A lactic acid bacterium and its use in the fermentation of goldenrod in canada

Abstract

The present application relates to the technical field of microbial inoculant, in particular to a kind of lactic acid bacteria and its application in Solidago Canadensis fermentation.The excellent lactic acid bacteria provided by the present application is identified as Lactobacillus plantarum, has the advantages of fast fermentation speed, strong acid production capacity, relatively strong acid tolerance and wide range of carbohydrate utilization, can reduce the number of mold, aerobic bacteria and yeast in Solidago Canadensis stem and leaf fermentation material, increase the number of lactic acid bacteria, effectively improve fermentation quality, and has good fermentation product production potential.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a lactic acid bacterium and its application in the fermentation of Canadian goldenrod. Background Technology

[0002] Canadian goldenrod is a highly invasive and widely distributed plant. Turning it into a valuable resource during its control can yield multiple benefits. Fermenting the harvested Canadian goldenrod plants can produce high-quality silage or other products such as enzymes.

[0003] Canadian goldenrod contains various allelochemicals, including flavonoids and terpenoids. These substances significantly inhibit the allelopathic effects of surrounding plants, but also suppress the growth and metabolic activity of fermenting microorganisms. A key challenge is balancing the utilization of allelochemicals with the avoidance of their inhibitory effects on microbial growth during fermentation. Therefore, it is necessary to screen strains that can tolerate the allelochemicals of Canadian goldenrod while efficiently degrading its macromolecules such as cellulose and starch.

[0004] During fermentation, the metabolism of attached lactic acid bacteria converts water-soluble carbohydrates into organic acids such as lactic acid and acetic acid, rapidly lowering the pH of the material. Acidic and anaerobic conditions help inhibit the activity of harmful microorganisms, reduce the risk of raw material spoilage, and preserve nutrients. Fermentation performance depends on many factors, including the characteristics of the raw materials, storage conditions, and the effectiveness of the lactic acid bacteria. The lactic acid bacteria attached to the surface of the fermenting material affect the final quality of the fermentation. When the number of epiphytic lactic acid bacteria is insufficient, they cannot become the dominant flora, and fermentation cannot effectively lower the pH or inhibit unfavorable bacteria, resulting in unsatisfactory fermentation and lower fermentation quality. Therefore, the abundance of lactic acid bacteria can be used as one of the indicators for predicting the success of fermentation.

[0005] Existing lactic acid bacteria strains suffer from slow fermentation rates and poor acid production in the fermentation of Canadian goldenrod. Therefore, developing a lactic acid bacteria strain that can efficiently promote the fermentation of Canadian goldenrod has become an urgent problem to be solved. Summary of the Invention

[0006] To address the technical problems of slow fermentation speed and poor acid production capacity of existing lactic acid bacteria in the fermentation of Canadian goldenrod, this invention provides a lactic acid bacteria and its application in the fermentation of Canadian goldenrod. The *Lactobacillus plantarum* provided by this invention has a fast fermentation speed, strong acid production capacity, strong acid resistance, and a wide range of carbohydrate utilization. It can reduce the number of molds, aerobic bacteria, and yeasts in the fermentation of Canadian goldenrod, increase the number of lactic acid bacteria, and effectively improve fermentation quality, demonstrating good potential for fermentation applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a lactic acid bacteria, wherein the lactic acid bacteria is *Lactobacillus plantarum* DCFColdC or *Lactobacillus plantarum* DCFY03; the preservation number of *Lactobacillus plantarum* DCFColdC is CCTCC NO: M2026974; and the preservation number of *Lactobacillus plantarum* DCFY03 is CCTCC NO: M2026975.

[0009] This invention provides a microbial inoculant, the effective components of which include *Lactobacillus plantarum* DCFColdC and / or *Lactobacillus plantarum* DCFY03 as described in the above technical solution.

[0010] Preferably, the viable count of the microbial agent is ≥5×10⁻⁶. 6 CFU / g.

[0011] The present invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps: culturing *Lactobacillus plantarum* in MRS liquid culture medium to obtain the microbial agent; wherein the *Lactobacillus plantarum* is one or more of *Lactobacillus plantarum* DCFColdC and *Lactobacillus plantarum* DCFY03 described in the above technical solution.

[0012] Preferably, the culture temperature is 15~35℃ and the pH value is 3.5~7.0.

[0013] This invention provides the application of *Lactobacillus plantarum* as described in the above-described technical solution, or the microbial agent as described in the above-described technical solution, or the microbial agent prepared by the preparation method described in the above-described technical solution, in the fermentation of *Solidago canadensis*.

[0014] Preferably, the fermentation raw material of Canadian goldenrod has a moisture content of 75% to 83%.

[0015] Preferably, the fermentation raw materials include the stems and leaves of Canadian goldenrod.

[0016] This invention provides a method for improving the fermentation quality of Canadian goldenrod, comprising the following steps:

[0017] The microbial agent described in the above technical solution is mixed with the stems and leaves of Canadian goldenrod and then fermented.

