Newly isolated strain of neocallimastix and its application in preparation of feed additive for degrading coumarin
Pretreatment of sweet clover with fermentation broth of *Syzygium spp.* LGM-ZA9 degraded coumarins, solving the problem of low resource utilization efficiency of sweet clover and achieving efficient degradation and effective resource utilization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING AGRICULTURAL UNIVERSITY
- Filing Date
- 2023-03-31
- Publication Date
- 2026-07-03
AI Technical Summary
How to effectively degrade coumarin, an anti-nutritional factor in sweet clover, improve its utilization efficiency in ruminant feed, and solve the problem of insufficient sweet clover resources.
Pretreatment of sweet clover with fermentation broth of *Eriocaulon buergerianum* LGM-ZA9 was carried out to degrade coumarin. Efficient degradation was achieved through optimization of fermentation broth preparation and anaerobic fermentation conditions.
It significantly improved the degradation efficiency of coumarin, from 14.1% to 78.6%, providing a new method for the application of sweet clover in ruminant feed and alleviating the current shortage of high-quality forage resources.
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Figure CN116555044B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biology, specifically relating to a strain of *Eriocaulon buergerianum* LGM-ZA9 and its application in the preparation of feed additives that degrade coumarin. Background Technology
[0002] High-quality forage can effectively replace the proportion of grain crops in cattle and sheep feed, alleviating the competition between humans and livestock for grain. However, my country lacks sufficient high-quality forage resources such as alfalfa, requiring large-scale imports. Therefore, the rational development of domestic unconventional forage resources is crucial. Sweet clover is a widely cultivated legume in my country, with high crude protein content and nutritional value comparable to alfalfa. It also exhibits strong tolerance to extreme environments such as drought, cold, and high salinity. However, due to the presence of the anti-nutritional factor coumarin, sweet clover has not been widely used as forage for ruminants. Coumarin is a plant secondary metabolite derived from the phenylpropane biosynthesis pathway. Under improper storage conditions, it can transform into dicoumarol, causing poisoning in ruminants. Therefore, the rational utilization of sweet clover is an urgent problem to be solved.
[0003] Plant secondary metabolites are naturally occurring compounds found within plants. These substances possess toxic or anti-nutritional effects, thus deterring grazing by herbivores. Through long-term evolution, ruminants have developed greater tolerance to toxic plant secondary metabolites due to the detoxification capabilities of rumen microorganisms. 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-ZA9 and its application in the preparation of feed additives that degrade coumarin. The method for degrading coumarin using the fermentation broth of *Eriocaulon buergerianum* LGM-ZA9 described in this invention is simple to operate and has the advantage of 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-ZA9, which is classified as Neocallimastix sp. and has the accession number CGMCC No.17592.
[0007] Furthermore, the optimal growth temperature for the *Lactobacillus neonicotinoides* LGM-ZA9 is 39°C, and the suitable growth pH is 6.0-7.2.
[0008] Furthermore, the first 24 hours of the *Lactobacillus neonicotinoides* LGM-ZA9 is the lag phase, followed by the logarithmic growth phase, reaching the end of the logarithmic growth phase at 72 hours, and the stable growth phase at 96 hours.
[0009] The present invention also provides a fermentation broth containing the aforementioned *Echinococcus neoformans* LGM-ZA9.
[0010] Furthermore, the fermentation broth is prepared by inoculating *Echinococcus neoformans* LGM-ZA9 into the fermentation broth and culturing it at 37-41°C for more than 72 hours. The fermentation broth includes basal culture medium, buffer solution containing trace elements, cell-free rumen fluid, substrate, redox agent, and redox indicator.
[0011] This invention also provides the application of *Eriocaulon buergerianum* LGM-ZA9 or its fermentation broth in the preparation of feed additives that degrade 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 liquid of *Eriocaulon buergerianum* LGM-ZA9 to the fermentation solution and anaerobic ferment at 37-41℃ for 72-196 h to obtain the product after degradation of coumarin.
[0015] Furthermore, the bacterial count of *Lactobacillus neonicotinoides* LGM-ZA9 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 amount of coumarin 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 contains 3.0 mM of coumarin, which results in the highest efficiency of coumarin degradation by the fermentation broth.
[0020] Furthermore, the fermentation broth in step (1) that is mixed with the coumarin-containing material to be degraded for fermentation includes a basal culture medium, a buffer solution containing trace elements, cell-free rumen fluid, an oxidizing agent, and an oxidizing-reduction 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: An anaerobic fungus, *Tetranychus neonicotinoides* LGM-ZA9, was isolated and purified from the stomach contents obtained from the rumen of goats, and the strain was preserved. The fermentation broth obtained by fermentation with this fungus degraded the anti-nutritional factor coumarin, and it was found that *Tetranychus neonicotinoides* LGM-ZA9 exhibited the highest efficiency in degrading coumarin at a coumarin concentration of 3.0 mM. The fermentation 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 alleviating 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 LGM-ZA9 is shown.
