Method for detecting diversity of fungal communities in fermentation process of Yi medicine fermentation liquor
Through the detection and analysis of the diversity of fungal communities during the fermentation of Yi medicine fermentation liquid, the problem of insufficient research on microbial diversity in the existing technology has been solved, better control of the fermentation process has been achieved, and product quality and effect have been improved.
Patent Information
- Application Number
- CN202411412005.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-03-29
- Filing Date
- 2024-10-11
- Publication Date
- 2025-02-18
AI Technical Summary
In the prior art, there are few researches on microbial diversity in the fermentation process of Yi medicine fermentation broth, and it is difficult to effectively control the fermentation process and improve product quality and effect.
A detection method is adopted, including sampling, genomic DNA extraction, PCR amplification, Miseq library construction and sequencing, data processing and analysis, and the diversity of fungal communities during the fermentation of Yi medicine fermentation liquid is carried out in detail.
By understanding the changes in microbial flora during the fermentation of Yi medicine fermentation broth, we can better control the fermentation process, thereby improving the quality and effect of Yi medicine fermentation broth.
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Figure CN119464538A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioinformatics and gene detection, and more specifically to a method for detecting fungal community diversity during 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 fungal 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 fungal community diversity 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 fungal community diversity during the fermentation process of Yi medicine fermentation liquid comprises the following steps:
[0007] (1) Sampling: According to the fermentation cycle, samples of 7 periods were selected from the Yi medicine fermentation liquid at different fermentation times, 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) Miseq library construction and sequencing;
[0011] (5) Data processing: OTU clustering is performed on non-repetitive and non-single sequences, and chimeras are removed during the clustering process to obtain representative sequences of OTUs;
[0012] (6) Data analysis: Perform taxonomic analysis on the OTU representative sequences obtained in step (5).
[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 seven periods in step (1) are: sampling on a monthly basis, the first month, the second month, the third month, the fourth month, the sixth month, the eighth month, and the tenth month from the start of fermentation.
[0015] Specifically, the primer set described in step (3) is universal primers ITS1 and ITS2.
[0016] Preferably, the similarity threshold of the OTU clustering in step (6) is set to 97%.
[0017] 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.
[0018] Preferably, the Alpha diversity index in step (6) includes a rarefaction curve, a Chao1 index and a Shannon index.
[0019] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for detecting the diversity of fungal communities during the fermentation process of Yi medicine fermentation liquid. By using bioinformatics methods, the fungal species, abundance and distribution between different samples are compared and analyzed, so as to understand the change pattern of the microbial flora during the fermentation process of Yi medicine fermentation liquid. 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
[0020] Figure 1 : Dilution curves of fungi in samples of Yi medicine fermentation broth at seven fermentation stages;
[0021] Figure 2 :Shannon-Wiener curves of fungal communities in seven fermentation stages of Yi medicine fermentation liquid;
[0022] Figure 3 : Venn diagram of fungal OTUs of samples from Yi medicine fermentation broth at seven fermentation stages;
[0023] Figure 4: Comparative box plot of Alpha diversity among sample groups at seven fermentation stages of Yi medicine fermentation broth; A is Chao1 index detection, B is Simpson index detection, and C is Shannon index detection;
[0024] Figure 5: Beta diversity analysis results of fungal communities of samples from Yi medicine fermentation broth at seven fermentation stages; Figure 5A is the fungal NMDS analysis result, and Figure 5B is the fungal PCoA analysis result;
[0025] Figure 6 : Relative abundance of fungal phyla in samples of Yi medicine fermented broth at seven fermentation stages;
[0026] Figure 7 : Relative richness of fungal genus level in samples of Yi medicine fermentation broth at seven fermentation stages. DETAILED DESCRIPTION
[0027] 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.
[0028] 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.
[0029] The following is an example description:
[0030] Yi medicine fermentation liquid (provided by Guizhou Jinqianguo Biotechnology Co., Ltd.);
[0031] DNA extraction kit ( Soil DNA Kit, Omega Bio, USA);
[0032] Nucleic acid purification kit (AgencourtAMPure XP, Beckman Coulter, USA);
[0033] DNA quality tester (Nanodrop 2000, Thermo Fisher Scientific, USA);
[0034] electrophoresis instrument (CaliperLabChip GX Touch HT, PerkinElmer, USA);
[0035] PCR amplification instrument (ABI 9700Qubit 2.0, Thermo Fisher Scientific, USA).
