A plant lactobacillus that rapidly lowers the pH of fermentation systems

Abstract

A plant-based Lactobacillus for rapidly lowering pH in silage, the plant-based Lactobacillus being classified as Lactobacillus plantarum (… Lactobacillus plantarum SUBS-LP12, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026798, dated April 23, 2026, is located at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The *Lactobacillus plantarum* SUBS-LP12 of this invention exhibits a certain degree of salt tolerance and demonstrates rapid acidification and good final fermentation quality in corn silage systems. By rapidly lowering the pH of the fermentation system, it helps to create an acidic fermentation environment. At the fermentation endpoint, it exhibits high lactic acid content and low butyric acid and NH3-N / TN levels, which contribute to improving silage fermentation quality and nutrient preservation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a plant lactobacillus that rapidly lowers the pH of silage. Background Technology

[0002] Silage is a common method of preserving feed through anaerobic fermentation, and its fermentation quality directly affects the nutritional value of the feed and animal health. Traditional lactic acid bacteria have limited effect on lowering pH within the first 72 hours of silage preparation, which can easily lead to the proliferation of putrefactive bacteria (such as molds and aerogenes), reducing feed safety. Therefore, there is an urgent need for a lactic acid bacteria strain that can rapidly lower pH, inhibit putrefactive bacteria, and improve feed hygiene and nutritional value in the early stages of silage preparation.

[0003] Silage is a stored feed made by fermenting fresh plant materials under anaerobic conditions using lactic acid bacteria. Its fermentation quality is closely related to the retention of nutrients, palatability, and storage stability. During silage fermentation, lactic acid bacteria utilize the soluble sugars in the raw materials to produce organic acids, causing a rapid decrease in the system's pH value, thereby inhibiting the growth and reproduction of putrefactive bacteria, Clostridium, and other harmful microorganisms. Therefore, in silage production, inoculation with lactic acid bacteria starter cultures is often used to improve silage quality. Commonly used strains include *Lactobacillus plantarum*, *Lactobacillus brunelli*, and *Pediococcus*. Furthermore, the growth and reproduction rate of lactic acid bacteria is also a key factor influencing whether they can become the dominant microbial community.

[0004] In existing technologies, *Lactobacillus plantarum* is widely used in silage fermentation due to its strong acid-producing ability and good adaptability; *Lactobacillus buchneri* is often used to improve the aerobic stability of silage in the later stages. Commercially available silage inoculants are usually added to silage raw materials in the form of single strains or compound strains to promote lactic acid fermentation, lower pH value, and reduce nutrient loss.

[0005] However, existing silage inoculants still have certain shortcomings in practical applications, especially in the early stages of silage fermentation. Some strains decrease the pH value slowly and cannot create a sufficiently acidic environment in a short time, allowing spoilage bacteria to still have a chance to grow, thus affecting the quality and storage safety of silage. Therefore, screening for a novel *Lactobacillus plantarum* strain that can lower the pH value more quickly within 72 hours before silage fermentation, preferentially establish a low-pH environment, and help create an acidic environment unfavorable to the growth of some harmful microorganisms, has significant application value. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Lactobacillus plantarum that can rapidly reduce pH during the fermentation process of silage.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A plant-based Lactobacillus for rapidly lowering pH in silage, characterized in that the plant-based Lactobacillus is classified as Lactobacillus plantarum (…). Lactobacillus plantarum SUBS-LP12, deposited at the China Center for Type Culture Collection, accession number CCTCC M 2026798, deposited on April 23, 2026, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] What distinguishes this *Lactobacillus plantarum* from other *Lactobacillus plantarum* species is that, during the fermentation of silage, it can rapidly lower the pH of the fermentation system in the early stages of fermentation, and by regulating the rate of pH change, it exhibits higher lactic acid content and lower butyric acid and NH3-N / TN levels at the end of fermentation, which helps to improve the quality of silage fermentation.

