Method for rapidly separating key target clostridium strains from alfalfa silage
By combining high-throughput sequencing technology and Hengate anaerobic rolling tube technology, the key target Clostridium strains are rapidly isolated from alfalfa silage, solving the problem of time-consuming and labor-intensive separation process in the prior art, and improving the separation efficiency and quality and safety control of silage.
Patent Information
- Application Number
- CN202411564577.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art has a time-consuming and labor-intensive problem when isolating Clostridium strains from alfalfa silage, and it is difficult to effectively solve the problem of butyric acid fermentation caused by Clostridium proliferation.
High-throughput sequencing technology was used to analyze the bacterial flora diversity, and the suspected Clostridium strain was initially isolated, and then mixed test was performed again through high-throughput sequencing, and further isolated and purified in combination with Hengate anaerobic rolling tube technology to obtain the purified Clostridium strain of target.
The isolation efficiency of the key target Clostridium strains in alfalfa silage was significantly improved, the ineffective separation workload was reduced, the testing cost was reduced, and the efficiency of silage quality and safety control was improved.
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Figure CN119506442A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of silage quality safety control, and in particular to a method for rapidly separating key target clostridium strains from alfalfa silage. Background Art
[0002] As people's living standards improve, the demand for livestock and poultry products such as meat, eggs and milk continues to increase. A stable supply of forage and feed is an important material basis for the development of animal husbandry.
[0003] Alfalfa ( Alfalfa L.) is an important and indispensable component of the diet of high-yielding dairy cows. With the rapid development of large-scale and standardized dairy industry, the demand for high-quality plant protein feed is increasing, and high-quality alfalfa has become an irreplaceable guaranteed forage. Silage is one of the important storage methods for alfalfa and other forages. Its basic principle is to use anaerobic fermentation of lactic acid bacteria to convert the sugar contained in the raw materials into organic acids mainly composed of lactic acid, increase the acidity of the system to inhibit the activity of corrupt microorganisms, and prevent the nutrients in the raw materials from being further decomposed or consumed by microorganisms, thereby preserving the nutrients of the raw materials. However, due to its high moisture content, low soluble carbohydrate content, and high buffering capacity, alfalfa is very prone to butyric acid fermentation caused by Clostridium when it is ensiled alone, which reduces the nutritional value of silage, causes the proliferation of pathogens and the accumulation of toxins, and endangers the health of humans and animals. Therefore, it is of great significance to the development of the modern forage industry to study the diversity of Clostridia in alfalfa silage, clarify the mechanism that leads to the proliferation of Clostridia during alfalfa silage and induces butyrate fermentation to reduce silage quality, develop new technologies to inhibit Clostridia, and build a safe and high-quality alfalfa silage preparation system.
[0004] In recent years, the rapid development of molecular biology technology has greatly enriched our understanding of Clostridium flora in silage. Researchers have used denaturing gradient gel electrophoresis, random amplification polymorphism, terminal restriction fragment polymorphism, 16SrDNA clone library, high-throughput sequencing and other technologies to identify Clostridium perfringens , C. tyrobutyricum , Clostridium sporogenes , Clostridium baratii , Clostridium butyricum , Clostridium paraputrificum , C. bifermentans and Clostridium sphenoidesClostridia related to butyric acid fermentation of alfalfa silage. Although modern molecular biology technology can quickly and accurately obtain the microbial ecosystem of a sample, it is difficult to determine the function at the strain level. Therefore, it is necessary to systematically analyze the physiological and biochemical characteristics of the dominant clostridia based on the strain level, and verify its mechanism of action in the butyric acid fermentation process of alfalfa silage through in situ inoculation technology. Rapidly obtaining the key target clostridia strains to be studied from alfalfa silage is a prerequisite for conducting this series of studies.
[0005] The commonly used Hungate anaerobic roller tube technology is to separate and purify Clostridium strains in silage based on traditional separation and pure culture technology. Although this technical method can obtain Clostridium strains, it has the serious defect of being time-consuming and labor-intensive due to unclear purpose. Summary of the invention
[0006] In order to solve the above-mentioned defects, the present invention provides a method for quickly isolating key target Clostridium strains from alfalfa silage, comprising: using high-throughput sequencing technology to perform diversity analysis on the bacterial flora in the alfalfa silage sample to determine whether the silage sample contains the key target Clostridium and its abundance; then using Hungate anaerobic rolling tube technology to preliminarily separate the suspected Clostridium strains; again using high-throughput sequencing technology to perform mixed testing on the suspected Clostridium strains, according to the mixed testing results, find the corresponding suspected Clostridium strains, and use Hungate anaerobic rolling tube technology to further separate and purify to obtain the purified key target Clostridium strains.
