Lactococcus lactis WZ04 and application thereof
By screening and applying Lactococcus lactis WZ04, the problem of insufficient lactic acid bacteria in high-fiber forage raw materials during silage was solved, achieving rapid fermentation and efficient nutrient preservation, and improving the quality and stability of silage.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-14
AI Technical Summary
In existing technologies, forage raw materials with high fiber, high protein, or high buffering capacity have a limited number of lactic acid bacteria during the silage process, resulting in weak competitiveness, slow or abnormal fermentation, affecting silage quality, and causing serious problems of harmful substance accumulation and spoilage.
A strain of Lactococcus lactis, WZ04, was screened and applied to establish a deeply acidic environment through rapid proliferation, aerobic consumption, and homologous fermentation, thereby inhibiting harmful bacteria and ensuring the stability and nutrient preservation of the silage process.
It significantly improves the quality of silage, rapidly reduces pH value, inhibits harmful bacteria, maintains nutrients, and improves feed preservation quality and nutritional value.
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Figure CN121852267A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Lactococcus lactis WZ04 and its applications. Background Technology
[0002] Silage technology is a traditional biological preservation method that utilizes an anaerobic environment to promote lactic acid bacteria-dominated fermentation. Its core objective is to rapidly generate large amounts of lactic acid, causing the pH value of the silage system to drop quickly to a level where putrefactive microorganisms cannot survive, thereby achieving long-term preservation of green fodder and providing a stable and high-quality feed source for animal husbandry.
[0003] However, for forage feed ingredients that are high in fiber, protein, or buffering capacity, the naturally attached lactic acid bacteria are often limited in number and weak in competitiveness, making it difficult to effectively inhibit harmful microorganisms in the early stages of fermentation. Slow or abnormal fermentation processes can lead to significant nutrient loss, accumulation of harmful metabolites such as butyric acid, and easy spoilage of the feed after opening the silage, seriously affecting the quality of silage.
[0004] Termites are highly efficient cellulose degraders in nature, and their hindgut is an extreme microbial ecosystem that has evolved over a long period. This environment is highly anaerobic and highly competitive, and the dominant microorganisms typically possess excellent metabolic efficiency, environmental adaptability, and competitive ability. Therefore, termite gut microbial resources provide a valuable source for screening superior lactic acid bacteria strains suitable for ensiling difficult-to-find raw materials.
[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 Lactococcus lactis WZ04 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 *Lactococcus lactis*, WZ04, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 3, 2025, with accession number CGMCC No. 35093, and its taxonomic name is *Lactococcus lactis*. Lactococcus lactis .
[0008] The 16S rDNA of Lactococcus lactis WZ04 of the present invention is shown in SEQ ID NO.1.
[0009] The screening and identification method for Lactococcus lactis WZ04 of the present 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=3.71 and OD value=1.811 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 Lactococcus lactis WZ04 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.
[0011] An antibacterial preparation, the active ingredient of which includes the above-mentioned Lactococcus lactis WZ04.
[0012] A silage additive, the active ingredient of which includes the above-mentioned Lactococcus lactis WZ04.
[0013] Furthermore, the aforementioned silage additives also include auxiliary materials known to those skilled in the art.
[0014] Furthermore, the dosage form of the above-mentioned silage additives is a suspension, dispersant, or solution.
[0015] A silage feed includes silage raw materials and the aforementioned lactococcus lactis WZ04.
[0016] The *Lactococcus lactis* strain of this invention possesses characteristics such as rapid proliferation, high oxygen consumption, and pure homofermentation, which can significantly improve the quality of silage. Its application effects are mainly reflected in three aspects: First, it rapidly consumes residual oxygen in the early stages of silage, inhibiting the reproduction of aerobic bacteria and creating a clean starting environment for anaerobic fermentation; second, through homofermentation, it efficiently converts soluble carbohydrates into lactic acid, rapidly lowering the pH without producing gas or losing dry matter, effectively inhibiting harmful bacteria and preserving nutrients; third, by rapidly establishing an acidic environment, it inhibits the activity of putrefactive bacteria, laying the foundation for the succession of acid-tolerant dominant bacteria in fermentation, thereby ensuring the stable and efficient progress of the silage process and ultimately significantly improving the preservation quality and nutritional value of the feed.
[0017] Furthermore, the silage raw material mentioned above is forage grass, preferably sorghum bicolor.
