Newly isolated strain of neocallimastix and its application in preparation of microbial agent for degrading anti-nutritional factor coumarin
By using the fermentation broth of *Syzygium spp.* LGM-ZA14 to anaerobic fermentate *Syzygium spp.*, the problem of the difficult degradation of coumarin in *Syzygium spp.* was solved, achieving efficient degradation and improving the forage utilization value of *Syzygium spp.*
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING AGRICULTURAL UNIVERSITY
- Filing Date
- 2023-03-31
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies are unable to effectively degrade coumarin, an anti-nutritional factor in sweet clover, which has prevented sweet clover from being widely used as forage for ruminants.
Sweet clover was pretreated with fermentation broth of *Eriocaulon buergerianum* LGM-ZA14 to degrade coumarin through anaerobic fermentation. The fermentation conditions were 39℃, pH 6.6-7.2, and fermentation time of 72-196 h. The fermentation broth contained basal culture medium, trace element buffer, cell-free rumen fluid, and redox agents.
It significantly improved the degradation efficiency of coumarin, from 18.4% to 84.9%, with a maximum degradation rate of 86.8%, providing a new method for the application of sweet clover as forage and alleviating the current shortage of high-quality forage.
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Figure CN116162553B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biology, specifically relating to a strain of *Eriocaulon buergerianum* LGM-ZA14 and its application in the preparation of a bacterial agent that degrades the anti-nutritional factor coumarin. Background Technology
[0002] Sweet clover is a widely cultivated legume in my country. It has a high crude protein content, comparable in nutritional value to alfalfa, and is highly tolerant of extreme environments such as drought, cold, and high salinity. However, due to the presence of the anti-nutritional factor coumarin, sweet clover is not widely used as forage for ruminants. Coumarin is a plant secondary metabolite derived from the phenylpropane biosynthesis pathway. Under improper storage conditions, it can be converted into dicoumarol, causing poisoning in ruminants. Therefore, how to rationally utilize sweet clover is an urgent problem to be solved.
[0003] Plant secondary metabolites are naturally occurring compounds found in plants. These substances possess toxic or anti-nutritional effects, thus deterring herbivores from grazing. During the long evolutionary process of ruminants, their rumen microorganisms, with their detoxification capabilities, have developed greater tolerance to toxic plant secondary metabolites. Pretreatment of sweet clover with anaerobic fungi to reduce its coumarin content would be an effective method. However, improving the efficiency of this method in degrading coumarin in sweet clover and selecting suitable fungal strains remain challenges that need to be addressed. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a strain of *Eriocaulon buergerianum* LGM-ZA14 and its application in the preparation of a bacterial agent for degrading the anti-nutritional factor coumarin. The method for degrading coumarin using the fermentation broth of *Eriocaulon buergerianum* LGM-ZA14 is simple to operate and has high degradation efficiency.
[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:
[0006] This invention provides a strain of Neocallimastix sp. LGM-ZA14, which is classified as Neocallimastix sp. and has the accession number CGMCC No.17594.
[0007] Furthermore, the optimal growth temperature for the *Euphorbia milii* LGM-ZA14 is 39°C, and the suitable growth pH is 6.6-7.2.
[0008] Furthermore, the first 24 hours of the *Echinococcus neoformans* LGM-ZA14 is the lag phase, followed by the logarithmic growth phase, reaching the end of the logarithmic growth phase at 72 hours, and the stationary phase at 96 hours.
[0009] The present invention also provides a fermentation broth containing the aforementioned *Euphorbia milii* LGM-ZA14, wherein the fermentation broth is prepared by culturing *Euphorbia milii* LGM-ZA14 in a fermentation broth at 39°C for 72 hours to obtain the fermentation broth.
[0010] Furthermore, the fermentation broth includes a basal culture medium, a buffer solution containing trace elements, cell-free rumen fluid, a substrate, an oxidizing-reducing agent, and an oxidizing-reducing indicator.
