Enterococcus avium WZ03 and application thereof

Abstract

The invention discloses enterococcus avium WZ03 and application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 3, 2025, the preservation number is CGMCC No.35092, and the strain is named as Enterococcus avium by taxonomy. The enterococcus avium WZ03 disclosed by the invention is screened and verified by a system, shows high protein preservation capacity and anti-mold property, has excellent fermentation performance and wide temperature and pH tolerance, and can adapt to variable environmental conditions. As an ensiling leavening agent, fermentation can still be stably started and the pH value can be continuously reduced under complex conditions such as temperature fluctuation or high-buffering raw materials, the putrefaction phenomenon caused by fermentation delay is effectively prevented, and an excellent strain resource is provided for improving the success rate and the stability of an ensiling process under adverse conditions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Enterococcus avium WZ03 and its applications. Background Technology

[0002] The core technology for long-term preservation of the nutritional value of forage—silage—involves physically treating freshly cut forage and placing it under sealed, anaerobic conditions to allow for acidic fermentation through microbial activity. The success of this biological preservation strategy hinges on the ability of lactic acid bacteria to rapidly convert sugars in plants into organic acids, creating a highly acidic environment that erects a formidable barrier against the proliferation of harmful microorganisms, thus ensuring the stable preservation of the feed.

[0003] However, in actual production, the biochemical characteristics of some silage raw materials (especially high-moisture, high-protein legumes or late-stage grasses) pose obstacles to natural fermentation. Their low fermentable carbohydrate content and strong acid-base buffering capacity often lead to delayed fermentation initiation and a slow pH decrease. This unfavorable situation can trigger a series of negative chain reactions, such as butyric acid fermentation leading to feed spoilage and excessive protein hydrolysis into ammonia nitrogen, ultimately resulting in a significant loss of nutrients and severe deterioration of feed value.

[0004] The digestive tract of livestock is an extremely complex micro-ecosystem, in which the colonizing microbial communities have evolved remarkable environmental adaptability and rapid metabolic rates to gain an advantage in fierce survival competition. This unique ecological niche is an ideal natural gene pool for screening and discovering lactic acid bacteria with high activity, strong tolerance, and the ability to rapidly establish population dominance.

[0005] Therefore, how to screen a novel and efficient silage fermentation strain is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a strain of Enterococcus avium WZ03 and its application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A strain of *Enterococcus avium* WZ03, deposited on July 3, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35092, is taxonomically named *Enterococcus avium*. Enterococcus avium .

[0008] The 16S rDNA of Enterococcus avium WZ03 of this invention is shown in SEQ ID NO.1.

[0009] The screening and identification method for Enterococcus avians WZ03 of this invention specifically includes the following steps: (1) Take termite gut samples, grind them homogenously with sterile PBS solution, dilute them, spread them on MRS solid medium for culture, pick out single colonies and continue to culture, and subculture for no less than 2 consecutive times to obtain purified colonies; (2) After culturing the purified colonies obtained in step (1) on a solid culture medium of lactic acid bacteria under constant temperature and anaerobic conditions, the lactic acid bacteria are identified by Gram staining and catalase contact reaction. The identified lactic acid bacteria are added to a liquid nutrient culture medium containing sterile glycerol and stored to obtain isolated and purified lactic acid bacteria. (3) After activating the isolated and purified lactic acid bacteria obtained in step (2), inoculate them into MRS liquid culture medium to obtain bacterial solution; measure the pH and OD value of the bacterial solution, and screen the bacterial solution with pH=3.89 and OD value=1.843 according to the measurement results to obtain the strain after preliminary screening. (4) After the strains obtained in step (3) are cultured at 37°C, DNA of a single strain is extracted and PCR amplification is performed using the extracted DNA as a template to obtain the amplification product (lactic acid bacteria). The PCR amplification process is as follows: The upstream primer is 5′-AGAGTTTGATCCTGGCTCAG-3′, and the downstream primer is 5′-GGTTACCTTGTTACGACTT-3′. These are specific primer pairs. The extracted DNA is used as a template for PCR amplification to obtain the amplification product. The PCR amplification reaction conditions are: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, repeated 30 times, and then extended at 72℃ for 5 min. (5) The amplification product (lactic acid bacteria) obtained in step (4) is identified as a sugar fermentation substrate and its ability to decompose different glycogens is determined.