[0007] The method of the present invention detects bacterial flora diversity of the Clostridium isolation source (alfalfa silage) by using high-throughput sequencing technology, and can discard silage samples that do not contain target Clostridium as soon as possible, greatly reducing the ineffective separation workload. By using high-throughput sequencing technology again to conduct mixed testing on suspected Clostridium strains, negative samples (non-target Clostridium) can be quickly discarded, and the ineffective separation workload can be reduced again. Through two rapid tests based on high-throughput sequencing technology, the blindness of strain isolation based on Hungate anaerobic rolling tube technology can be greatly reduced, and the testing cost can be saved.
[0008] Preferably, the method further comprises: isolating, purifying and identifying the purified key target Clostridium strain after obtaining it.
[0009] Preferably, the method further comprises: preserving the strain after identification.
[0010] Preferably, the alfalfa silage sample is an alfalfa silage extract; preferably, the method for preparing the alfalfa silage extract comprises: mixing alfalfa silage with sterile water, treating the mixture with a sterile homogenizer, and filtering the mixture to obtain the extract.
[0011] Preferably, the step of performing diversity analysis on bacterial flora in alfalfa silage samples using high-throughput sequencing technology comprises: centrifuging alfalfa silage extract to collect bacterial precipitates, extracting bacterial genomic DNA, and performing 16S rDNA diversity analysis on the bacterial flora using single-molecule real-time sequencing technology.
[0012] Preferably, the step of preliminarily isolating the suspected Clostridium strains comprises: treating the alfalfa silage extract containing the key target Clostridium in a water bath at 75-85° C., and then preliminarily isolating the suspected Clostridium strains using the Hungate anaerobic rolling tube technique.
[0013] Preferably, the mixed testing step includes: culturing the suspected Clostridium strains using the Hungate anaerobic tube technique, then mixing the culture fluids of multiple Clostridium strains into a mixed testing sample to be tested, extracting bacterial genomic DNA, and using single-molecule real-time sequencing technology to perform mixed testing on the suspected Clostridium strains.
[0014] Preferably, after obtaining the purified key target Clostridium strain, it is cultured using the Hungate anaerobic roller tube technique, the culture fluid is centrifuged to collect the bacterial precipitate, the genomic DNA is extracted, and the strain is identified based on 16S rDNA sequence homology analysis.
[0015] Preferably, the alfalfa silage is spoiled alfalfa silage or unspoiled alfalfa silage.
[0016] Preferably, lactic acid bacteria or sucrose is added to the alfalfa silage; the lactic acid bacteria include Lactobacillus plantarum and Lactobacillus buchneri.
[0017] Furthermore, the present invention provides application of the method in silage quality and safety control.
[0018] Most preferably, the method comprises the steps of: (1) Preparation of alfalfa silage extract: Take 10 g of alfalfa silage sample that has been thoroughly mixed and put it into a sterile conical bottle. Add 90 mL of sterile deionized water in a clean bench, treat it with a sterile homogenizer for 1 min, and then filter it through 4 layers of sterile gauze to obtain the silage extract. (2) High-throughput sequencing of alfalfa silage bacterial flora: The extract was centrifuged at 10,000 rpm and 4°C for 15 min, and the bacterial precipitate was collected. The bacterial genomic DNA was extracted, and the single-molecule real-time sequencing technology was used to analyze the diversity of the bacterial flora to determine whether the silage sample contained key target Clostridia and their abundance; (3) Preliminary isolation of suspected Clostridium strains: The alfalfa silage extract containing the key target Clostridium was treated in an 80°C water bath for 10 min to kill the vegetative bodies and activate spore germination. The pretreated extract was inoculated into a solid Clostridium selective counting medium and cultured at 37°C for 3-5 days using the Hungate anaerobic rolling tube technique. The black or brown colonies formed by anaerobic sulfite reduction were picked up with an inoculation loop, and the suspected Clostridium strains were initially isolated; (4) Mixed testing of suspected Clostridium strains: Suspected Clostridium strains are inoculated into liquid fortified Clostridium culture medium and cultured at 37°C for 12-24 hours using Hungate anaerobic tube technology. The culture fluids of multiple Clostridium strains are mixed into one sample to be tested. Bacterial genomic DNA is extracted, and single-molecule real-time sequencing technology is used to perform mixed testing of suspected Clostridium strains to determine whether the mixed sample contains key target Clostridium; (5) Isolation and purification of key target Clostridium strains: Based on the mixed test results, the corresponding suspected Clostridium strains are found and further isolated and purified using the Hungate anaerobic rolling tube technique to obtain purified Clostridium strains; (6) Identification and preservation of key target Clostridium strains: The purified Clostridium strains were identified by 16S rDNA homology analysis. The identified key target Clostridium strains were stored in a fortified Clostridium culture medium containing 15% glycerol and stored at -80°C for future use.