[0010] Lactobacillus plantarum SUBS-LP12 is milky white, round, opaque, and raised. It is a Gram-positive bacterium with rod-shaped cells. Compared to other tested lactic acid bacteria strains, its OD... 600 The changes in pH value indicate rapid growth and strong acidification ability.

[0011] In addition, Lactobacillus plantarum SUBS-LP12 has a significant salt tolerance. When the NaCl concentration is 6% and 8%, the survival rate of the strain is 91% and 78%, respectively. When the NaCl concentration is further increased to 10%, the survival rate still reaches 63%.

[0012] Application of Lactobacillus plantarum SUBS-LP12 in the fermentation and preparation of silage.

[0013] The present invention has the following technical effects: The *Lactobacillus plantarum* SUBS-LP12 strain described in this invention exhibits a certain degree of salt tolerance in NaCl-containing media and demonstrates rapid acidification and good final fermentation quality in corn silage systems. By rapidly lowering the pH of the fermentation system, it helps to create an acidic fermentation environment. At the fermentation endpoint, it exhibits high lactic acid content and low butyric acid and NH3-N / TN levels, which contribute to improved silage fermentation quality and nutrient preservation. Attached Figure Description

[0014] Figure 1 : Colony morphology diagram of Lactobacillus plantarum SUBS-LP12 of this invention.

[0015] Figure 2 Gram-stained image of Lactobacillus plantarum SUBS-LP12 of this invention.

[0016] Figure 3 The phylogenetic tree of Lactobacillus plantarum SUBS-LP12 of this invention.

[0017] Figure 4 The pH change pattern of Lactobacillus plantarum SUBS-LP12 in this invention is compared with that of other lactic acid bacteria.

[0018] Figure 5 The present invention relates to *Lactobacillus plantarum* SUBS-LP12 and other related lactic acid bacteria OD. 600 Patterns of change.

[0019] Figure 6 The present invention relates to the acid resistance of Lactobacillus plantarum SUBS-LP12.

[0020] Figure 7 The present invention relates to the salt tolerance of Lactobacillus plantarum SUBS-LP12.

[0021] Figure 8 The pH changes of Lactobacillus plantarum SUBS-LP12 during 72 hours of silage fermentation were investigated in this invention. Detailed Implementation

[0022] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0023] Example 1 Isolation and identification of strains 1. Culture medium formulation MRS-Calcium Carbonate Medium Composition: Calcium carbonate 20 g / L, peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, solvent is pure water.

[0024] Salt-tolerant medium composition: Based on commercially available MRS medium, 2%, 4%, 6%, 8%, and 10% sodium chloride were added respectively, with pure water as the solvent.

[0025] 2. Isolation of strains Take 1 mL from traditional fermented food and inoculate it into MRS medium; culture it in a shaker at 37℃ and 160 rpm for 48 h; isolate the strain by plate dilution plating method, and take 100 μL of culture solution with different dilution gradients and spread it on MRS-calcium carbonate medium plates, and culture it in a constant temperature incubator at 37℃ for 24 h.

[0026] Through enrichment culture, isolation, and purification, a strain of *Lactobacillus plantarum* with a relatively fast acid-producing rate was finally screened from traditional foods and named SUBS-LP12. Its colony morphology on calcium carbonate-MRS solid medium is as follows: Figure 1 As shown, the main physiological characteristics are: colony morphology is milky white, round, with clear edges and a slightly raised center. Microscopically, it is Gram-positive, and the cells are rod-shaped.

[0027] 3. Identification of strains Gram staining and bacterial 16S rDNA sequencing were used for identification. The sequencing results were compared with sequences in the NCBI database for homology analysis, and a phylogenetic tree was constructed. (See attached image) Figure 3 The results showed that strain SUBS-LP12 belongs to Lactobacillus plantarum ( Lactobacillus plantarum The 16S rDNA sequence alignment results showed that SUBS-LP12 had 100% sequence similarity to the Lactobacillus plantarum-related model strains.