[0010] Furthermore, the screening and identification method for Enterococcus avium WZ03 also includes step (6): the amplification product (lactic acid bacteria) obtained in step (4) is analyzed by physicochemical determination, different temperature test, salt tolerance test, acid tolerance test and sugar fermentation test evaluation test.

[0011] In silage applications, *Enterococcus avianus* plays multiple key roles: First, thanks to its rapid growth and tolerance to residual oxygen, it quickly occupies a niche advantage in the early stages of silage, effectively inhibiting aerobic bacteria and laying the foundation for high-quality fermentation; second, through a purely homofermentative fermentation pathway, *Enterococcus avianus* efficiently converts soluble carbohydrates into lactic acid, significantly accelerating pH reduction, minimizing dry matter loss (without producing CO2), and rapidly creating an acidic inhibitory environment; finally, this rapid acidification process strongly inhibits the activity of putrefactive microorganisms such as Clostridium and Enterobacteriaceae, fundamentally reducing protein degradation and ammonia nitrogen production, thus preserving the nutritional value and fermentation quality of the feed.

[0012] An antibacterial preparation, the active ingredient of which includes the above-mentioned Enterococcus avianus WZ03.

[0013] A silage additive, the active ingredient of which includes the above-mentioned Enterococcus avium WZ03.

[0014] Furthermore, the aforementioned silage additives also include auxiliary materials known to those skilled in the art.

[0015] Furthermore, the dosage form of the above-mentioned silage additives is a suspension, dispersant, or solution.

[0016] A silage feed includes silage raw materials and the aforementioned Enterococcus avium WZ03.

[0017] Furthermore, the silage raw material mentioned above is forage grass, preferably sorghum bicolor.

[0018] Furthermore, the dosage of the aforementioned Enterococcus avianus WZ03 is (1.0-5.0) × 10⁻⁶. 6 CFU / g silage raw material.

[0019] A method for preparing the above-mentioned silage includes the following steps: mixing the silage raw material with the above-mentioned Enterococcus avium WZ03 and fermenting it to obtain the silage.

[0020] Furthermore, the fermentation temperature was room temperature, and the time was 60 days.

[0021] This invention also claims protection for the use of the above-mentioned Enterococcus avium WZ03 in the preparation of antibacterial agents, silage additives, and silage.

[0022] This invention also claims protection for the use of the above-mentioned silage additive in the preparation of silage.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: The *Enterococcus avianus* WZ03 strain of this invention, after systematic screening and verification, exhibits high protein preservation capacity and antifungal properties, along with excellent fermentation performance and broad temperature and pH tolerance, enabling it to adapt to varying environmental conditions. As a silage starter, it can stably initiate fermentation under complex conditions such as temperature fluctuations or highly buffered raw materials, continuously lowering the pH value and effectively preventing spoilage caused by delayed fermentation. This provides an excellent strain resource for improving the success rate and stability of silage processes under adverse conditions. Attached Figure Description

