is, the product can be any one, two, or three of the test strips, reagent kits, and microfluidic chips, all within the scope of this invention. The aforementioned various detection products combine the portability of rapid on-site screening with the high efficiency of automated laboratory testing, meeting the needs of different application scenarios.

[0021] In some embodiments, the product includes a kit, which comprises at least one of a real-time quantitative PCR kit, a multiplex PCR / nucleic acid microarray kit, a digital PCR kit, a high-throughput sequencing kit, a colloidal gold immunochromatographic kit, and an enzyme-linked immunosorbent assay (ELISA) kit. These kits are suitable for different detection methods and can flexibly balance detection sensitivity, accurate quantification, and high-throughput screening requirements.

[0022] In some embodiments, the product includes a kit comprising primers and probes, wherein the primers and probes are nucleic acids, and the primers and probes are formulated to target specific nucleic acids. Fibrobacter , Sharpea , Bradymonadales , Mogibacterium The primers and probes described above can be used to rapidly identify the types and levels of the aforementioned rumen microbial markers, facilitating rapid detection.

[0023] In some embodiments, the method includes the following steps: S1, providing Chinese Black Wagyu beef cattle to be tested and obtaining the types and levels of their rumen microbial markers; S2, comparing the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested with the rumen microbial markers of known A1-2, A3, A4, and A5 grades of Chinese Black Wagyu beef cattle to determine the grade of the Chinese Black Wagyu beef cattle to be tested.

[0024] Among them, the rumen microbial markers of Chinese Black Sheepflake beef cattle of grades A1-2, A3, A4, and A5 can be obtained by the following methods: (1) A large number of rumen fluid samples of Chinese Black Sheepflake beef cattle of four meat quality grades (A1-2, A3, A4, and A5) are selected, total microbial DNA is extracted from the rumen fluid samples, the hypervariable region of 16S rRNA gene V3-V4 is amplified by PCR, and sequencing is performed using the Illumina NovaSeq platform; (2) The raw data is quality controlled using the QIIME2 process, and amplicon sequence variants (ASVs) are obtained by DADA2 denoising, splicing, and chimera removal, and species annotation is completed based on the SILVA database; (3) Alpha diversity analysis is performed using Chao1 and Shannon indices, and Beta diversity is assessed by NMDS analysis based on Bray-Curtis distance; (4) Rumen microbial markers are screened by LEfSe analysis with an LDA threshold > 2. Since the number of samples selected in this application is large, the accuracy is high and can represent the status of most grades of Chinese Black Sheepflake beef cattle.

[0025] In some embodiments, in step S2, the determination method includes: if the rumen microbial markers of the Chinese Black-haired Snowflake beef cattle to be tested contain... Fibrobacter If the absolute abundance is ≥106.19, it is classified as A1-2 grade Chinese Black Sheep marbled beef cattle; if the rumen microbial markers of the Chinese Black Sheep marbled beef cattle being tested contain... Sharpea If the absolute abundance is ≥111.54, it is classified as A3 grade Chinese Black Sheep marbled beef cattle; if the rumen microbial markers of the Chinese Black Sheep marbled beef cattle being tested contain... Bradymonadales If the absolute abundance is ≥30.23, it is classified as A4 grade Chinese Black Sheep marbled beef cattle; if the rumen microbial markers of the Chinese Black Sheep marbled beef cattle being tested contain... Mogibacterium If the absolute abundance is ≥105.38, it is determined to be A5 grade Chinese Black Sheep marbled beef cattle. The determination method provided by this invention is relatively simple and fast.

[0026] This invention also provides the application of rumen microbial markers in identifying the quality grade of Chinese Black Marble beef, wherein the rumen microbial markers include... Fibrobacter , Sharpea , Bradymonadales , Mogibacterium At least one of them.

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0028] Example 1: Selection and preparation of Chinese Black Wagyu beef cattle samples 1. Laboratory animals and grouping Purebred Chinese Black Wagyu beef cattle from Qingdao Longming Cattle Industry Co., Ltd. in Shandong, China, were selected and divided into four groups (A1-2, A3, A4, and A5) according to the marbling pattern of the longissimus dorsi muscle and the standards of the Japan Meat Grading Association, with sample sizes of 138, 370, 261, and 221 head respectively. The feeding and management of these cattle complied with the ethical requirements for laboratory animal welfare and were approved by the institution's Animal Care and Use Committee.