[0011] The present invention also provides the application of the aforementioned *Eriocaulon buergerianum* LGM-ZA14 or its fermentation broth in the preparation of a microbial agent that degrades the anti-nutritional factor coumarin.
[0012] Furthermore, the specific steps for the fermentation broth to degrade the anti-nutritional factor coumarin are as follows:
[0013] (1) The product containing coumarin to be degraded is mixed with the fermentation broth and then sterilized to obtain a fermentation solution;
[0014] (2) Add the fermentation solution to the fermentation liquid and anaerobic ferment at 38-41℃ for 72-196h to obtain a product with low coumarin content.
[0015] Furthermore, the bacterial count of *Eriocaulon simonii* LGM-ZA14 in the fermentation broth is 2.0 × 10⁻⁶. 6 ~2.0×10 7 per mL.
[0016] Furthermore, in step (2), the mass ratio of fermentation liquid to fermentation solution is 1:5 to 80.
[0017] Furthermore, the optimal conditions for anaerobic fermentation to degrade coumarin in step (2) are anaerobic fermentation at 39°C for 168 hours.
[0018] Furthermore, when the coumarin content in the fermentation solution is 2.0-4.0 mM, the fermentation broth has a higher efficiency in degrading coumarin.
[0019] Furthermore, the fermentation solution exhibits the highest efficiency in degrading coumarin when the coumarin content is 3.0 mM.
[0020] Furthermore, the fermentation broth includes a basal culture medium, a buffer solution containing trace elements, cell-free rumen fluid, an oxidizing-reducing agent, and an oxidizing-reducing indicator.
[0021] Furthermore, the redox agent is L-cysteine hydrochloride, and the redox indicator is 0.1 w / v% resazurin.
[0022] Furthermore, the cell-free rumen fluid is obtained by preliminarily filtering and low-temperature high-speed centrifugation of rumen fluid from the rumen of ruminants.
[0023] Furthermore, the ruminants include cattle and sheep.
[0024] Compared with existing technologies, this invention has the following advantages and beneficial effects: This invention isolates and purifies an anaerobic fungus, *Spirodactylum neocarpa* LGM-ZA14, from the stomach contents obtained from the rumen of goats. The fermentation broth obtained by this fungus degrades the anti-nutritional factor coumarin, and it was found that *Spirodactylum neocarpa* LGM-ZA14 exhibits the highest efficiency in degrading coumarin at a coumarin concentration of 3.0 mM. The aforementioned bacterial broth can efficiently degrade coumarin, and the preparation method is simple and highly operable. The fermentation broth used is easy to prepare, and the raw materials are readily available. This method not only significantly reduces the coumarin content but also discovers an extracellular enzyme that can degrade coumarin, providing a new method for subsequent application in forage pretreatment and helping to alleviate the current shortage of high-quality forage in my country. Attached Figure Description
[0025] Figure 1 For morphological identification of anaerobic fungi.
[0026] Figure 2 The growth curve of *Lactobacillus neonatorum* LGM-ZA14 is shown.
[0027] Figure 3 The degradation rate of coumarin by *Eriocaulon buergerianum* LGM-ZA14 is shown.
[0028] Figure 4 The degradation rate of different concentrations of coumarin by *Eriocaulon buergerianum* LGM-ZA14 is shown. Detailed Implementation
[0029] The technical solution of the present invention will be further described in detail below with reference to specific examples. However, those skilled in the art will readily understand that the content described in the embodiments is only for illustrating the present invention and should not and will not limit the present invention as described in detail in the claims. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0030] Example 1: Screening, identification and culture of *Neopycium nigra* LGM-ZA14
[0031] 1. Screening of *Neococcus neoformans* LGM-ZA14
[0032] Rumen contents were obtained from the goat rumen using a fistula method and cultured in fermentation medium supplemented with specific antibiotics (penicillin + streptomycin sulfate) as shown in Table 1. The enriched cultures were serially diluted 10-fold to reduce bacterial and protozoan contamination. Bacterial-free anaerobic fungal enriched cultures were obtained through continuous subculturing. The enriched cultures were inoculated into agar-containing roller tubes, and pure anaerobic fungi were obtained through continuous roller tube culture.