[0036] Universal primers ITS1 and ITS2 (Shanghai Sangon Biotechnology Co., Ltd.)
[0037] Example 1 Method
[0038] (1) The fermentation cycle was 10 months, and sampling was carried out on a monthly basis. Samples were selected from the first month, second month, third month, fourth month, sixth month, eighth month, and tenth month after the start of fermentation, a total of seven periods, numbered A, B, C, D, E, F, and G, respectively. Five samples were collected in each period.
[0039] (2) Using a DNA extraction kit, extract the genomic DNA of the microorganisms in the fermentation sample of step (1).
[0040] (3) The universal primers ITS1 and ITS2 were used to amplify the microbial genomic DNA in each sample extracted in step (2), 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.
[0041] (4) The final library was constructed and finally high-throughput sequencing was performed by Beijing Aovison Gene Technology Co., Ltd. through the platform.
[0042] (5) OTU clustering was performed on non-repetitive sequences (excluding single sequences) according to 97% similarity. Chimeras were removed during the clustering process to obtain representative sequences of OTU.
[0043] (6) Perform taxonomic analysis on the OTU representative sequences obtained in step (5).
[0044] Example 2 Results and Analysis
[0045] 1. Quality assessment of microbial sequencing during the fermentation process of Yi medicine fermentation broth
[0046] After removing low-quality sequences and chimeras for the fungal community, valid sequences were obtained for each sample, and 6,014,445 high-quality sequences were obtained from all samples. Rarefaction curves were constructed based on the number of OTUs and sequencing quantities (see Figure 1 ), and the Shannon-Wiener curve was constructed based on the Shannon index and the number of sequencing (see Figure 2 ), which can present the species diversity and richness of the samples.
[0047] from Figure 1-2 It can be seen that as the number of sequencing increases, the slope of the dilution curve gradually decreases, and the Shannon index curve quickly enters a plateau phase, indicating that the sequencing depth is sufficient to basically reflect most of the microbial information in the sample and meet the requirements of subsequent analysis.
[0048] In addition, common and unique OTUs of microorganisms in Yi medicine fermentation broth at different fermentation stages were found. Figure 3 ,Depend on Figure 3 It can be seen that the fungal communities in the samples of Yi medicine fermentation broth at the beginning of fermentation for 1 month, 2 months, 3 months, 4 months, 6 months, 8 months and 10 months recorded 579, 796, 503, 51, 4, 220 and 114 fungal OTUs, respectively, and there were 101 OTUs in all seven fermentation stages. This shows that the microbial community structure in Yi medicine fermentation broth showed differences in different fermentation stages.
[0049] 2. Analysis of Alpha Diversity of Fungal Communities at Different Fermentation Stages of Yi Medicine Fermentation Broth
[0050] Chao1, Shannon and Simpson indices were selected to represent the abundance and diversity of fungal communities in Yi medicine fermentation broth, as shown in Table 1.
[0051] Table 1 Analysis results of fungal diversity during Yi medicine fermentation
[0052]
[0053] Different lowercase letters indicate P < 0.05, indicating significant difference
[0054] As can be seen from Table 2, the coverage of each group of samples is above 99%, indicating that the probability of sequences in the samples being sequenced is extremely high and close to saturation. Currently, sequencing can cover most of the species in the samples, and the sequencing depth meets the requirements of subsequent analysis.
[0055] The Chao1 index can reflect the number of OTUs in samples of Yi medicine fermentation broth at each stage of fermentation. The larger the index, the greater the number of fungal species. Figure 4 A (Chao1 index) shows that the Chao1 index of Yi medicine fermented liquid shows a downward trend in the fermentation stage from 2 to 6 months, and its Chao1 index tends to be the same in the fermentation stage from 8 to 10 months. The Chao1 index of the sample fermented for 2 months is the largest, and the Chao1 index of the sample fermented for 5 months is the smallest.