[0018] Furthermore, the dosage of the aforementioned Lactococcus lactis WZ04 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 lactococcus lactis WZ04 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 Lactococcus lactis WZ04 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 Lactococcus lactis strain of this invention possesses excellent acid-producing capacity and protein preservation characteristics, which can reduce the pH and ammonia nitrogen content of feed to a low level. By establishing a deeply acidic environment, it effectively blocks the protein hydrolysis pathway, providing an efficient microbial resource for producing high-quality silage with high protein retention. Attached Figure Description
[0024] Figure 1 This is an optical microscope image of Lactococcus lactis WZ04 of the present invention; Figure 2The graph shows the changes in dry matter content and pH of silage raw material (Sorghum bicolor Gd) after different treatments; where: LL represents the Lactococcus lactis WZ04 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 Lactococcus lactis Lactococcus lactis The screening and identification method for WZ04 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=3.71 and OD value=1.811 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 lactococci Lactococcus lactis WZ04 was deposited at the China General Microbiological Culture Collection Center on July 3, 2025, with accession number CGMCC No. 35093, and its taxonomical name is *Lactococcus lactis*. Lactococcus lactis .
[0028] Lactococcus lactis Lactococcus lactis Optical microscope image of WZ04 as shown Figure 1 As shown. By Figure 1 It can be seen that the bacterial cells of this strain are spherical or oval in shape, mostly arranged in pairs or short chains, and do not have spores, which is consistent with the typical morphological characteristics of the Lactococcus genus.
[0029] Lactococcus lactis Lactococcus lactis The 16S rRNA of WZ04 is shown in SEQ ID NO.1 of the sequence listing, specifically as follows:
[0030] Example 2 Lactococcus lactis Lactococcus lactis Physicochemical properties of WZ04 (hereinafter referred to as "WZ04") Test at different temperatures: WZ04 was inoculated into MRS liquid medium at an inoculation 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: WZ04 was inoculated into MRS liquid medium containing 3.0% (w / v), 6.5% (w / v), and 18% (w / v) NaCl at an inoculation volume percentage of 3%, and after culturing at 30 °C for 2 days, its salt tolerance was measured. The results are shown in Table 1.
[0032] Acid tolerance test: WZ04 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 WZ04
[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 WZ04 and LPC are Gram-positive (+), catalase-negative (-), non-gas-producing from glucose (-), and homofermentative bacteria. The most significant difference between the two lies in their shape: WZ04 is spherical, while LPC is rod-shaped. In terms of temperature adaptability, the two perform similarly and can both grow well (+) at 20 °C and 37 °C, while showing weak growth (W) at low temperatures of 4 °C, 10 °C, and 15 °C. In terms of salt tolerance, both can grow well (+) at 3% NaCl and show weak growth (W) at 18% NaCl; however, under the condition of 6.5% NaCl, LPC can grow well (+), while WZ04 only shows weak growth (W), indicating that LPC has better tolerance to medium salt concentration. In terms of pH tolerance, neither can grow at pH 3 (-), but both can grow well (+) within the range of pH 4 to 7.
[0036] Example 3 Lactococcus lactis Lactococcus lactis Sugar fermentation characteristics of WZ04 (hereinafter referred to as "WZ04") Sugar fermentation test: The sugar fermentation characteristics of WZ04 were analyzed through the sugar fermentation test. The results are shown in Table 2.
[0037] Table 2 Sugar fermentation characteristics of WZ04
[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 WZ04 and LPC exhibited similar fermentation abilities for the tested carbohydrate substrates. Both could ferment inulin, urate, sorbitol, sucrose, D-mannitol, maltose, and D-glucose (all positive), but neither could ferment xylose (all negative). For cellobiose, both showed weak fermentation ability (both W).
[0040] Example 4 Lactococcus lactis Lactococcus lactis Silage fermentation characteristics of WZ04 (hereinafter referred to as "WZ04") 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). Lactococcus lactis was then introduced according to the method described in Example 1. Lactococcus lactis WZ04 (LL), Commercial Lactococcus lactis (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 CK, both additives significantly improved the fermentation quality of silage, with LL additive showing particularly outstanding performance in acidification capacity, inhibition of protein degradation and mold growth.