[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention organically combines advanced molecular biology technology with traditional separation and pure culture technology, greatly reducing the blindness of traditional separation and pure culture technology in the process of microbial strain screening, significantly improving the separation efficiency of key target Clostridium strains in alfalfa silage, and has broad application prospects in the field of silage quality and safety control technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is the species-level structure of bacterial flora in alfalfa silage in different treatment groups; CK is the control group; LB is the lactic acid bacteria group; and SC is the sucrose group.
[0021] Figure 2 It is the species level structure of the clostridial community in the mixed bacterial solution.
[0022] Figure 3 This is the phylogenetic tree of 16S rDNA sequences of Clostridium strains. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are 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.
[0024] If no specific techniques or conditions are specified in the examples, all are conventional methods or are carried out according to the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.
[0025] Example 1 Rapid Isolation of Key Target Clostridium from Deteriorated Alfalfa Silage Clostridium sporogenes
[0026] (1) Alfalfa silage preparation and fermentation quality analysis: On July 17, 2023, the third crop of early flowering alfalfa (Sanditi variety) was cut in the experimental field of the National Precision Agriculture Research Demonstration Base of Beijing Academy of Agricultural and Forestry Sciences, leaving a stubble height of 10 cm. The alfalfa was quickly brought back to the laboratory and cut into 1-2 cm with a paper cutter. The chopped alfalfa raw materials were divided into a control group, a lactic acid bacteria group, and a sucrose group. The lactic acid bacteria group was a lactic acid bacteria agent (mainly composed of: Lactobacillus plantarum (≥1.3×10 10 cfu / g) and Lactobacillus buchneri (≥7.0×10 9 cfu / g)) were dissolved in sterile deionized water and then 6 cfu / g FM was added to the alfalfa raw material. The sucrose group was prepared by dissolving sucrose in sterile deionized water and adding it to the alfalfa raw material at a ratio of 20 g / kg FM. The control group was added with an equal amount of sterile deionized water. Each treatment group had 3 replicates. After the alfalfa was fully mixed, it was packed into polyethylene bags for silage, each bag was about 500 g, and was quickly vacuumed and sealed with a vacuum packaging machine. It was stored at room temperature and away from light for 56 days. The fermentation quality analysis results (Table 1) showed that the alfalfa silage in the control group underwent strong butyric acid fermentation and was seriously corrupted.
[0027] Table 1 Fermentation quality of alfalfa silage in different treatment groups
[0028] Note: DM, dry weight; TN, total nitrogen; FM, fresh weight; ND, not detected; different letters in the same row indicate significant differences (P<0.05).
[0029] (2) Preparation of alfalfa silage extract: Take 10 g of the thoroughly mixed alfalfa silage sample and put it into a sterile conical bottle. Add 90 mL of sterile deionized water in a clean bench and process it with a sterile homogenizer for 1 min. Then filter it through 4 layers of sterile gauze to obtain the silage extract.