[0056] The Simpson and Shannon indices can be used to assess microbial diversity, with larger index values indicating higher community diversity. Figure 4 B (Simpson index) and Figure 4 C (Shannon index) shows that the fungal community diversity changes in the samples of Yi medicine fermentation broth at each stage of fermentation are the same as the species abundance changes, first rising, then falling, and then rising again. The Simpson and Shannon indexes of the Yi medicine fermentation broth samples fermented for 2 months are the largest, and the index is the smallest when fermented to the 6th month. There is a significant difference between the two (P﹤0.05).
[0057] 3. Beta diversity analysis of fungal communities at different fermentation stages of Yi medicine fermentation broth
[0058] Beta diversity is an analytical method for comparing the composition of microbial communities between different samples. The microorganisms in the Yi medicine fermentation broth were classified according to the number of OTUs detected, and then nonmetric multidimensional scaling (NMDS) analysis was performed based on the classification results at each level. The results are as follows: Figure 5A shown.
[0059] Depend on Figure 5A It can be seen that as the fermentation progresses, the fungal community continues to change. In terms of spatial distribution, the difference in fungal community is reflected in the fermentation period of Yi medicine fermentation liquid in the first and second months and the fermentation period from March to October; in the middle of fermentation, the fungi are more concentrated during the fermentation process from March to August, indicating that the degree of change of microbial flora is low and tends to be the same within a certain range. The same result was obtained by principal coordinates analysis (PCoA) (see Figure 5B ).
[0060] 4. Community structure analysis
[0061] Using QIIME software, the OTU representative sequences were compared with the Unite8.2 database using the BLAST algorithm, and the e-value threshold was set to 1e-5 to obtain the species classification information corresponding to each OTU. The fungal classification results showed that a total of 14 phyla, 48 classes, 128 orders, 281 families, 639 genera, and some unclassified sequences were detected. The differences at the fungal phylum level are shown in Figure 6 , the differences at the genus level are shown in Figure 7 .
[0062] Depend on Figure 6 It can be seen that the fungal community during the fermentation of Yi medicine fermentation liquid is mainly Ascomycota, Basidiomycota and (unidentified). Only at the 2nd month of fermentation, there are 19.2% Basidiomycota and 4.3% unidentified in the fermentation stage samples. In the fermentation stage of the remaining groups of samples, the proportion of Ascomycota microorganisms exceeded 86.5%.
[0063] Depend on Figure 7It can be seen that the fungal community in the fermentation process of Yi medicine fermentation liquid is mainly Saccharomyces, Xeromyces, unidentified, Candida, Papiliotrema, Kazachstania, Colacogloea, Wallemia and Sphaerulina. After 1 month of fermentation, Saccharomyces was the dominant species in the sample, with a relative abundance of 49.6%. After 2 months of fermentation, the relative abundance of Saccharomyces in the sample of Yi medicine fermentation liquid decreased to 20.8%. After 3 to 8 months of fermentation, Saccharomyces was the main dominant species in the sample, with a relative abundance of more than 91.8%. After 10 months of fermentation, the abundance of Saccharomyces cerevisiae in the sample decreased to 65%, among which the abundance of unidentified, Kazachstania and Wallemia species increased to 6.9%, 4.8% and 4.9%, respectively.
[0064] In summary, the present invention provides a method for detecting the diversity of fungal communities during the fermentation process of Yi medicine fermentation liquid, and for the first time, conducts a detailed analysis of the fungal flora 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.
[0065] 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.
[0066] 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 fungal community diversity during the fermentation process of Yi medicine fermentation liquid, characterized in that: The following steps are involved: (1) Sampling: According to the fermentation cycle, samples of 7 periods were selected from the Yi medicine fermentation liquid at different fermentation times, 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) Miseq library construction and sequencing; (5) Data processing: OTU clustering is performed on non-repetitive and non-single sequences, and chimeras are removed during the clustering process to obtain representative sequences of OTUs; (6) Data analysis: Perform taxonomic analysis on the OTU representative sequences obtained in step (5).
2. The method for detecting fungal 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 fungal community diversity during the fermentation process of Yi medicine fermentation liquid according to claim 1, characterized in that: The seven periods described in step (1) are: sampling on a monthly basis, the first month, the second month, the third month, the fourth month, the sixth month, the eighth month, and the tenth month from the start of fermentation.
4. The method for detecting fungal 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 primers ITS1 and ITS2.
5. The method for detecting fungal 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 (5) was set to 97%.
6. The method for detecting fungal 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.
7. The method for detecting fungal 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.