Correlation analysis method for microbial flora structures in liquid milk of horses in different regions

Abstract

The invention discloses a correlation analysis method for microbial flora structures in liquid milk of local horses in different regions, and relates to the technical field of microbial resource analysis of the liquid milk of the local horses, and the method is technically characterized by comprising the following steps: S1, sample collection and treatment: collecting fresh horse milk and naturally fermented horse milk samples of the local horses from at least three regions, mixing samples in the same region; s2, metagenome sequencing: extracting total DNA of a sample to construct a sequencing library, and performing metagenome sequencing by adopting an Illumina double-end sequencing platform; and S3, carrying out bioinformatics analysis on the genome. The invention provides a method for accurately analyzing microbial flora resources of local horse fresh milk and traditional fermented liquid milk, which is simple to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial resource analysis of local horse liquid milk, and specifically relates to a method for analyzing the correlation of microbial flora structures in local horse liquid milk from different regions. Background Art

[0002] Horse milk is a dairy product resource with high nutritional value and special fermentation characteristics. In recent years, with the increasing demand for functional dairy products, the composition, function of the microbial flora in horse milk and its relationship with the geographical environment have gradually become research hotspots.

[0003] During the fermentation process of horse milk, different microbial flora will affect the fermentation process. Currently, there are few studies on the regional differences analysis of the microbial flora in horse milk from different regions, and there is an urgent need for a method for analyzing the correlation of microbial flora in local horse liquid milk. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide a method for analyzing the correlation of microbial flora structures in local horse liquid milk from different regions.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] The present invention provides a method for analyzing the correlation of microbial flora structures in local horse liquid milk from different regions, including the following steps:

[0007] Step S1: Sample collection and processing: Collect fresh horse milk and naturally fermented horse milk samples of local horses from at least three regions, and mix and process the samples from the same region;

[0008] Step S2: Metagenomic sequencing: Extract the total DNA of the sample to construct a sequencing library, and perform metagenomic sequencing using the Illumina paired-end sequencing platform;

[0009] Step S3: Perform bioinformatics analysis on the genome.

[0010] The present invention is further set as: The specific sub-steps of the said Step S2 are:

[0011] Step S21: Use a genomic DNA extraction kit to extract the DNA of the sample;

[0012] Step S22: After fragmenting the DNA to 300bp, perform gene library preparation;

[0013] Step S23: Use Qubit4.0 for library quantification and Qsep400 to detect the inserted fragments of the library;

[0014] Step S24: After the library passes the inspection, different libraries are mixed for sequencing according to the requirements of molar concentration and the target output data volume, and then sequenced using Illumina PE150.

[0015] The present invention is further configured as: The specific sub-steps of step S3 are as follows:

[0016] Step S31: Calculate the dilution curves of Core genes and Pan genes, and analyze the distribution characteristics of common genes and total genes among samples;

[0017] Step S32: Conduct Alpha diversity analysis, and calculate Obs index, Chao1 index, Shannon index, and Simpson index;

[0018] Step S33: Perform species annotation through the NR database, and statistically analyze the relative abundances at the phylum, family, genus, and species classification levels;

[0019] Step S34: Use LEfSe analysis to screen for differential species with an LDA value > 4.

[0020] The present invention is further configured as: The Core gene is defined as the set of common genes in all samples, and the Pan gene is defined as the union of the gene sets of all samples.