[0030] (3) High-throughput sequencing of alfalfa silage bacterial flora: The extract was centrifuged at 10,000 rpm and 4°C for 15 min, and the bacterial precipitate was collected. ® Bacterial genomic DNA was extracted using soil DNA kit (Omega Bio-tek, USA). The extraction quality of DNA was tested by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by NanoDrop2000. The full length of 16S rDNA was amplified by PCR using 27F (5'-AGRGTTYGATYMTGGCTCAG-3') and 1492R (5'-RGYTACCTTGTTACGACTT-3'). The PCR products of the same sample were mixed and recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), detected by 2% agarose gel electrophoresis, and detected and quantified using Quantus™ Fluorometer (Promega, USA). SMRT bell ® Express Template Prep Kit 2.0 (Pacific Biosciences, USA) was used to construct the library. Single-molecule real-time sequencing was performed using the Pacbio Sequel IIe System (Pacific Biosciences, USA). Using SILVA as the reference database, the naive Bayesian classifier combined with the alignment method was used to annotate the characteristic sequences taxonomically, and the species classification information corresponding to each characteristic was obtained. Then, the community composition of each sample was statistically analyzed at each level (phylum, class, order, family, genus, species). The results showed that the alfalfa silage in the control group contained a large amount of Clostridium sporogenes ( Figure 1 ), which can be used to separate Clostridium sporogenes strains.
[0031] (4) Preliminary isolation of suspected Clostridium strains: The alfalfa silage extract of the control group was treated in an 80°C water bath for 10 min and then diluted to an appropriate dilution. The extract was inoculated into a melted 45°C solid Clostridium selective counting medium (Nissui Pharmaceutical Co., Ltd.) Hungate anaerobic tube, gently shaken, and quickly rolled on ice. After the medium solidified, it was cultured in a 37°C constant temperature biochemical incubator for 3-5 days. The black or brown colonies formed by anaerobic sulfite reduction were picked up with an inoculation loop, and the suspected Clostridium strains were preliminarily isolated.
[0032] (5) Mixed testing of suspected Clostridium strains: The suspected Clostridium strains were numbered in sequence (1, 2, 3...), and then inoculated into Hungate anaerobic tubes of liquid fortified Clostridium culture medium (Difco Laboratories, USA), and cultured in a constant temperature biochemical incubator at 37°C for 12-24 hours. The culture fluids of every 10 Clostridium strains were mixed into a sample to be tested and numbered in sequence (H1, H2, H3...). After extracting the genomic DNA of the mixed strains, high-throughput sequencing analysis of the Clostridium flora was performed according to the above-mentioned operating procedures. The results showed that the four mixed samples H3, H4, H7 and H8 contained a large amount of Clostridium sporogenes ( Figure 2 ), the mixed test result was positive.
[0033] (6) Key target Clostridium Clostridium sporogenes Strain isolation and purification: The suspected Clostridium strains corresponding to the four mixed samples, H3, H4, H7 and H8, were inoculated into melted 45°C solid Clostridium selective counting medium Hengate anaerobic tubes, gently shaken, and quickly rolled on ice. After the culture medium solidified, it was cultured in a 37°C constant temperature biochemical incubator for 3-5 days. The black or brown colonies formed by anaerobic sulfite reduction were picked with an inoculation loop. After multiple isolation and pure culture until the strains had consistent morphology, the Clostridium strains were obtained and numbered sequentially (C1, C2, C3...).
[0034] (7) Key target Clostridium Clostridium sporogenes Strain identification and preservation: The purified Clostridium strains were inoculated into liquid fortified Clostridium medium Hengate anaerobic tubes and cultured in a 37°C constant temperature biochemical incubator for 12-24 h. The enriched culture solution was centrifuged at 10,000 rpm and 4°C for 15 min, the bacterial precipitate was collected, the genomic DNA was extracted, and the strains were identified based on 16S rDNA sequence homology analysis. The results showed that strains C28, C29, and C30 were Clostridium sporogenes ( Figure 3 ). C. sporogenes C28, Clostridium sporogenes C29 and Clostridium sporogenes The C30 strain was stored in a fortified Clostridium medium containing 15% glycerol and stored at -80°C until use.