[0024] Figure 1 This is an optical microscope image of Enterococcus avium WZ03 of the present invention; Figure 2 The graph shows the changes in dry matter content and pH of silage raw material (Gd) after different treatments; where: EA represents the Enterococcus avium WZ03 treatment group, LPC represents the commercial lactic acid bacteria treatment group, and CK represents the blank control group. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1 Enterococcus avianus Enterococcus avium The screening and identification method for WZ03 specifically includes the following steps: (1) Take termite gut samples, grind them homogenously with sterile PBS solution, dilute them, spread them on MRS solid medium for culture, pick out single colonies for further culture, and subculture twice to obtain purified colonies; (2) After culturing the purified colonies obtained in step (1) on a solid culture medium of lactic acid bacteria under constant temperature and anaerobic conditions, the lactic acid bacteria are identified by Gram staining and catalase contact reaction. The identified lactic acid bacteria are added to a liquid nutrient culture medium containing sterile glycerol and stored to obtain isolated and purified lactic acid bacteria. (3) After activating the isolated and purified lactic acid bacteria obtained in step (2), inoculate them into MRS liquid culture medium to obtain bacterial solution; measure the pH and OD value of the bacterial solution, and screen the bacterial solution with pH=3.64 and OD value=1.732 according to the measurement results to obtain the strain after preliminary screening. (4) After the strains obtained in step (3) are cultured at 37°C, DNA of a single strain is extracted and PCR amplification is performed using the extracted DNA as a template to obtain the amplification product. The PCR amplification process is as follows: The upstream primer is 5′-AGAGTTTGATCCTGGCTCAG-3′, and the downstream primer is 5′-GGTTACCTTGTTACGACTT-3′. These are specific primer pairs. The extracted DNA is used as a template for PCR amplification to obtain the amplification product. The PCR amplification reaction conditions are: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, repeated 30 times, and then extended at 72℃ for 5 min. (5) The amplification product obtained in step (4) is subjected to sugar fermentation substrate identification and its utilization ability for different glycogen is determined. (6) The amplification product obtained in step (4) was analyzed by physicochemical determination, different temperature test, salt tolerance test, acid tolerance test and sugar fermentation test.

[0027] The final selected Enterococcus avianus Enterococcus avium WZ03 was deposited at the China General Microbiological Culture Collection Center on July 3, 2025, with accession number CGMCC No. 35092, and its taxonomic name is Enterococcus avium. Enterococcus avium .

[0028] Enterococcus avianus Enterococcus avium Optical microscope image of WZ03 as shown Figure 1 As shown. By Figure 1 It is known that the bacterial cells of this strain are spherical or oval in shape, and the cells are often arranged in pairs or short chains, but single cells can also be seen.

[0029] Enterococcus avianus Enterococcus avium The 16S rRNA of WZ03 is shown in SEQ ID NO.1 of the sequence listing, specifically as follows:

[0030] Example 2 Enterococcus avium Enterococcus avium Physicochemical properties of WZ03 (hereinafter referred to as "WZ03") Test at different temperatures: WZ03 was inoculated into MRS liquid medium at an inoculum volume percentage of 3%, and cultured under the temperature conditions in Table 1, and its growth status was observed. The results are shown in Table 1.

[0031] Salt tolerance test: WZ03 was inoculated into MRS liquid medium containing 3.0% (w / v), 6.5% (w / v), and 18% (w / v) NaCl at an inoculum volume percentage of 3%, and cultured at 30 °C for 2 days, and its salt tolerance was measured. The results are shown in Table 1.

[0032] Acid tolerance test: WZ03 was cultured in MRS liquid medium under the pH conditions in Table 1 at 30 °C for 7 days, and its acid tolerance was measured. The results are shown in Table 1.

[0033] Table 1 Physicochemical properties of WZ03

[0034] Note: +: Growth or positive; -: No growth or negative; W: Weak growth (OD value < 0.2 is recorded as no growth, 0.2 < OD value < 0.3 is recorded as weak growth, 0.3 < OD value is recorded as growth), LPC: Commercial lactic acid bacteria (Gaofuji Biotechnology Co., Ltd.).

[0035] As shown in Table 1, both WZ03 and LPC are Gram-positive (+), catalase-negative (-), non-gas-producing from glucose (-), and homofermentative bacteria. The most significant difference between the two is their shape: WZ03 is spherical, while LPC is rod-shaped. In terms of temperature adaptability, both can grow well (+) at 20 °C and 37 °C; but at low temperatures, WZ03 can grow well (+) at 10 °C and 15 °C, while LPC only shows weak growth (W) at these temperatures, indicating that WZ03 has stronger low-temperature adaptability. In terms of salt tolerance, both can grow well (+) at 3% and 6.5% NaCl, and both show weak growth (W) at 18% NaCl. In terms of pH tolerance, both can grow well (+) within the range of pH 4 to 7, but neither can grow (-) under strong acid conditions of pH 3.