[0033] 2. Identification of *Neopycium nigra* LGM-ZA14
[0034] The obtained pure anaerobic fungus was named LGM-ZA14, and its species was identified using morphological methods. Morphological identification included rhizoid, mycelium, flagella identification, and DAPI staining. The morphological identification results showed that strain LGM-ZA14 had the highest homology with Neocallimastix. Figure 1 Therefore, the strain LGM-ZA14 was identified as the anaerobic fungus *Neopterinaria*.
[0035] The selected Neocallimastix sp. sp. was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 12, 2019. The species name is Neocallimastix sp., and the accession number for Neocallimastix sp. sp. sp. is CGMCC No. 17594.
[0036] 3. Growth assay and physiological and biochemical characteristics of *Neisseria gonorrhoeae* LGM-ZA14
[0037] The *Echinococcus neoformans* LGM-ZA14 bacterial culture was inoculated into the fermentation broth (pH = 6.68) shown in Table 1, and cultured statically in a 39℃ incubator for 120 h. Gas production was measured every 8 h, and a growth curve was plotted. Figure 2 As shown, LGM-ZA14 exhibits a lag phase during the first 16 hours of cultivation, followed by a logarithmic growth phase. After 72 hours of cultivation, it reaches the end of the logarithmic growth phase, reaches a stationary phase at 96 hours, and then enters the decline phase, thus completing the entire growth cycle.
[0038] The aforementioned *Lactobacillus neonicotinoides* LGM-ZA14 requires a strictly anaerobic environment to grow, with a growth temperature of 39°C and an optimal pH of 6.5-7.0.
[0039] Table 1. Fermentation Broth Preparation Table (1L)
[0040]
[0041] The cell-free rumen fluid can be purchased or prepared, and is obtained by preliminary filtration and low-temperature high-speed centrifugation of rumen fluid from ruminants. The buffer solution containing trace elements is prepared separately as buffer A and buffer B. When preparing the fermentation broth, the components in Table 1 above are mixed to obtain the fermentation broth.
[0042] Example 2: Study on the anaerobic fermentation degradation of coumarin
[0043] 1. Coumarin (Shanghai Maclean Biochemical Co., Ltd., China) was dissolved in dimethyl sulfoxide and prepared into a 1 mol / L stock solution, which was then filtered through a 0.22 μm filter membrane for sterilization before use.
[0044] 2. Prepare the fermentation medium according to the formula in Table 1 and autoclave it. The preparation method of cell-free rumen fluid is as follows: obtain rumen fluid from the rumen of ruminants, filter it through four layers of gauze, bring the filtrate back to the laboratory for high-speed centrifugation (12000 rpm, 20 min, 4℃), take the clear cell-free rumen fluid from the top layer, and store it in a -20℃ refrigerator for later use.
[0045] 3. Anaerobic fungus *Eriocaulon simonii* LGM-ZA14 can be used for formal fermentation after being cultured in the fermentation broth shown in Table 1 for 72 hours.
[0046] 4. Add coumarin to 90 mL of fermentation broth to make a final concentration of 5.0 mM, add 10 mL of fermentation bacteria, and anaerobic ferment at 39 °C for 168 h. This group is designated as the treatment group.
[0047] 5. A control group was set up at the same time, with the same conditions as the treatment group, except that the additive was replaced with an equal volume of dimethyl sulfoxide.
[0048] After 168 hours of fermentation, the treatment group showed a significant increase in the degradation rate of coumarin. Figure 3 By comparing the treatment group and the control group, it was found that after adding the fermentation broth, the proportion of coumarin degraded by the anaerobic fungus *Eriocaulon simonii* LGM-ZA14 increased from 18.4% to 84.9%, a 4.6-fold increase, compared with traditional hydrolyzed coumarin.