[0035] Example 2 Rapid Isolation of Key Target Clostridium from High-Quality Alfalfa Silage of Example 1 C. perfringens
[0036] The fermentation quality analysis results of Example 1 showed (Table 1) that the fermentation quality of alfalfa silage in the sucrose group was good, butyrate fermentation did not occur, and the concentration of Clostridium was lower than the detection limit of the Hungate anaerobic roller tube technique. The high-throughput sequencing results of the bacterial flora showed that ( Figure 1 ), sucrose group alfalfa silage contains a small amount of Clostridium perfringens , which can be used to separate C. perfringens In order to use Hungate anaerobic roller tube technology to isolate the C. perfringens To isolate the strains, it is necessary to enrich and culture the Clostridium in the extract. Use a sterile syringe to take 10 mL of the extract and add it to the deoxygenated anaerobic tube. After treating it in an 80°C water bath for 10 min, use a sterile syringe to draw 1 mL of the extract and inoculate it into an anaerobic tube containing 9 mL of pre-reduced fortified Clostridium culture medium. Place it in a constant temperature shaking incubator at 37°C and 200 rpm to culture for 3-5 days. Use the enriched culture fluid according to the operating procedures described in Example 1 to continue to carry out preliminary isolation of suspected Clostridium strains, mixed testing of suspected Clostridium strains, and key target Clostridium. Clostridium perfringens Strain isolation and purification, key target Clostridium Clostridium perfringens The results showed that strains C356, C357, and C359 were Clostridium perfringens ( Figure 3 ). Clostridium perfringens C356, Clostridium perfringens C357 and Clostridium perfringens The C359 strain was stored in a fortified Clostridium medium containing 15% glycerol and stored at -80°C until use.
[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for rapidly isolating key target Clostridium strains from alfalfa silage, characterized in that: include: High-throughput sequencing technology was used to conduct diversity analysis of the bacterial flora in alfalfa silage samples to determine whether the silage samples contained key target Clostridia and their abundance. The Hungate anaerobic tube rolling technique was then used to preliminarily isolate suspected Clostridium strains. High-throughput sequencing technology was again used to conduct mixed testing on the suspected Clostridium strains. Based on the mixed testing results, the corresponding suspected Clostridium strains were found, and the Hungate anaerobic tube rolling technique was used for further separation and purification to obtain the purified key target Clostridium strains.
2. The method according to claim 1, characterized in that Also includes: After obtaining the purified key target Clostridium strain, it is isolated, purified and identified.
3. The method according to claim 1, characterized in that The alfalfa silage sample is an alfalfa silage extract; preferably, the preparation method of the alfalfa silage extract comprises: mixing alfalfa silage with sterile water, treating the mixture with a sterile homogenizer, and filtering the mixture to obtain the alfalfa silage extract.
4. The method according to claim 1, characterized in that: The steps of using high-throughput sequencing technology to perform diversity analysis on bacterial flora in alfalfa silage samples include: centrifuging alfalfa silage extract to collect bacterial precipitates, extracting bacterial genomic DNA, and using single-molecule real-time sequencing technology to perform 16S rDNA diversity analysis on the bacterial flora.
5. The method according to claim 1, characterized in that: The steps of preliminarily isolating suspected Clostridium strains include: treating the alfalfa silage extract containing the key target Clostridium in a 75-85°C water bath, and then using the Hungate anaerobic rolling tube technology to preliminarily isolate the suspected Clostridium strains.
6. The method according to claim 1, characterized in that The mixed testing step includes: culturing the suspected Clostridium strains using the Hungate anaerobic tube technique, then mixing the culture fluids of multiple Clostridium strains into a mixed testing sample to be tested, extracting bacterial genomic DNA, and using single-molecule real-time sequencing technology to perform mixed testing on the suspected Clostridium strains.
7. The method according to claim 1, characterized in that After obtaining the purified key target Clostridium strain, it was cultured using the Hungate anaerobic roller tube technique, the culture fluid was centrifuged to collect the bacterial precipitate, the genomic DNA was extracted, and the strain was identified based on the 16SrDNA sequence homology analysis.
8. The method according to claim 1, characterized in that The alfalfa silage is deteriorated alfalfa silage or undeteriorated alfalfa silage.
9. The method according to claim 1, characterized in that: The alfalfa silage is added with lactic acid bacteria or sucrose; the lactic acid bacteria include Lactobacillus plantarum and Lactobacillus buchneri.
10. Use of the method according to any one of claims 1 to 9 in silage quality and safety control.