[0028] The above-mentioned strain was deposited at the China Center for Type Culture Collection on April 23, 2026, with accession number CCTCCM 2026798, and classified as follows: Lactobacillus plantarum SUBS-LP12, deposited at: China Center for Type Culture Collection, Wuhan, China.

[0029] Effect of strain growth on pH test SUBS-LP12 and other lactic acid bacteria were inoculated separately into MRS liquid medium at an inoculum volume of 2%, and incubated at 37°C for 24 hours. The pH value of the bacteria was measured every 2 hours. The results are shown in the figure. Figure 4 .pass Figure 4 It can be seen that the Lactobacillus plantarum treatment group showed the largest pH decrease within the first 10 hours of fermentation and the lowest final pH value, indicating that it has a faster early acid-building ability and a stronger overall acidification effect.

[0030] The growth of Lactobacillus plantarum is as follows: Figure 5 As shown, the OD of Lactobacillus plantarum SUBS-LP12 in the first 12 hours of fermentation 600 The largest increase indicates that it can rapidly proliferate and establish a growth advantage in the early stages of fermentation. Combined with the pH change results, it can be seen that this strain, while growing rapidly, can also cause the system pH to drop more quickly, thus preferentially creating an acidic environment. Furthermore, at the end of fermentation, the OD of this treatment group... 600 The pH value was still higher than that of other experimental groups, and the final pH value was lower than that of other experimental groups, indicating that the *Lactobacillus plantarum* described in this invention has a strong continuous growth ability and acidification ability, which is beneficial to inhibiting the growth of putrefactive bacteria and improving the fermentation effect.

[0031] Example 2 Acid resistance test of strains The activated bacterial culture was transferred to fresh culture medium at a 2% inoculum size, centrifuged at 4000 r / min for 10 min, and resuspended in PBS to obtain 1×10⁻⁶ cells / mL. 10 CFU / mL bacterial suspension. Inoculate 2% of the culture medium into MRS liquid medium at pH 2, 2.5, and 3 (adjusted with 1M HCl), and incubate statically at 37℃ for 6 h. Samples are taken every 3 h to determine the viable cell count and assess the acid tolerance of the lactic acid bacteria. The laboratory-preserved strain *Lactobacillus plantarum* type strain JCM1149 is selected as a control.

[0032] The results are as follows Figure 6 As shown, under different acidic conditions, the viable counts of both lactic acid bacteria decreased with prolonged treatment time, and the rate of decrease increased with decreasing pH. Compared with JCM1149, SUBS-LP12 maintained a higher viable count at pH 2.0, 2.5, and 3.0, especially after 6 hours of treatment at pH 2.0, it remained at 10. 6 A concentration of CFU / mL or higher indicates that it has a strong tolerance to acid stress.

[0033] Example 3 Effect of NaCl concentration on bacterial growth SUBS-LP12 was inoculated at a 2% inoculum into MRS medium containing different NaCl concentrations (0%, 2%, 4%, 6%, 8%, 10%) and incubated at 37°C for 24 h.

[0034] The measurement results are as follows Figure 7 As shown in the experimental results, the *Lactobacillus plantarum* described in this invention exhibits good salt tolerance. Under 24-hour culture conditions, the survival rate of the strain was 100% when the NaCl concentration was 0%–4%; the survival rates were 91% and 78% when the NaCl concentration was 6% and 8%, respectively. Even when the NaCl concentration was further increased to 10%, the survival rate still reached 63%, indicating that the strain can maintain high activity under high salt concentrations. These results demonstrate that the *Lactobacillus plantarum* described in this invention has strong salt tolerance within the 10% NaCl range, meeting the application requirements under certain salt stress conditions and suitable for salt-stressed fermentation environments under specific conditions.

[0035] Finally, its ability to rapidly lower pH makes it effective in silage fermentation. Rapidly lowering pH is crucial during silage fermentation, helping to create an acidic environment unfavorable to the growth of certain harmful microorganisms, reducing nutrient loss, improving silage fermentation quality, and maintaining good nutrient preservation.

