Method for detecting bacterial community diversity in fermentation process of Yi medicine fermentation liquor

Abstract

The invention discloses a method for detecting bacterial community diversity in the fermentation process of Yi medicine fermentation liquor, and belongs to the technical field of biological information and gene detection. According to the method, 16S rRNA gene sequences of microorganisms of Yi medicine fermentation liquor at different fermentation times are analyzed through a high-throughput sequencing technology, and 16S rRNA gene sequence information of various bacteria existing in a sample is obtained. A bioinformatics method is utilized to compare and analyze bacterial species, abundance and distribution conditions among different samples, so that the change rule of microbial flora of the Yi medicine fermentation liquor in the fermentation process is known, and the change of the microbial flora is monitored and analyzed, so that the Yi medicine fermentation liquor can be obtained. The method is of great significance in better controlling the fermentation process to improve the quality and effect of the Yi medicine fermentation liquor.

Description

Technical Field

[0001] The invention relates to the technical field of bioinformatics and gene detection, and more specifically to a method for detecting the diversity of bacterial communities in the fermentation process of Yi medicine fermentation liquid. Background Art

[0002] Chinese ethnic medicine is an important part of traditional medicine, among which Yi medicine has a long history and rich theories. After long-term practical verification, its efficacy is accurate and reliable, and it has made positive contributions to the development of my country's medical and health care. Yi medicine fermentation liquid is a fermentation liquid prepared in a low-temperature, metal-free environment. Its uniqueness lies in that it combines a variety of natural plants with microorganisms for co-fermentation to obtain rich and diverse nutrients, which has certain health care and disease prevention and treatment effects.

[0003] However, there are few studies on the microbial diversity in the fermentation process of Yi medicine fermentation broth. Therefore, providing a method for detecting the diversity of bacterial communities in the fermentation process of Yi medicine fermentation broth is of great significance for better controlling the fermentation process and improving the quality and effect of Yi medicine fermentation broth. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for detecting the diversity of bacterial communities during the fermentation process of Yi medicine fermentation liquid.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid comprises the following steps:

[0007] (1) Sample pretreatment: According to the fermentation cycle, samples were collected from 7 batches of Yi medicine fermentation broth with different fermentation times, and samples were collected from 5 sampling points in each batch; each batch of samples was mixed, sealed in sterile bottles, and placed in an ice box at -80°C;

[0008] (2) Extraction of genomic DNA: extracting genomic DNA of microorganisms from each sample in step (1);

[0009] (3) PCR amplification: using a primer set to amplify the microbial genomic DNA in each sample extracted in step (2), detecting the size of the amplified target band by 1% agarose gel electrophoresis, and purifying the PCR product;

[0010] (4) Illumina MiSeq high-throughput sequencing: The PCR amplification products were subjected to Illumina MiSeq high-throughput sequencing;

[0011] (5) Illumina MiSeq high-throughput sequencing data processing: filtering and splicing the high-throughput sequencing raw data obtained in step (4) to obtain high-quality sequences;

[0012] (6) Data analysis: The upparse algorithm in Vsearch software was used to perform OTU clustering on high-quality sequences and perform taxonomic analysis.

[0013] Specifically, the Yi medicine fermentation liquid is prepared by crushing or slicing 5 to 100 parts of roxburghii, 5 to 100 parts of honey, 1 to 30 parts of emblica, 1 to 30 parts of walnuts and 1 to 30 parts of lemons, adding 0.1 to 5% yeast and mixing evenly, and taking the supernatant after aerobic fermentation to obtain the liquid.

[0014] Preferably, the sampling of the seven batches in step (1) is specifically carried out on a monthly basis, namely, the first month, the second month, the third month, the fourth month, the sixth month, the eighth month and the tenth month at the beginning of fermentation.

[0015] Specifically, the primer set described in step (3) is: universal primer 338F / 806R.

[0016] Preferably, before the Illumina MiSeq high-throughput sequencing in step (4), the concentration of the detection library needs to be measured.

[0017] Preferably, the filtering and splicing described in step (5) are specifically as follows: using Pear software to remove N bases and low-quality parts, and setting the minimum overlap to 10 bp and the p-value to 0.0001; using Vsearch software to remove sequences less than 230 bp in length, and using the uchime method to compare with the Gold Database to remove chimera sequences.

[0018] Preferably, the similarity threshold of the OTU clustering in step (6) is set to 97%.

[0019] Preferably, the taxonomic analysis in step (6) is specifically: evaluating the abundance and diversity of the microbial community by the Alpha diversity index; and comparing the differences in community composition between different samples by Beta diversity analysis.