[0036] Example 3 Enterococcus avium Enterococcus avium Sugar fermentation properties of WZ03 (hereinafter referred to as "WZ03") Sugar fermentation test: The sugar fermentation characteristics of WZ03 were analyzed by sugar fermentation test. The results are shown in Table 2.

[0037] Table 2 Sugar fermentation characteristics of WZ03

[0038] Note: +: available; -: not available; W: weakly available; LPC: commercial lactic acid bacteria (Gao Fu Ji Biotechnology Co., Ltd.).

[0039] As shown in Table 2, strains WZ03 and LPC exhibited similar fermentation abilities for most tested sugars. Both fermented inulin, urate, sorbitol, sucrose, D-mannitol, maltose, and D-glucose (all positive), but neither fermented xylose (all negative). The only difference lay in their fermentation ability for cellobiose: WZ03 failed to ferment cellobiose (negative), while LPC showed a weak fermentation ability (W).

[0040] Example 4 Enterococcus avianus Enterococcus avium Silage fermentation characteristics of WZ03 (hereinafter referred to as "WZ03") Sorghum was collected from Hefeng Town, Jianyang City, Sichuan Province, and cut into pieces approximately 20 mm in length to obtain fresh samples. 300 g of fresh sample was taken from each treatment and placed in a plastic bag (25 × 35 cm; Aodeju, China). Enterococcus avium from Example 1 was then added. Enterococcus avium WZ03 (EA), commercial Lactobacillus plantarum (LPC, Gao Fu Ji Biotechnology Co., Ltd.) 1.0 × 10 6 CFU / g fresh material and an equal volume of sterile water (CK) were added to the silage, then vacuum-sealed and fermented at room temperature. Each treatment group had three replicates. After 60 days of silage, the bags were opened, and the chemical composition of the fresh samples and the samples after 60 days, as well as the pH and dry matter content for each day, were measured. The results are shown in Table 3. Figure 2 The data shown (data was analyzed using Tukey's HSD method for multiple comparisons) P <0.05 is considered a significant difference.

[0041] Table 3 Chemical composition and fermentation quality of sorghum silage

[0042] Note: Different lowercase letters after the same row of data indicate that the difference is significant at the 5% level. Microbial count units are converted to 1g CFU / g.

[0043] As shown in Table 3, compared with the blank control group (CK), both LPC and EA treatment groups significantly improved the fermentation mode of silage, and the EA group was particularly outstanding in terms of nutrient preservation and mold inhibition.

[0044] Regarding fermentation quality, the additives LPC and EA significantly promoted lactic acid fermentation. The pH values ​​of both treatment groups (3.79 and 3.83, respectively) were significantly lower than those of the control group (4.20). P <0.05%, which is directly attributed to their higher lactate accumulation: the lactate content in the LPC and EA groups (10.12% and 11.06%, respectively) was significantly higher than that in the CK group (6.73%). P <0.05%. Meanwhile, the additive treatment significantly reduced propionic acid content (LPC: 0.05%, EA: 0.03% vs CK: 0.23%). P The butyric acid content in all groups remained at extremely low levels (0.01%), while the butyric acid content in all groups remained at very low levels. These results indicate that LPC and EA successfully guided fermentation towards a highly efficient and high-quality fermentation pathway dominated by lactic acid, and effectively inhibited the activity of miscellaneous bacteria that produce undesirable volatile fatty acids.

[0045] In terms of nutrient preservation, the EA (WZ03) treatment group exhibited significant advantages. Firstly, regarding crude protein retention, the EA group achieved a high content of 11.92%, significantly higher than the CK group (10.53%) and the LPC commercial inoculum group (10.19%) (P<0.05), indicating that the WZ03 strain can most effectively protect plant-derived proteins from degradation during fermentation. Secondly, the EA group also performed excellently in preserving readily available energy, with a water-soluble carbohydrate (WSC) content of 14.16%, significantly higher than the LPC group (12.26%) and the blank control group (11.54%) (P<0.05). This demonstrates that WZ03 rapidly establishes the acidity required for preservation while minimizing the consumption of substrate sugars, exhibiting superior fermentation efficiency. Furthermore, microbial analysis showed that the number of molds in the EA group (5.73 lg CFU / g) was significantly lower than that in the LPC group (6.13 lg CFU / g) (P<0.05), further confirming that WZ03 has a significant advantage in inhibiting the growth of fungi that cause feed spoilage.