[0018] Preferably, based on the wet weight of the stems and leaves of Canadian goldenrod, the ratio of the microbial inoculant to the fermentation raw material is ≥5.0 × 10⁻⁶. 6 CFU: 1kg.

[0019] Beneficial effects:

[0020] This invention provides lactic acid bacteria with fast fermentation speed and strong acid production capacity. The lactic acid bacteria are Lactobacillus plantarum DCFColdC and Lactobacillus plantarum DCFY03. The preservation number of Lactobacillus plantarum DCFColdC is CCTCC NO: M2026974; the preservation number of Lactobacillus plantarum DCFY03 is CCTCC NO: M2026975.

[0021] This invention utilizes traditional culture methods to isolate 40 lactic acid bacteria strains from fermented Solidago canadensis L. plants. Six strains of lactic acid bacteria with rapid growth and high acid production efficiency were screened and identified. The physiological and biochemical characteristics of these strains, as well as their impact on the fermentation quality of Solidago canadensis, were analyzed to screen for superior lactic acid bacteria suitable for fermentation of Solidago canadensis stems and leaves. All six lactic acid bacteria strains were able to grow well in MRS liquid medium with a NaCl concentration of 3.0 wt.%, at 15–35°C and a pH of 3.5–7.0, and were identified as homosporogenic Lactobacillus plantarum.

[0022] These six *Lactobacillus plantarum* strains were able to rapidly multiply and produce acid within 12 hours. All six strains improved the fermentation quality of *Solidago canadensis*, significantly increasing soluble carbohydrates and lactic acid bacteria counts in the fermentation substrate, while significantly reducing pH, isobutyric acid content, ammonia nitrogen content, aerobic bacteria count, and yeast count. Four of these strains were used to verify their effectiveness. By setting different addition concentrations, varying withering times (to achieve different moisture contents), and comparing with commercial inoculants, it was found that all four strains effectively improved the fermentation quality of *Solidago canadensis*, outperforming commercial inoculants. Finally, strains DCFColdC and DCFY03 were preserved.

[0023] Biological Preservation Instructions

[0024] The strain of Lactobacillus plantarum, DCFColdC, was deposited on May 15, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC No: M2026974.

[0025] Lactobacillus plantarum strain DCFY03 was deposited on May 15, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC No: M2026975. Detailed Implementation

[0026] This invention provides *Lactobacillus plantarum* species with rapid growth and strong acid-producing capacity, namely *Lactobacillus plantarum* DCFColdC and *Lactobacillus plantarum* DCFY03; the preservation number of *Lactobacillus plantarum* DCFColdC is CCTCC NO: M2026974; and the preservation number of *Lactobacillus plantarum* DCFY03 is CCTCC NO: M2026975.

[0027] This invention involves fermenting fresh Canadian goldenrod to isolate lactic acid bacteria from naturally fermented Canadian goldenrod of excellent quality. Lactic acid bacteria strains are screened by examining their growth and acid-producing properties. The strains are then identified and their physiological and biochemical activities are analyzed to study their impact on fermentation quality during the Canadian goldenrod fermentation process. The *Lactobacillus plantarum* provided by this invention exhibits rapid fermentation speed, strong acid-producing capacity, strong acid tolerance, and a wide range of carbohydrate utilization. It can reduce the number of molds, aerobic bacteria, and yeasts in fermented Canadian goldenrod while increasing the number of lactic acid bacteria, effectively improving fermentation quality and nutritional quality. It possesses excellent fermentation potential and provides effective strains and scientific basis for the development of lactic acid bacteria agents for the fermentation of special materials.

[0028] This invention also provides a microbial inoculant, wherein the effective component of the microbial inoculant includes one or more of *Lactobacillus plantarum* DCFColdC and *Lactobacillus plantarum* DCFY03 as described in the above-mentioned technical solutions. In this invention, the effective component of the microbial inoculant is preferably *Lactobacillus plantarum* DCFY03 as described in the above-mentioned technical solutions; the viable count of the microbial inoculant is preferably ≥5 × 10⁻⁶. 6 CFU / g, more preferably 5×10 6 CFU / g.

[0029] The present invention also provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps: culturing *Lactobacillus plantarum* in MRS liquid culture medium to obtain the microbial agent; wherein the *Lactobacillus plantarum* is one or more of *Lactobacillus plantarum* DCFColdC and *Lactobacillus plantarum* DCFY03 described in the above technical solution.

[0030] In this invention, the culture temperature is preferably 15-35℃, more preferably 15-25℃; the pH value is preferably 3.5-7.0, more preferably 4-6. Both *Lactobacillus plantarum* strains provided by this invention can grow well in MRS liquid medium with a NaCl concentration of 3.0 wt.%, at 15-35℃ and a pH of 3.5-7.0. These six *Lactobacillus plantarum* strains can rapidly multiply and produce acid within 12 hours. Both *Lactobacillus plantarum* strains can improve the fermentation quality of *Solidago canadensis*, including significantly increasing the soluble carbohydrates and lactic acid bacteria count in the fermentation substrate, and significantly reducing the pH value, isobutyric acid content, ammonia nitrogen content, aerobic bacteria count, and yeast count in the fermentation substrate.