[0020] Preferably, the Alpha diversity index in step (6) includes a rarefaction curve, a Chao1 index and a Shannon index.

[0021] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for detecting the diversity of bacterial communities during the fermentation process of Yi medicine fermentation liquid, and obtains the 16S rRNA gene sequence information of various bacteria present in the sample by analyzing the 16S rRNA gene sequences of microorganisms at different fermentation times of Yi medicine fermentation liquid through high-throughput sequencing technology. By using bioinformatics methods, the bacterial species, abundance and distribution between different samples are compared and analyzed, so as to understand the change law of the microbial flora during the fermentation process of Yi medicine fermentation liquid, and by monitoring and analyzing the changes in the microbial flora, it is of great significance to better control the fermentation process to improve the quality and effect of Yi medicine fermentation liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Dilution curve of bacteria in each sample of Yi medicine fermentation broth at different fermentation times;

[0023] Figure 2 :Shannon-Wiener curve of bacterial community in Yi medicine fermentation broth at different fermentation times;

[0024] Figure 3 :Bacterial community distribution at the phylum level during the fermentation of Yi medicine fermentation liquid;

[0025] Figure 4 : Distribution of bacterial communities at the genus level in the fermentation process of Yi medicine fermentation liquid;

[0026] Figure 5 :The principal component analysis results of Yi medicine fermentation broth based on genus level samples;

[0027] Figure 6 : OTU-Venn diagram of each sample of Yi medicine fermentation broth at different fermentation times;

[0028] A represents the first batch of sampling, B represents the second batch of sampling, C represents the third batch of sampling, D represents the fourth batch of sampling, E represents the fifth batch of sampling, F represents the sixth batch of sampling, and G represents the seventh batch of sampling. The numbers 1-5 after the same letter represent 5 different collection points in the same batch of samples. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0031] The following is an example description:

[0032] Yi medicine fermentation liquid (Guizhou Jinqianguo Biotechnology Co., Ltd.);

[0033] DNA extraction kit ( Soil DNA Kit, Omega Bio, USA);

[0034] Nucleic acid purification kit (AgencourtAMPure XP, Beckman Coulter, USA);

[0035] High-throughput sequencer (Illumina Miseq PE300, Illumina, USA);

[0036] DNA quality tester (Nanodrop 2000, Thermo Fisher Scientific, USA);

[0037] electrophoresis instrument (CaliperLabChip GX Touch HT, PerkinElmer, USA);

[0038] Fluorescence PCR instrument (ABI 9700 PCR instrument, Applied Biosystems, USA).

[0039] Example 1 Method

[0040] (1) Samples were collected from 7 batches of Yi medicine fermentation liquid at different fermentation times, and samples were collected from 5 sampling points for each batch. Fermentation began in September, and the first batch was sampled in October; the second batch was sampled in November; the third batch was sampled in December; the fourth batch was sampled in January of the following year; the fifth batch was sampled in February of the following year; the sixth batch was sampled in April of the following year; and the seventh batch was sampled in June of the following year. All batches of "Jieben Liquid" samples were mixed separately, sealed in sterile bottles, placed in ice boxes and brought back to the laboratory, and stored in a -80℃ refrigerator.

[0041] (2) Using a DNA extraction kit, extract the genomic DNA of the microorganisms in the fermentation sample of step (1).

[0042] (3) The microbial genomic DNA in each sample extracted in step (2) was amplified using universal primers 338F / 806R on a fluorescent PCR instrument, and the size of the amplified target band was detected by 1% agarose gel electrophoresis (170 V voltage, 30 minutes). The PCR product was automatically purified using a nucleic acid purification kit.

[0043] (4) After PCR amplification, the library was tested (2% agarose gel electrophoresis) and the library concentration was measured (Qubit 2.0 fluorescence quantification instrument). Finally, Beijing Aoweisen Gene Technology Co., Ltd. was commissioned to perform Illumina MiSeq high-throughput sequencing on the PCR amplification products.

[0044] (5) The raw double-end data obtained by high-throughput sequencing contains primers, adapters, and barcode labels. The data needs to be filtered and spliced. Specifically, the Pear software is used to remove N bases and low-quality parts of the data, and the minimum overlap is set to 10 bp and the p-value is set to 0.0001; the Vsearch software is used to remove sequences less than 230 bp in length, and the uchime method is used to compare with the Gold Database to remove chimeric sequences.