[0027] Figure 3 The degradation rate of coumarin by *Lactobacillus neonicotinoides* LGM-ZA9 is shown.
[0028] Figure 4 The degradation rate of different concentrations of coumarin by *Eriocaulon buergerianum* LGM-ZA9. 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 *Neococcus neoformans* LGM-ZA9
[0031] 1. Screening of LGM-ZA9 of *Echinococcus neoformans*
[0032] Rumen contents were obtained from the goat rumen using a fistula method and added to a 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-ZA9
[0034] The obtained pure anaerobic fungus was named LGM-ZA9, 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-ZA9 had the highest homology with Neocallimastix. Figure 1 Therefore, the strain LGM-ZA9 was identified as the anaerobic fungus *Neopterinaria*.
[0035] The Neocallimastix spLGM-ZA9 strain obtained through screening 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 accession number for Neocallimastix spLGM-ZA9 is CGMCC No. 17592.
[0036] 3. Growth assay and physiological and biochemical characteristics of *Neisseria gonorrhoeae* LGM-ZA9
[0037] The *Echinococcus neoformans* LGM-ZA9 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-ZA9 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 *Lactobacillus neonicotinoides* LGM-ZA9 requires a strictly anaerobic environment to grow, with a growth temperature of 39°C and an optimal pH of 6.0-7.2.
[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: Experiment on 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. The anaerobic fungus *Eriocaulon simonii* LGM-ZA9 was cultured in the fermentation broth shown in Table 1 at 39°C for 72 hours to obtain the fermentation broth, which can then be used for formal fermentation.
[0046] 4. Add coumarin to 90 mL of fermentation broth to make a final concentration of 5.0 mM, then add 10 mL of the fermentation culture, 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 experimental results showed that the treatment group significantly increased 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-ZA9 increased from 14.1% to 78.6% compared with traditional hydrolyzed coumarin, which is 5.6 times higher, and the degradation efficiency of coumarin was significantly improved.
[0049] Example 3: Experiment 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. The anaerobic fungus *Eriocaulon simonii* LGM-ZA9 was cultured in the fermentation broth shown in Table 1 for 72 hours to obtain the fermentation broth, which can then be used for formal fermentation.
[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-ZA9 exhibited the highest degradation efficiency of coumarin, approximately 84.2%. 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 new strain of C. canis ( Neocallimastix sp. LGM-ZA9, characterized in that, Its accession number is CGMCCNo.17592.
2. The *Neopterinaria* LGM-ZA9 according to claim 1, characterized in that, The optimal growth temperature for the *Lactobacillus neonicotinoides* LGM-ZA9 is 39℃, and the suitable pH for growth is 6.0-7.
2.
3. A fermentation broth containing *Euphorbia neonicotinoides* LGM-ZA9 as described in claim 1, wherein the fermentation broth is prepared by inoculating *Euphorbia neonicotinoides* LGM-ZA9 into a fermentation broth culture medium and culturing it at 37-41°C for more than 72 hours, wherein the fermentation broth culture medium 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.
4. The application of the *Eriocaulon buergerianum* LGM-ZA9 as described in claim 1 or the fermentation broth as described in claim 3 in the preparation of feed additives that degrade coumarin.
5. The application according to claim 4, characterized in that, The specific steps for the degradation of coumarin in the fermentation broth are as follows: (1) The product containing coumarin to be degraded was mixed with the fermentation broth culture medium and then sterilized to obtain a fermentation solution; (2) Add the fermentation liquid of *Eriocaulon buergerianum* LGM-ZA9 to the fermentation solution and anaerobic ferment at 38-41℃ for 72-196 h to obtain the product after degradation of coumarin.
6. The application according to claim 5, characterized in that, The content of the new strain of Neocallimastix LGM-ZA9 in the fermentation broth is 2.0×10 6 ~2.0×10 7 individuals / mL.
7. The application according to claim 5, characterized in that, In step (2), the mass ratio of the fermentation broth of *Eriocaulon simonii* LGM-ZA9 to the fermentation product solution is 1:5 to 80.
8. The application according to claim 5, characterized in that, When the amount of coumarin in the fermentation solution is 2-4 mM, the fermentation broth has a high efficiency in degrading coumarin.
9. The application according to claim 5, characterized in that, The optimal conditions for anaerobic fermentation to degrade coumarin in step (2) are 39°C for 168 hours.