[0029] 2. Feeding and Management The total breeding cycle is approximately 40 months, with nutritional regulation beginning during the cow's gestation period and continuing until the calf is slaughtered; 0 Supplementing 9-month-old calves with vitamins and minerals promotes rumen development; 18 20-month-old high-concentrate fattening, 13 At 20 months of age, vitamin A intake is reduced to promote intramuscular fat cell hypertrophy; from 21 months of age to slaughter, probiotics are added to increase feed intake. This is divided into the lactation period (0... 3 months old), weaning period (3 months) 6 months old), growth period (6 months) 18 months old), fattening period (18 months) (30 months old, divided into three stages: early, middle, and late).

[0030] 3. Sample collection Experimental cattle were slaughtered at 30±1 months of age. Rumen fluid was collected through an incision in the rumen wall using a sterile syringe, filtered through four layers of sterile gauze, flash-frozen in liquid nitrogen, and then transferred to… Store at 80℃.

[0031] Example 2: Rumen Microbial 16S rRNA Sequencing and Bioinformatics Analysis 1. DNA extraction and sequencing Total DNA was extracted from rumen fluid using the MagPure Soil DNA LQ Kit (Magan). The hypervariable region of the 16S rRNA gene V3-V4 was amplified using primers 343F / 798R. The PCR products were purified and sequenced on the Illumina NovaSeq platform.

[0032] 343F primer sequence: 5'-TACGGRAGGCAGCAG-3'; 798R primer sequence: 5'-AGGGTATCTAATCCT-3'.

[0033] 2. Data Preprocessing The raw data was processed using the QIIME2 workflow, and DADA2 was used for denoising, splicing, and removal of chimeras to obtain ASVs; species classification annotation was completed based on the SILVA database.

[0034] 3. Diversity Analysis Alpha diversity: The Chao1 and Shannon indices were calculated, and the results are as follows: Figure 1 As shown, the Kruskal-Wallis test shows the differences between groups.

[0035] Beta diversity: NMDS (Non-metric Multidimensional Scaling) analysis was performed based on Bray-Curtis distance, and the results are as follows: Figure 2 As shown, the PERMANOVA test was used to determine the significance of differences in community structure.

[0036] 4. Community composition analysis The absolute abundance of species at the phylum and genus levels was statistically analyzed, and the results are as follows: Figure 3 As shown.

[0037] The results showed that there were no significant differences in the Alpha diversity index among the four groups; NMDS analysis showed no significant differences in community structure; there were no differences among the dominant bacterial groups at the phylum level, while differences at the genus level were concentrated in low-abundance genera, with Bacteroidetes and Firmicutes accounting for over 70% of the total abundance at the phylum level; at the genus level... Prevotella The dominant bacteria were those with absolute abundance, while low-abundance bacterial groups showed intergroup differences. Muribaculaceae The abundance of the Chinese Black Wagyu beef gradually increases with the increase of meat quality grades A1-2 → A3 → A4 → A5.

[0038] Example 3: Screening and Validation of Rumen Microbial Markers 1. LEfSe analysis and absolute abundance analysis Setting the LDA threshold > 2, the results of screening for grade-specific marker microorganisms are as follows: Figure 4 As shown: Group A1-2 showed significant enrichment. Fibrobacter Group A3 was significantly enriched Sharpea Group A4 was significantly enriched Bradymonadales Group A5 was significantly enriched Mogibacterium The absolute abundance of the above four microorganisms in Chinese Black Wagyu beef cattle of different meat quality grades is shown in Table 1.

[0039] Table 1. Absolute abundance of bovine-specific rumen marker microorganisms at different meat quality grades

[0040] The values ​​in Table 1 represent the absolute abundance of microorganisms. Absolute abundance refers to the proportion of the effective sequencing read length corresponding to the target microorganism to the total effective sequencing read length of rumen microorganisms. It is statistically obtained based on the high-throughput sequencing results of the V3-V4 hypervariable region of the 16S rRNA gene. The sequencing work was commissioned to Nanjing Shadow Gene Biotechnology Co., Ltd.

[0041] The experimental data in the table above show that: FibrobacterThe absolute abundance was highest in A1-2 grade and bovine rumen samples; Sharpea The abundance reached its peak value in the A3 level samples. Bradymonadales The absolute abundance was the highest among all groups in the A4 level samples; Mogibacterium The absolute abundance gradually increased with beef quality grade from A1-2 to A5, reaching its maximum at grade A5. The above four types of microorganisms showed significant differences in abundance distribution among different grades of Wagyu cattle, and can serve as specific rumen microbial markers for distinguishing different marbling grades of Wagyu cattle.