[0036] Example 4 Applying the strain to silage fermentation: After the corn raw material was chopped, 0.5% urea and 1% molasses were added by weight, and the mixture was then subjected to the following treatments: 1. *Lactobacillus plantarum* treatment group: inoculated with *Lactobacillus plantarum* of the present invention; 2. Commercially available inoculant treatment group (the commercially available inoculant was a commercially available silage inoculant composed of *Lactobacillus plantarum* and *Lactobacillus bruneri*, activated according to the recommended method in the instructions, and inoculated with the same final viable count as the SUBS-LP12 treatment group); 3. Blank control group (CK): no inoculant was added. After treatment, each group was placed in a silage fermentation container, compacted, and sealed, and anaerobic fermented at 25-30℃. pH changes were measured during fermentation, with samples taken every 6 hours. The solid-liquid ratio was 1:10, and the mixture was thoroughly stirred. The juice was extracted, and after sedimentation, the supernatant was taken to determine the pH. The inoculation treatment group was calculated based on the fresh weight of the silage raw material to ensure a final inoculation amount of 1×10⁻⁶. 6 CFU / g fresh weight; the blank control group was not inoculated with inoculum. The initial raw material dry matter was 40%, and the pH was 6.7. Each treatment had an independent silage fermentation container set up at each sampling time point. After the corresponding fermentation time was reached, the container was opened and samples were taken to determine the pH.

[0037] To compare the acid-producing capacity of the strains under high-sugar substrate conditions, lactic acid bacteria were inoculated into a medium with a glucose concentration of 100 g / L and cultured at 37°C for 72 h, with CaCO3 added as a buffer. The titratable acid content of the fermentation broth was determined by acid-base titration, and the acid-producing capacity was expressed as lactic acid equivalent. The results showed that the acid production of SUBS-LP12 was 86.6 g / L, which was close to the acid production capacity of 84.2 g / L of the commercially available inoculum. Although the titratable acid content of the two strains was similar under in vitro high-sugar culture conditions, SUBS-LP12 showed a faster pH decrease and higher lactic acid accumulation in the corn silage system, indicating that it has better adaptability to the silage substrate environment.

[0038] During silage fermentation, a rapid decrease in pH helps shorten the window for harmful microorganisms to multiply, inhibits undesirable fermentation, and reduces nutrient loss. Results are as follows... Figure 8As shown in the examples, the results indicate that during corn silage fermentation, at 12 hours of fermentation, the pH value of the *Lactobacillus plantarum* SUBS-LP12 group was 5.64, lower than the 6.29 of the blank group and the 5.85 of the commercially available inoculant group. At 24 hours of fermentation, the pH of the *Lactobacillus plantarum* SUBS-LP12 treatment group was 4.66, significantly lower than the 5.60 of the blank control group and the 4.85 of the commercially available inoculant group. At 48 hours of fermentation, the pH of the *Lactobacillus plantarum* SUBS-LP12 treatment group further decreased to 3.90, lower than the 4.20 of the commercially available inoculant group and the 4.60 of the blank control group. At 72 hours of fermentation, the pH of the *Lactobacillus plantarum* treatment group decreased to 3.7, still lower than the 3.95 of the commercially available inoculant group and the 4.20 of the blank control group. These results demonstrate that the *Lactobacillus plantarum* of this invention has a strong and sustained acidification capacity during silage fermentation, can establish a low-pH fermentation environment more quickly within 48 hours, and maintain a better acidification effect at 72 hours, exhibiting superior application potential compared to commercially available inoculants. This indicates that the *Lactobacillus plantarum* of the present invention can promote system acidification, with a rapid decrease in pH in the early stage. Corresponding to lower NH3-N / TN and butyric acid content, this helps reduce protein degradation and undesirable fermentation. This suggests superior silage quality. 72 h was used for early pH dynamics, and 30 d was used for final quality evaluation.