[0021] Compared with the prior art, the beneficial effects of this solution: The present invention reveals the influence of geographical factors on the mare milk microbiota through the mixing of samples from multiple regions and cross-regional comparison, integrating Qubit-Qsep400 joint quality control and Illumina PE150 sequencing, combined with Core / Pan gene analysis and LEfSe screening. The present invention provides a simple and accurate method for analyzing mare milk microbiota. Description of the Drawings

[0022] Figure 1 It is the core-pan gene dilution curve diagram in the embodiment of the present invention, where (a) is the core gene dilution curve diagram, and (b) is the pan gene dilution curve diagram;

[0023] Figure 2 It is the bar chart of the relative abundances of species at the phylum (a), family (b), genus (c), and species (d) levels of mare milk from different regions in the embodiment of the present invention. Among them, Figure a is the bar chart of the relative abundances of species at the phylum level, Figure b is the bar chart of the relative abundances of species at the family level, Figure c is the bar chart of the relative abundances of species at the genus level, and Figure d is the bar chart of the relative abundances of species at the species level;

[0024] Figure 3It is a bar chart of the LDA values of differential species in fresh horse milk (a) and fermented horse milk (b) from different regions in the embodiments of the present invention. Among them, a is the bar chart of the LDA values of differential species in fresh horse milk from different regions, and b is the bar chart of the LDA values of differential species in fermented horse milk from different regions;

[0025] Figure 4 It is a bar chart of the relative abundances of bacteria (a) and fungi (b) in horse milk from different regions in the embodiments of the present invention. Among them, a is the bar chart of the relative abundance of bacteria at the species level, and b is the bar chart of the relative abundance of fungi at the species level. Detailed implementation manners

[0026] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0027] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0028] Embodiment:

[0029] 1.1 Sample treatment

[0030] The samples were collected from Altay, Tacheng, and Yili regions in northern Xinjiang. Four sampling points were selected in each region, and 3 batches of fresh horse milk (fermented for 0 h) and naturally fermented horse milk (fermented for 48 h) were collected at each sampling point. A total of 12 batches of samples were collected, and each batch of samples was a mixed milk of 10 horses, with a total of 120 horses. The fresh horse milk and fermented horse milk from the same region were mixed separately, and three replicates were set, resulting in a total of 18 samples for systematic analysis of the microbial results in horse milk from the same region. See Table 1 for details

[0031] Table 1 Information of horse milk samples in different regions Table 1 information of horse milk samples in different regions

[0032]

[0033] 1.2 Total DNA extraction, library construction, and high-throughput sequencing analysis

[0034] The genomic DNA of 18 mare milk samples from three regions was extracted using a genomic DNA extraction kit. The extracted genomic DNA was detected by 1% agarose gel electrophoresis. The DNA samples were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for metagenomic sequencing. The qualified DNA samples were fragmented into fragments of about 300 bp in length by enzymatic digestion. The whole library preparation was completed through steps such as end repair, A-tailing, addition of sequencing adapters, purification, and PCR amplification. After the library construction was completed, Qubit 4.0 was first used for preliminary quantification, and then Qsep 400 was used to detect the insert size of the library. The insert size met the expectation and the quality inspection was qualified. After the library inspection was qualified, different libraries were pooled according to the requirements of molar concentration and the target output data volume, and then Illumina PE150 sequencing was carried out.

[0035] 2.1 Analysis of the microbial community structure of mare milk in different regions of northern Xinjiang

[0036] As Figure 1 shown, based on the gene abundance table in each sample, the gene number information of each sample can be obtained. By randomly selecting different numbers of samples, the gene numbers between different sample combinations can be obtained, and thus the dilution curves of Core and Pan genes are constructed and drawn. The abscissa is the number of randomly selected samples, and the ordinate is the number of core genes and pan genes in the sample combination. Core genes are the common genes of all samples, the intersection. As the number of samples increases, the common genes become fewer and fewer; Pan genes are the genes contained in all samples, the union, and also increase as the number of samples increases.