[0045] (6) The uprease algorithm in the Vsearch software was used to perform OTU clustering on high-quality sequences (the similarity threshold was set at 97%), and taxonomic analysis was performed. Specifically, the abundance and diversity of the microbial community were evaluated using the Alpha diversity index, which included parameters such as the rarefaction curve, Chao1 index, and Shannon index; and the differences in community composition between different samples were compared using Beta diversity analysis.

[0046] Example 2 Results and Analysis

[0047] 1. Data optimization and statistics

[0048] Yi medicine fermentation broth samples were sequenced using the Illumina Miseq sequencing platform, and 4,617,727 raw data were obtained. After splicing and filtering, the number of valid sequences was 4,488,113. These sequences were clustered into OTUs (Operational Taxonomic Units) at a similarity of 97%, and the structure and diversity information of the microbial community was obtained. After screening, a total of 3,373 OTUs were generated, and after leveling, the number was optimized to 3,044 OTUs. The results are shown in Table 2

[0049] Table 1 Sequencing results and OTU clustering results of each sample in high-throughput sequencing

[0050]

[0051]

[0052] 2. Dilution curve and diversity curve analysis

[0053] The dilution curves of bacteria in each sample are shown in Figure 1 , the Shannon-Wiener curve of bacterial community is shown in Figure 2 By comparison Figure 1-2 The changes in OTU species between different samples show that the bacterial community in the Yi medicine fermentation broth has a high diversity, and as the number of sequencing increases, the growth rate of the number of OTU species gradually slows down and eventually approaches stability. This shows that despite the increasing amount of sequencing data, the number of bacterial species in the Yi medicine fermentation broth has gradually reached saturation. Figure 2 It can be seen that the bacterial microbial community in the Yi medicine fermentation liquid has a low β-diversity. This indicates that the bacterial community composition differences between different samples are small, which may be due to the uniform distribution and relative stability of bacterial species in the "Jieben Liquid".

[0054] 3. Analysis of α-diversity of bacterial communities in different fermentation processes of Yi medicine fermentation broth

[0055] The α-diversity analysis of the bacterial community during the fermentation process of "Jieben Liquid" was carried out, mainly including the abundance of the bacterial community and the number of bacterial species. The results are shown in Table 2.

[0056]

[0057] As shown in Table 2, in each batch of Yi medicine fermentation liquid samples, the bacterial sequencing depth index Coverage exceeded 0.99. These data results show that the sequencing depth used is sufficient and most fungal microbial information has been detected, indicating that the data obtained in this sequencing is reasonable and sufficient in quantity, and can provide sufficient data support for the α-diversity analysis and β-diversity analysis of bacterial microorganisms in "Jieben Liquid".

[0058] The number of OTUs displayed in the Chao1 index sample was used to analyze the richness of the bacterial community. Generally, the Chao1 index increases when the number of bacterial species increases. According to the results in Table 2, the Chao1 index decreased in the fermentation samples in December, January of the following year, and June of the following year, indicating that the richness of the bacterial community decreased during the fermentation process. This may be because as the fermentation time goes by, some bacterial species have an advantage in the competition for survival, while other bacteria are gradually eliminated.

[0059] The number of bacterial species in the samples was analyzed by the Simpson index and the Shannon index. The more bacterial species there were, the higher the Shannon value and the lower the Simpson index. On the contrary, the fewer bacterial species there were, the lower the Shannon value and the higher the Simpson index. According to the results in Table 2, as the Shannon index of the fermented samples in December gradually increased, the Simpson index also showed the same growth trend, indicating that the diversity of the microbial community was gradually increasing; when the Shannon index of the fermented samples in January of the following year decreased, the Simpson index also showed a corresponding decreasing trend, indicating that the diversity of the microbial community was decreasing. This may be because the abundance of certain specific microbial species in the community decreased, resulting in a decrease in overall diversity.

[0060] 4. Analysis of β-diversity of bacterial communities in different fermentation processes of Yi medicine fermentation broth

[0061] (1) Analysis of bacterial flora structure in different fermentation processes of Yi medicine fermentation broth based on phylum level

[0062] The top 20 dominant bacteria ranked at the phylum level were selected for comparative analysis of relative abundance, and those with lower relative abundance were classified into one category (Others). The results are shown in Figure 3 .