[0039] The fermentation quality indicators of silage from different treatment groups were determined: 1. Take 10 g of silage sample, add 90 mL of deionized water, shake thoroughly to mix, allow to stand for extraction, centrifuge, and filter the supernatant through a 0.22 μm filter membrane. Determine the contents of lactic acid, acetic acid, propionic acid, and butyric acid using high-performance liquid chromatography (HPLC), in g / kg DM. The results are shown in Table 1. An Agilent TC-C18 column was used, with methanol as mobile phase A, 0.01 mol / L KH₂PO₄ as mobile phase B (adjusted to pH 2.7 with phosphoric acid), A:B (mobile phase 10:90), 20 μL injection, UV detection wavelength 210 nm, flow rate 0.7 mL / min, and column temperature. After fermentation, silage samples from each treatment were used for fermentation quality and nutrient composition determination. Samples were dried at 65℃ to constant weight, and the dry matter content was determined. The samples were then pulverized and sieved for later use. Each treatment had three replicates. Crude protein content was determined using the Kjeldahl method, and total nitrogen was calculated as total nitrogen × 6.25. Neutral and acidic detergent fibers were tested using the filter bag method, and the results are expressed on a dry matter basis. Ammonia nitrogen content was determined using the phenol-sodium hypochlorite colorimetric method, and total nitrogen content was determined using the Kjeldahl method, and calculated as NH3-N / TN = ammonia nitrogen content / total nitrogen content × 100%.

[0040] Table 1:

[0041] As shown in Table 1, in the corn silage system with added molasses and urea, different treatment groups showed significant differences at the fermentation endpoint. Among them, the Lactobacillus plantarum treatment group of the present invention showed better overall performance in terms of acidification effect, organic acid composition and nitrogen preservation.

[0042] Regarding the fermentation acidification effect, the final pH of the *Lactobacillus plantarum* treatment group of this invention was 3.70, which was lower than 3.85 of the commercially available inoculant treatment group and 4.20 of the blank control group. High-quality corn silage usually requires a final pH below about 4.2. Therefore, the *Lactobacillus plantarum* treatment group of this invention has achieved a relatively ideal acidification level, while the CK group was at a critically high level.

[0043] Regarding the organic acid composition, the lactic acid content of the *Lactobacillus plantarum* SUBS-LP12 treatment group of this invention was 61 g / kg DM, which was higher than the 50 g / kg DM of the commercially available inoculant treatment group and the 34 g / kg DM of the blank control group; the acetic acid content was 10 g / kg DM, which was lower than the 12 g / kg DM of the commercially available inoculant treatment group and the 15 g / kg DM of the blank control group. Meanwhile, the propionic acid and butyric acid contents of the *Lactobacillus plantarum* treatment group of this invention were only 0.40 g / kg DM and 0.234 g / kg DM, respectively, which were significantly lower than those of the commercially available inoculant treatment group and the blank control group. Lactic acid should generally be the dominant organic acid in silage, while elevated butyric acid often indicates poor fermentation. Therefore, the above results show that the *Lactobacillus plantarum* treatment group of this invention formed a high-quality fermentation mode dominated by lactic acid, with moderate acetic acid and controlled butyric and propionic acids. It should be noted that, from the comparison of the self-acid production capacity of the two groups of microbial agents and the changes in the final lactic acid content in the silage, it can be found that when the in vitro acid production capacity of the two groups of microbial agents is similar, the SUBS-LP12 treatment group showed a higher lactic acid content in the silage system, indicating that it has better fermentation adaptability and lactic acid accumulation effect in this silage system.

[0044] Regarding nitrogen retention, the crude protein content of the *Lactobacillus plantarum* treatment group of this invention was 8.92% DM, higher than the 8.59% DM of the commercially available inoculant treatment group and the 7.40% DM of the blank control group; simultaneously, its ammonia nitrogen / total nitrogen ratio was 6% TN, lower than the 7% TN of the commercially available inoculant treatment group and the 11% TN of the blank control group. A lower NH3-N / TN ratio generally indicates less protein degradation and better fermentation quality. This result demonstrates that the *Lactobacillus plantarum* treatment group of this invention exhibited a lower level of protein degradation and better nitrogen retention while promoting rapid pH reduction.