[0042] The threshold values ​​used in this study uniformly adopted the industry-standard arithmetic mean method of adjacent feature groups (critical midpoint method). Combined with the characteristic that each marker bacterium exhibited abundance peaks at a single meat quality grade in this study, the arithmetic mean of the average abundance of the target grade and adjacent grades was used as the critical value. This effectively offset the influence of individual differences in beef cattle, sequencing errors, and sample pretreatment, ensuring stable and reliable judgment results. The sample sizes for each group in this experiment were A1-2: 138 head, A3: 370 head, A4: 261 head, and A5: 221 head, respectively. The data were statistically representative, and all threshold values ​​were calculated based on actual measured data without any manual experience-based assignment.

[0043] 2. Trend Microbial Analysis Muribaculaceae The abundance of the beef cattle increased continuously with the improvement of the grade of Chinese Black Wagyu beef cattle.

[0044] Results: Rumen microbial biomarkers at levels A1-2 to A5 of Chinese Black-haired Snowflake beef cattle were successfully constructed.

[0045] In summary, this invention, based on real experimental data, constructs a combination of rumen microbial markers related to the grade of Chinese Black Wagyu beef cattle, objectively revealing community characteristics, the distribution and increasing patterns of marker microorganisms; this invention does not involve meat quality testing, identification, or grading methods, and is authentic, objective, and compliant.

[0046] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. Use of a rumen microbial marker in the manufacture of a product for identifying the quality grade of Chinese black hair snow flower beef, characterized in that, The rumen microbial markers include at least one of Fibrobacter , Sharpea , Bradymonadales , Mogibacterium .

2. Use of the rumen microbial marker of claim 1 in the manufacture of a product for identifying the quality grade of Chinese black hair yak beef, characterized in that, The product includes at least one of test strips, reagent kits, and microfluidic chips.

3. Use of the rumen microbial marker of claim 2 in the manufacture of a product for identifying the quality grade of Chinese black hair yak beef, characterized in that, The product includes a kit, which includes at least one of the following: real-time quantitative PCR kit, multiplex PCR / nucleic acid microarray kit, digital PCR kit, high-throughput sequencing kit, colloidal gold immunochromatography kit, and enzyme-linked immunosorbent assay kit.

4. The application of the rumen microbial marker as described in claim 2 in the preparation of products for identifying the quality grade of Chinese Black Marble beef, characterized in that, The products include kits comprising primers and probes, the primers and probes being nucleic acids, the primers and probes comprising primers and probes for at least one of Fibrobacter , Sharpea , Bradymonadales , Mogibacterium .

5. The application of the rumen microbial marker as described in claim 1 in the preparation of products for identifying the quality grade of Chinese Black Marble beef, characterized in that, Includes the following steps: S1. Provide Chinese Black Wagyu beef cattle to be tested and obtain the types and levels of their rumen microbial markers; S2. Compare the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested with the rumen microbial markers of known A1-2, A3, A4, and A5 grades of Chinese Black Wagyu beef cattle to determine the grade of the Chinese Black Wagyu beef cattle to be tested.

6. The application of the rumen microbial marker as described in claim 5 in the preparation of products for identifying the quality grade of Chinese Black Marble beef, characterized in that, In step S2, the determination method includes: If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain Fibrobacter If the absolute abundance is ≥106.19, it is determined to be a Chinese Black Sheep marbled beef cattle of grade A1-2; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain... Sharpea If the absolute abundance is ≥111.54, it is determined to be A3 grade Chinese Black Wagyu beef cattle; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain... Bradymonadales If the absolute abundance is ≥30.23, it is judged to be A4 grade Chinese Black Sheep Snowflake Beef Cattle; If the rumen microbial markers of the Chinese Black Wagyu beef cattle to be tested contain Mogibacterium If the absolute abundance is ≥105.38, it is determined to be A5 grade Chinese Black Wagyu beef cattle.

7. The application of a rumen microbial marker in identifying the quality grade of Chinese Black Hair marbled beef, characterized in that, The rumen microbial markers include at least one of Fibrobacter , Sharpea , Bradymonadales , Mogibacterium .

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