[0049] Example 3: Study on anaerobic fermentation of different concentrations of coumarin
[0050] 1. Coumarin (Shanghai Maclean Biochemical Co., Ltd., China) was dissolved in dimethyl sulfoxide and prepared into a 1 mol / L stock solution, which was then filtered through a 0.22 μm filter membrane for sterilization before use.
[0051] 2. Prepare the fermentation medium according to the formula in Table 1 and autoclave it. The preparation method of cell-free rumen fluid is as follows: obtain rumen fluid from the rumen of ruminants, filter it through four layers of gauze, bring the filtrate back to the laboratory for high-speed centrifugation (12000 rpm, 20 min, 4℃), take the clear cell-free rumen fluid from the top layer, and store it in a -20℃ refrigerator for later use.
[0052] 3. Anaerobic fungus *Eriocaulon simonii* LGM-ZA14 can be used for formal fermentation after being cultured in the fermentation broth shown in Table 1 for 72 hours.
[0053] 4. Add different amounts of coumarin to 90 mL of fermentation broth to make final concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mM respectively, add 10 mL of fermentation culture, and anaerobic ferment at 39℃ for 168 h. The above are set as treatment groups.
[0054] 5. A control group was set up at the same time, with the same conditions as the treatment group, except that the additive was replaced with an equal volume of dimethyl sulfoxide.
[0055] After 168 hours of fermentation, it was found that the degradation of coumarin initially increased and then decreased with increasing coumarin concentration. When the coumarin concentration was 3.0 mM, *E. neomycetes* LGM-ZA14 exhibited the highest degradation efficiency of coumarin, approximately 86.8%. Figure 4 ).
[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.
Claims
1. A strain of *Neotrichum candida* LGM-ZA14, characterized in that, Its classification name is Neomyxobolus ( Neocallimastix (sp.), deposited at: China General Microbiological Culture Collection Center, China; address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 17594.
2. A fermentation broth containing *Eriocaulon buergerianum* LGM-ZA14 as described in claim 1, characterized in that, The fermentation broth was prepared by inoculating *Eriocaulon simonii* LGM-ZA14 into the fermentation broth and culturing it at 37-41 ℃ for more than 72 h.
3. The application of the *Eriocaulon buergerianum* LGM-ZA14 as described in claim 1 or the fermentation broth as described in claim 2 in the preparation of a microbial agent that degrades the anti-nutritional factor coumarin.
4. The application according to claim 3, characterized in that, The specific steps for the degradation of coumarin in the fermentation broth are as follows: (1) The coumarin-containing product to be degraded is mixed with the fermentation broth and then sterilized to obtain a fermentation solution; (2) Add the fermentation solution to the fermentation liquid and anaerobic ferment at 38-41 ℃ for 72-196 h to obtain a product with low coumarin content.
5. The application according to claim 4, characterized in that, In step (2), the bacterial count of *Eriocheir sinensis* LGM-ZA14 in the fermentation broth is 2.0 × 10⁻⁶. 6 ~2.0×10 7 per mL.
6. The application according to claim 4, characterized in that, In step (2), the mass ratio of fermentation liquid to fermentation solution is 1:5~80.
7. The application according to claim 4, characterized in that, The optimal conditions for anaerobic fermentation to degrade coumarin in step (2) are 39 °C for 168 h.
8. The application according to claim 4, characterized in that, When the coumarin content in the fermentation solution is 2-4 mM, the fermentation broth has a high efficiency in degrading coumarin.
9. The application according to claim 4, characterized in that, The fermentation broth includes a basal culture medium, a buffer solution containing trace elements, cell-free rumen fluid, an oxidizing-reducing agent, and an oxidizing-reducing indicator.