[0046] Microbiological analysis further supports the improvement in fermentation quality. All treatments effectively inhibited the growth of *E. coli* and yeast (both <2.00 lg CFU / g). Notably, the mold count in the EA group (5.73 lg CFU / g) was significantly lower than that in the LPC group (6.13 lg CFU / g). P <0.05, indicating superior antifungal activity, which is crucial for improving the aerobic stability of silage. The final lactic acid bacteria count in the EA group (5.22 lg cfu / g) was slightly lower than that in the CK and LPC groups ( P<0.05), which may reflect the natural inhibitory effect of the low pH environment on the microbial community in the later stage of fermentation, but does not affect its efficient acid production in the early stage.

[0047] Regarding structural composition, there were no significant differences in dry matter, neutral detergent fiber (NDF), and acid detergent fiber content among the treatment groups. P >0.05), indicating that under these fermentation conditions, the main contribution of the additives lies in optimizing fermentation quality and preserving nutrients.

[0048] according to Figure 2 Microbial diversity analysis showed that LPC and EA additives significantly reshaped the silage microbial ecosystem. (Principal coordinate analysis diagram) Figure 2 Alpha diversity analysis (A) showed that the microbial community structure of the two additive treatment groups (LPC and EA) was significantly altered compared to the naturally fermented control group (CK), and the community structures of the two groups were very similar. Figure 2 B, Figure 2 C, Figure 2 The study (D) revealed the nature of this change: the microbial species richness (Chao1 index) and overall diversity (Shannon and Simpson indices) of the control group (CK) were significantly higher than those of the two treatment groups (P<0.05). In silage fermentation, this reduction in diversity is a positive signal, indicating that the additives successfully promoted the absolute dominance of a few beneficial bacteria (such as lactic acid bacteria), thereby inhibiting the proliferation of a large number of miscellaneous bacteria and creating a stable and controllable fermentation environment. This is also the microbiological basis for the superior fermentation quality achieved by the LPC and EA groups.

[0049] In conclusion, both LPC and EA additives can significantly optimize the silage fermentation process. However, EA additive, while achieving efficient acidification, demonstrates more comprehensive and superior performance in inhibiting protein degradation, preserving soluble carbohydrates, and suppressing mold growth, making it the better choice for producing high-nutritional-value silage.

[0050] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Enterococcus avians WZ03, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center on July 3, 2025, with accession number CGMCC No. 35092, and its taxonomic name is Enterococcus avium. Enterococcus avium .

2. An antibacterial agent, characterized in that, The active ingredient includes Enterococcus avianus WZ03 as described in claim 1.

3. A silage additive, characterized in that, The active ingredient includes Enterococcus avianus WZ03 as described in claim 1.

4. A type of silage, characterized in that, It includes silage raw materials and Enterococcus avium WZ03 as described in claim 1.

5. The silage according to claim 4, characterized in that, The silage material is forage grass.

6. The silage according to claim 4, characterized in that, The dosage of Enterococcus avianus WZ03 is (1.0-5.0)×10⁻⁶. 6 CFU / g silage raw material.

7. A method for preparing silage as described in any one of claims 4-6, characterized in that, Specifically, the following steps are included: The silage raw material is mixed with Enterococcus avium WZ03 as described in claim 1 and fermented to obtain the silage feed.

8. The method for preparing silage according to claim 7, characterized in that, The fermentation was carried out at room temperature for 60 days.

9. The use of Enterococcus avium WZ03 as described in claim 1 in the preparation of antibacterial agents, silage additives, and silage.

10. The application of the silage additive as described in claim 3 in the preparation of silage.

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