[0063] Figure 3 It can be seen that Cyanobacteria (55.61%), Proteobacteria (36.24%), and Firmicutes (4.48%) are the dominant phyla. The sum of the relative abundances of these three bacterial phyla in each fermentation sample is greater than 96%, which fully demonstrates that at the phylum level, the bacterial flora structure of Yi medicine fermentation broth in different fermentation processes is stable. In the early and middle and late stages of fermentation, Cyanobacteria is the absolute dominant phylum, while Proteobacteria is the dominant phylum in the fermentation process of the December fermentation sample. Proteobacteria gradually increases its relative abundance in the early stage of fermentation and shows an overall upward trend, reaching the highest value of 57.74% in the fermentation stage of the December fermentation sample. The relative abundance of Cyanobacteria was relatively stable, reaching a minimum of 18.08% in the fermentation stage of the fermented samples from December to January of the following year, and a maximum of 76.04% in the fermentation stage of the fermented samples from January to December of the following year. The relative abundance of Firmicutes fluctuated slightly, ranging from 1.15% to 15.08%, indicating the stability and relative equilibrium of this phylum in the fermentation environment. This stability may be related to its strong ecological adaptability and high tolerance to environmental changes.

[0064] (2) Analysis of bacterial flora structure of fermented mash in different fermentation processes of Yi medicine fermentation liquor at genus level

[0065] The top 20 dominant bacteria ranked at the genus level were selected for comparative analysis of relative abundance, and those with lower relative abundance were classified into one category (Others). The results are shown in Figure 4 .

[0066] Figure 3 It can be seen that Chloroplast (55.54%) is the dominant genus in the sample; Mitochondria (9.89%), Pantoea (6.36%), and Others (8.78%) are relatively dominant genera; the genera that account for more than 1% include Acinetobacter (2.73%), Herbaspirillum (1.95%), Gluconobacter (1.51%), Lactobacillus (1.38%), Pseudomonas (1.36%), and Sphingomonas (1.02%). The sum of the relative abundance of relatively dominant bacterial genera in different fermentation samples is 90.52%, indicating their important position in the fermentation process.

[0067] In addition, by Figure 4 The results showed that Chloroplast was the dominant genus in the fermentation process of Yi medicine fermentation liquid. Its relative abundance showed a trend of first decreasing and then increasing in the early stage of fermentation. In the fermentation stage of the fermentation sample in January of the following year, the relative abundance of Chloroplast reached the highest value of 76.03%. This shows that at this stage, Chloroplast began to regain its dominant position. This may be because at this stage, the fermentation environment changed, which was conducive to the growth of Chloroplast. During the fermentation stage of the December fermentation samples, the bacterial community structure underwent obvious dynamic changes. The relative abundances of Chloroplast and Mitochondri were 17.84% and 0.22%, respectively, which were the lowest values ​​in different fermentation processes, indicating that these two genera may have been inhibited during the fermentation process of the December fermentation samples; at the same time, the abundances of some other genera also showed obvious changes in this stage. The changes in the two genera Pantoea and Others showed similar trends. There was no obvious change in the early stage of fermentation, but in the fermentation stage of the December fermentation samples, the relative abundance of Pantoea dropped to the lowest value of 2.21%, while Others rose to 25%, the highest value, indicating that the fermentation environment at this stage provided more suitable growth conditions for Others, which affected the survival and reproduction of Pantoea, resulting in a significant decrease in its relative abundance during the fermentation stage of the December fermentation samples.

[0068] (3) Principal component analysis of fermentation samples during the fermentation of Yi medicine fermentation broth based on genus level

[0069] Using R software, principal component analysis (PCA) was performed on samples from the fermentation process of "Jieben Liquid" at the genus level. The results are shown in Figure 5 , OTU-Venn diagram of each sample is shown in Figure 6 .

[0070] In PCA, the greater the distance between points, the greater the difference in their bacterial communities; conversely, the closer the sample points are, the more similar the two species compositions are. Figure 5 The PCA results show that the bacterial principal coordinate PC1 is 46.73% and the ordinate PC2 is 13.15%. These principal components represent most of the variable information, which can reflect the significant differences in samples with different fermentation times. Among them, the distances between the October fermentation samples, the November fermentation samples, the January fermentation samples of the following year, the February fermentation samples of the following year, and the April fermentation samples of the following year are relatively close, indicating that the bacterial community differences between these samples are small, which indicates that during the fermentation process of "Jieben Liquid", the bacterial community composition of these samples at the genus level is highly similar. However, the December fermentation sample is the farthest from the other samples, indicating that the bacterial community of this sample is quite different from that of other samples, which indicates that the bacterial community composition of this sample at the genus level is significantly different from that of other samples.