[0044] From the perspective of fermentation quality, the LL treatment group exhibited the best acidification ability. Its final pH value was as low as 3.61, significantly lower than the LPC group (3.79) and the CK group (4.20) (P<0.05), indicating that the WZ04 strain successfully constructed the strongest acid barrier in the silage system. Regarding nutrient preservation, the LL group showed particularly outstanding protection of protein. Ammonia nitrogen (NH3-N) is a key indicator for measuring the degree of protein degradation; the NH3-N content in the LL group was only 0.11 g / kg DM, significantly lower than the LPC group (0.15 g / kg DM) and the CK group (0.17 g / kg DM) (P<0.05). This result demonstrates that the WZ04 strain effectively inhibited the activity of protein-degrading microorganisms such as Clostridium by rapidly establishing a low pH environment, thereby maximizing the preservation of the true protein nutritional value in the feed.
[0045] Microbiological analysis further supports the improvement in fermentation quality. All treatment groups effectively inhibited the growth of *E. coli* and yeast (all <2.00 lg CFU / g). The mold count in the LL treatment group (5.66 lg CFU / g) was significantly lower than that in the CK (5.96 lg CFU / g) and LPC (6.13 lg CFU / g) groups (P<0.05), indicating that the LL additive has superior antifungal activity, which is crucial for preventing feed spoilage and ensuring feed safety. The final lactic acid bacteria count in the LL group (5.22 lg CFU / g) was significantly lower than that in the CK group (5.39 lg CFU / g) (P<0.05), which may reflect the inhibitory effect of its extremely low pH environment on the survival of the microbial community in the later stages, but this does not affect its high-efficiency acid production capacity in the early stages.
[0046] Regarding chemical composition, there were no significant differences in dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content among the treatment groups (P>0.05), indicating that under the experimental conditions, the main function of the additives was to optimize the fermentation process rather than degrade structural fibers. However, the soluble carbohydrate (WSC) content in the LL treatment group (13.61%) was significantly higher than that in the CK group (11.54%) (P<0.05), indicating that the LL additive consumed relatively less sugar when reaching the final stable pH, or had a better sugar retention effect, thus preserving more readily available energy for the final feed product. Regarding crude protein (CP), although there were no statistically significant differences among the three groups, the results for ammonium nitrogen (NH3-N) are noteworthy. The ammonium nitrogen content in the LL group (0.11 g / kg DM) was significantly lower than that in the CK (0.17 g / kg DM) and LPC (0.15 g / kg DM) groups (P<0.05). Ammonium nitrogen is a key indicator of protein degradation, and its lowest content in the LL group strongly suggests that the LL additive can most effectively inhibit protein hydrolysis and has the best protein preservation potential.
[0047] according to Figure 2 Microbial diversity analysis showed that LPC and LL additives significantly reshaped the silage microbial ecosystem. (Principal coordinate analysis diagram) Figure 2 Alpha (A) showed that the microbial communities in both treatment groups were significantly altered compared to the control group (CK) (P = 0.008), and the community structures of the LPC and LL groups were more similar. The Alpha diversity index further revealed the nature of this change: the control group (CK) maintained a significantly higher species richness (Chao1 index). Figure 2 (B) and overall diversity (Shannon and Simpson indices, Figure 2 C and Figure 2 (D). This sharp reduction in diversity is a sign of successful fermentation, indicating that the additives have enabled a small number of beneficial bacteria to become the dominant population, effectively suppressing a large number of miscellaneous bacteria, thus creating a stable and controllable low-diversity fermentation environment, which corresponds to its superior fermentation quality.
[0048] In conclusion, while both LPC and LL additives effectively improve silage fermentation, LL additives exhibit more comprehensive advantages across several key indicators: it not only achieves the lowest pH value but also most effectively inhibits protein degradation (as indicated by the lowest ammonium nitrogen), better preserves soluble carbohydrates, and most significantly inhibits mold growth. Therefore, LL is a superior choice for producing high-quality silage.
[0049] 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 Lactococcus lactis WZ04, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center on July 3, 2025, with accession number CGMCC No. 35093, and its taxonomical name is *Lactococcus lactis*. Lactococcus lactis .
2. An antibacterial agent, characterized in that, The active ingredient includes Lactococcus lactis WZ04 as described in claim 1.
3. A silage additive, characterized in that, The active ingredient includes Lactococcus lactis WZ04 as described in claim 1.
4. A type of silage, characterized in that, It includes silage raw materials and Lactococcus lactis WZ04 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 the lactococcus lactis WZ04 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 Lactococcus lactis WZ04 as described in claim 1 and fermented to obtain the silage feed.
8. A method for preparing silage according to claim 7, characterized in that, The fermentation was carried out at room temperature for 60 days.
9. The application of Lactococcus lactis WZ04 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.