[0045] Regarding fiber quality, the NDF and ADF of the *Lactobacillus plantarum* treatment group of this invention were 34% DM and 24% DM, respectively, both lower than those of the commercially available inoculant treatment group and the blank control group, indicating that this treatment also has a certain improving effect on fiber composition. Considering that NDF and ADF in corn silage are usually affected by both the maturity of the raw materials and the fermentation process, the *Lactobacillus plantarum* treatment group of this invention showed lower fiber indicators, indicating that it helps maintain better feed value while improving fermentation quality.

[0046] Although the commercially available inoculant treatment group had slightly higher dry matter content, the *Lactobacillus plantarum* treatment group exhibited a lower endpoint pH, higher lactic acid content, and lower ammonia nitrogen / total nitrogen, butyric acid, and propionic acid content. This indicates that the *Lactobacillus plantarum* strain of this invention still possesses stronger fermentation regulation capabilities and superior endpoint fermentation quality under reasonable dry matter conditions. In summary, the *Lactobacillus plantarum* strain of this invention, in a corn silage system with added molasses and urea, can effectively promote lactic acid accumulation, with the organic acid composition leaning more towards lactic acid accumulation, lower butyric acid and propionic acid levels, reduce system pH and NH3-N / TN, and improve crude protein and fiber indicators. This demonstrates its strong sustained acidification ability, excellent fermentation regulation capabilities, and good application potential, making it suitable for use as a corn silage fermentation strain.

[0047] In a corn silage system with added molasses and urea, the *Lactobacillus plantarum* treatment group of this invention showed superior fermentation quality at the 30-day endpoint. Specifically, the pH was maintained at around 3.70 (3.70), the lactic acid content was higher (61 g / kg DM), the acetic acid content was moderate (10 g / kg DM), and the crude protein content was higher (8.92%). Overall, it was superior to the commercially available inoculant treatment group and the blank control group, indicating that this strain has a strong continuous acidification ability and a good nutrient retention ability.

[0048] At the 30-day fermentation endpoint, the crude protein content of the *Lactobacillus plantarum* treatment group was 8.92%, higher than the 8.59% of the commercially available inoculant treatment group and the 7.4% of the blank control group. Simultaneously, its ammonia nitrogen / total nitrogen ratio was 6% TN, lower than the 7% TN of the commercially available inoculant treatment group and the 11% TN of the blank control group. The results indicate that the *Lactobacillus plantarum* treatment group not only exhibits strong and sustained acidification capabilities during corn silage fermentation but also effectively reduces protein degradation, demonstrating better nitrogen retention.

[0049] This *Lactobacillus plantarum* strain exhibits superior sustained acidification capabilities and promising application potential in corn silage systems supplemented with molasses and urea compared to commercially available inoculants. Specifically, it rapidly lowers the pH during the initial fermentation stage of corn silage fermentation and demonstrates superior sustained acidification capabilities at the fermentation endpoint. Specifically, it rapidly lowers the pH within the initial 12-48 hours of fermentation and maintains a low pH level for 72 hours, resulting in an even lower endpoint pH, higher lactic acid content, moderate acetic acid content, lower butyric and propionic acid content, and a lower ammonia nitrogen / total nitrogen ratio. Furthermore, it exhibits good crude protein retention, demonstrating its potential as a strain for silage fermentation.

Claims

1. Lactobacillus plantarum for use in rapidly lowering the pH in silage, characterized in that, The classification name of the plant lactobacillus is *Lactobacillus plantarum* (…). Lactobacillus plantarum SUBS-LP12, deposited at the China Center for Type Culture Collection, accession number CCTCC M 2026798, deposited on April 23, 2026, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application of Lactobacillus plantarum SUBS-LP12 as described in claim 1 in the preparation of silage fermentation agents.

3. The application of Lactobacillus plantarum SUBS-LP12 as described in claim 1 in rapidly reducing the pH of a silage fermentation system.

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