[0037] 2.2 Alpha diversity analysis

[0038] Alpha diversity reflects the microbial diversity and richness of mare's milk in different regions. The Obs index represents the number of species directly observed; the Chao1 index is an index used to estimate the number of species contained in a sample by the chao1 algorithm. The larger the Chao1 index, the higher the richness of the community; the Shannon index indicates that the uncertainty of microbial species is large. The greater the uncertainty, the more unknown factors there are in the sample microbial community, that is, the higher the diversity; the Simpson index characterizes the diversity and evenness of species distribution within a microbial community. Based on Illumina high-throughput sequencing, using the paired-end sequencing method, a total of 295,926 reads were obtained from fresh mare's milk and naturally fermented koumiss samples collected from three regions in northern Xinjiang. Among them, 153,661 were clean reads. As shown in Table 2, both the Obs index and Chao1 index of microorganisms in the fresh mare's milk samples from Altay were significantly higher than those of other mare's milk samples, indicating that the number of microbial species in the fresh mare's milk from Altay was the largest, that is, the richness was the highest. While the Shannon index and Simpson index of microorganisms in the fresh mare's milk samples from Tacheng were significantly higher than those of other mare's milk samples, indicating that the diversity and evenness of the microbial community in the fresh mare's milk from Tacheng were the highest. The reason why the Shannon index and Simpson index were not the highest in the fresh mare's milk samples from Altay may be due to the uneven distribution of microorganisms.

[0039] Table 1 Microbial diversity of mare's milk in different regions

[0040]

[0041] 2.3 Microbial composition in mare's milk from different regions

[0042] Based on gene-based taxonomic annotation and alignment with the NR database, species and abundance information at each taxonomic level (kingdom, phylum, class, order, family, genus, species) in each sample were obtained. A total of 60 phyla, 90 classes, 170 orders, 315 families, 777 genera, and 2,995 species were identified through NR species annotation.

[0043] As Figure 2 shown, the microbial abundance and diversity in fresh milk and naturally fermented milk directly affect their quality, flavor, and safety. To better understand the microbial flora structure in mare's milk from different regions, starting from the relative abundance table at different taxonomic levels, the top 10 species with the largest relative abundance in each sample were selected, and the remaining species were set as Others. The relative abundance bar charts of the species annotation results corresponding to each sample at different taxonomic levels were drawn.

[0044] As Figure 2As can be seen, the dominant phylum of mare's milk in all three regions is Bacillota, with a relative abundance of over 65%. Among them, the relative abundance of Tacheng koumiss is the highest. The second most dominant phylum is Pseudomonadota, while the second most dominant phylum of Yili koumiss is Ascomycota. Thus, although the microbial flora of mare's milk from different regions shows similarities at the phylum level, there are significant differences in the microbial flora of Yili koumiss at the phylum level. Figure 2 As can be seen from b, the main family of mare's milk in all three regions is Lactobacillaceae. The second most dominant family of Tacheng fresh mare's milk and Yili fresh mare's milk is Streptococcaceae, while the second most dominant family of Altay fresh mare's milk is Acetobacteraceae. Figure 2 As can be seen from c, the main genus of mare's milk in all three regions is Lactobacillus. The second most dominant genus of Tacheng fresh mare's milk and Yili fresh mare's milk is Lactococcus, while the second most dominant genus of Altay fresh mare's milk is Acetobacter. It can be seen from the family level and genus level that the microbial flora of fresh mare's milk from different regions is more diverse, indicating that the grassland environment, temperature, altitude, and water source in different regions directly affect the microbial flora in fresh mare's milk. Figure 2 As can be seen from d, the top seven species in terms of richness are Lactobacillus helveticus, Lactococcus lactis, Lactobacillus orientalis, Kluyveromyces marxianus, Lactobacillus durans, Acetobacter pasteurianus, and Lactobacillus kefiranofaciens. The dominant species of mare's milk in all three regions is Lactobacillus helveticus. Among them, the distribution of the top seven species in terms of richness in Tacheng fresh mare's milk is more uniform. In addition, all three regions' mare's milk samples contain foodborne pathogenic bacteria such as Enterococcus, Enterobacter, Klebsiella, etc. The presence of pathogenic bacteria is due to the fact that traditional fermented yak dairy products are usually produced in family workshops using an open natural fermentation production process, which is easily affected by milking methods, the physiological conditions and feeding methods of yaks, and the dairy product production methods. Improving the environmental health conditions during the production process helps reduce or avoid infection by pathogenic bacteria and stabilize the quality and safety of yak dairy products.