[0071] Figure 6 It can reflect the dynamic changes of the flora of Yi medicine fermentation liquid during the fermentation process, as well as the number of unique and common bacterial species among multiple groups of fermentation samples. Based on NMDS analysis, these fermentation samples were divided into seven groups, which had a total of 140 OTUs. Among them, the number of OTUs that were the same between the fermentation samples in October and November, the fermentation samples in November and December, the fermentation samples in December and the fermentation samples in January of the following year, the fermentation samples in January of the following year and the fermentation samples in February of the following year, and the fermentation samples in February of the following year and the fermentation samples in April of the following year were 70, 16, 16, 26, 122, and 3, respectively. This result shows that there are certain connections and similarities between these groups, representing different fermentation conditions or different combinations of fermentation flora. At the same time, samples in different groups may be affected by different microbial communities, resulting in differences in fermentation products. The number of unique OTUs in these groups also shows the characteristics and differences of each group.

[0072] In addition, by Figure 6It can be seen that during the fermentation process of Yi medicine fermented liquid, the similarity between the fermented samples in October and November, and between the fermented samples in February of the following year and the fermented samples in April of the following year was high, indicating that the bacterial community composition between these samples was relatively close, and these bacterial communities may play similar functions and roles in the fermentation process, jointly providing the necessary substances and energy for the fermentation of Yi medicine fermented liquid, promoting the fermentation of Yi medicine fermented liquid and forming similar flavors and tastes. However, the similarity of the bacterial communities of the samples fermented in April of the following year and the samples fermented in June of the following year was low, indicating that there were large differences in the bacterial community composition of the two samples, and these differences may play different roles in the fermentation process, thus further affecting the fermentation process and final product of Yi medicine fermented liquid.

[0073] In summary, the present invention provides a method for detecting the diversity of bacterial communities during the fermentation process of Yi medicine fermentation liquid, and for the first time, conducts a detailed analysis of the bacterial community structure and its changes during the fermentation process of Yi medicine fermentation liquid, providing basic data for further exploring the relationship between the formation of flavor substances in Yi medicine fermentation liquid and the characteristics of microbial communities. At the same time, it lays a foundation for screening dominant microbial strains for brewing Yi medicine fermentation liquid, and provides new ideas and data support for realizing the modernization, scale and standardized production of Yi medicine fermentation liquid.

[0074] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0075] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid, characterized in that: The following steps are involved: (1) Sample pretreatment: According to the fermentation cycle, samples were collected from 7 batches of Yi medicine fermentation broth with different fermentation times, and samples were collected from 5 sampling points in each batch; each batch of samples was mixed, sealed in sterile bottles, and placed in an ice box at -80°C; (2) Extraction of genomic DNA: extracting genomic DNA of microorganisms from each sample in step (1); (3) PCR amplification: using a primer set to amplify the microbial genomic DNA in each sample extracted in step (2), detecting the size of the amplified target band by 1% agarose gel electrophoresis, and purifying the PCR product; (4) Illumina MiSeq high-throughput sequencing: The PCR amplification products were subjected to Illumina MiSeq high-throughput sequencing; (5) Illumina MiSeq high-throughput sequencing data processing: filtering and splicing the high-throughput sequencing raw data obtained in step (4) to obtain high-quality sequences; (6) Data analysis: The upparse algorithm in Vsearch software was used to perform OTU clustering on high-quality sequences and perform taxonomic analysis.

2. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The Yi medicine fermentation liquid is prepared by crushing or slicing 5-100 parts of roxburghii, 5-100 parts of honey, 1-30 parts of emblica, 1-30 parts of walnut and 1-30 parts of lemon, adding 0.1-5% yeast and mixing evenly, and taking supernatant after aerobic fermentation to obtain the liquid.

3. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The sampling of the seven batches described in step (1) is specifically as follows: sampling is carried out on a monthly basis, namely, the first month, the second month, the third month, the fourth month, the sixth month, the eighth month and the tenth month at the beginning of fermentation.

4. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The primer set described in step (3) is: universal primer 338F / 806R.

5. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: Before the Illumina MiSeq high-throughput sequencing described in step (4), the concentration of the detection library needs to be measured.

6. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The filtering and splicing described in step (5) are specifically as follows: using Pear software to remove N bases and low-quality parts, and setting the minimum overlap to 10 bp and the p-value to 0.0001; using Vsearch software to remove sequences less than 230 bp in length, and using the uchime method to compare with the Gold Database to remove chimera sequences.

7. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The similarity threshold of OTU clustering in step (6) was set to 97%.

8. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The taxonomic analysis in step (6) is specifically as follows: evaluating the abundance and diversity of the microbial community through the Alpha diversity index; and comparing the differences in community composition between different samples through Beta diversity analysis.

9. The method for detecting bacterial community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The Alpha diversity index in step (6) includes a rarefaction curve, a Chao1 index and a Shannon index.

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