[0045] 2.4 LEfSe analysis

[0046] Through LEfSe analysis, taking the characteristic bacteria with an LDA greater than 4 as representatives, Figure 3 it can be seen that the differential species in Altay fresh mare's milk are Alphaproteobacteria, Acetobacteraceae, Rhodospirillales, Acetobacter, Acetobacter_orientalis, Lactobacillus, Lactobacillus_helveticus, and Lactobacillaceae.

[0047] The differential species in Tacheng fresh mare's milk are s__Kluyveromyces_marxianus, f__Enterococcaceae, g__Kluyveromyces, g__Enterococcus, g__Enterobacter, c__Gammaproteobacteria, and s__Enterococcus_durans.

[0048] The differential species in Ili fresh mare's milk are c__Bacilli, o__Lactobacillales, g__Lactococcus, f__Streptococcaceae, p__Bacillota, s__Lactococcus_lactis, and k__Bacteria.

[0049] The differential species in Altay fermented mare's milk are p__Pseudomonadota, o__Rhodospirillales, c__Alphaproteobacteria, s__Acetobacter_pasteurianus, f__Acetobacteraceae, g__Acetobacter, s__Lacticaseibacillus_paracasei, f__Lactobacillaceae, and c__Bacilli.

[0050] The differential species in Tacheng fermented mare's milk are c__Gammaproteobacteria, g__Lactococcus, s__Lactococcus_lactis, o__Enterobacterales, s__Kazachstania_unispora, c__Actinomycetes, and p__Actinomycetota.

[0051] The differential species of Ili kefir are p__Ascomycota, o__Saccharomycetales, g__Brettanomyces, g__Kluyveromyces, c__Saccharomycetes, s__Kluyveromyces_marxianus, k__Eukaryota, f__Saccharomycetaceae.

[0052] The above specific embodiments are only explanations of the present invention, and they are not limitations on the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without creative contributions as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A correlation analysis method for the microbial flora structure in the liquid milk of local horses from different regions, characterized by: The following steps are involved: Step S1: Sample collection and processing: fresh mare's milk and naturally fermented mare's milk samples of local horses are collected from at least three regions, and samples from the same region are mixed and processed; Step S2: Metagenomic sequencing: extract total DNA from samples to construct sequencing libraries, and use Illumina paired-end sequencing platform for metagenomic sequencing; Step S3: Perform bioinformatics analysis on the genome.

2. The method for correlation analysis of microbial flora structure in local horse liquid milk from different regions as claimed in claim 1, characterized in that: The specific sub-steps of step S2 are: Step S21: extracting sample DNA using a genomic DNA extraction kit; Step S22: After the DNA is fragmented to 300 bp, a gene library is prepared; Step S23: Use Qubit4.0 to quantify the library and Qsep400 to detect the inserted fragments of the library; Step S24: After the library is qualified, different libraries are mixed and sequenced according to the molar concentration and the target data volume, and then sequenced using Illumina PE150.

3. The method for correlation analysis of microbial flora structure in local horse liquid milk from different regions as claimed in claim 1, characterized in that: The specific sub-steps of step S3 are: Step S31: Calculate the dilution curves of the Core gene and the Pan gene, and analyze the distribution characteristics of the common genes and total genes among the samples; Step S32: performing Alpha diversity analysis and calculating Obs index, Chao1 index, Shannon index and Simpson index; Step S33: Annotate species through the NR database and count the relative abundance of the phylum, family, genus, and species classification levels; Step S34: LEfSe analysis was used to screen differential species with LDA values ​​> 4.

4. The method for correlation analysis of microbial flora structure in local horse liquid milk from different regions as claimed in claim 3, characterized in that: The Core gene is defined as a set of genes common to all samples, and the Pan gene is defined as the union of all sample gene sets.

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