[0031] This invention also provides the application of *Lactobacillus plantarum* or the microbial agent described in the above-described technical solutions, or the microbial agent prepared by the preparation method described in the above-described technical solutions, in fermentation. In this invention, the moisture content of the fermentation raw material is preferably 75%~83%, more preferably 75.04%~83.13%; the fermentation raw material preferably includes *Solidago canadensis*. The *Lactobacillus plantarum* provided by this invention can rapidly multiply and produce acid within 12 hours, and can be used for the fermentation of *Solidago canadensis*. It can increase the soluble carbohydrate content and lactic acid bacteria count of the fermented material, and reduce the pH value, isobutyric acid content, ammonia nitrogen content, aerobic bacteria count, and yeast count of the fermented feed, thereby improving the fermentation quality and nutritional quality of the fermented material.

[0032] The present invention also provides a method for improving fermentation quality, comprising the following steps:

[0033] The microbial agent and fermentation raw materials described in the above technical solution are mixed and fermented.

[0034] In this invention, based on the wet weight of the fermentation raw materials, the ratio of the microbial inoculant to the fermentation raw materials is preferably ≥5.0 × 10⁻⁶. 6 CFU: 1 kg, more preferably 5.0 × 10 6 CFU: 1kg.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a group of excellent lactic acid bacteria for fermentation of high-moisture materials provided by the present invention and their applications, should not be construed as limiting the scope of protection of the present invention.

[0036] Statistical analysis of the embodiments of this invention was performed using SPSS 13.0 (IBM, Chicago, IL, USA) software. The LSD test was used (P < 0.05).

[0037] Example 1

[0038] 1.1 Isolation of lactic acid bacteria and screening of superior strains

[0039] Canadian goldenrod was collected from wasteland in Jiangsu Agricultural Expo Park. After being chopped, it was placed in polyethylene bags and fermented at room temperature in the dark for 2 months before being sampled for the isolation of lactic acid bacteria.

[0040] Weigh 10.0 g of fermented Canadian goldenrod and place it in a sealed Erlenmeyer flask containing 90.0 mL of sterile physiological saline. Shake at 120 rpm at room temperature for 1 h. Filter through a single layer of sterile gauze and perform serial dilutions of the filtrate. Select 10 g of the fermented goldenrod. -5 10 -6 10 -7 Three gradients of dilution, 100 μL each, were plated onto MRS medium and incubated upside down at 37°C for 48 h. Larger, well-grown single colonies were selected from the MRS solid medium, streaked twice for purification, and then streaked again on MRS solid medium. The colonies were incubated at 37°C for 48 h, and their morphology was observed. Typical colonies were Gram-stained and examined microscopically; the presence of a single purple cell and a negative catalase reaction confirmed them as lactic acid bacteria. The isolated strains were purified twice on MRS medium, yielding a total of 40 lactic acid bacteria strains, which were then preserved in glycerol.

[0041] Forty isolated and purified lactic acid bacteria strains were activated and added to 5 mL of MRS liquid medium at an inoculum rate of 1% (V / V). The cultures were incubated statically at 37°C for 24 h, and the OD of the bacterial culture was measured using a spectrophotometer. 600 nm, pH of fermentation supernatant was directly measured using a pH meter, OD 600 nm The pH of the fermentation broth was between 0.57 and 1.67, and the pH ranged from 4.34 to 3.53, with OD... 600 nm There were 37 strains of lactic acid bacteria with a pH >1.50 and a pH <3.80. The strain numbers are shown in Table 1.

[0042] 1.2 Lactic acid bacteria fermentation of Canadian goldenrod pollen

[0043] Fresh Canadian goldenrod was blanched at 105℃ for 30 min, dried at 65℃ to constant weight, and then pulverized through a 0.63 mm sieve to obtain the Canadian goldenrod pollen to be tested. 1.0 g of Canadian goldenrod pollen was weighed and placed in a resealable bag, mixed with 2 mL of sterile water, and then 300 μL of a bacterial suspension of 37 lactic acid bacteria obtained from the initial screening in step 1.1 was added. The OD of the bacterial suspension was... 600 nm The control group (CK) was treated with an added volume of sterile water, with three replicates per treatment. After mixing, air was removed, the mixture was sealed, and incubated at 30°C for 48 h. Then, 27 mL of sterile water was added, and the pH was measured. A lower pH value indicates a stronger ability of the lactic acid bacteria strain to acidify Canadian goldenrod pollen. Results are shown in Table 1.

[0044] Table 1. pH values ​​after 48 h of fermentation with 37 strains of lactic acid bacteria from Canadian goldenrod pollen.

[0045]

[0046] Table 1 shows that the pH of Canadian goldenrod pollen before fermentation was 6.02, the pH of the control group (CK) after 48 h of fermentation was 6.49, and the pH of the treatment group after 48 h of fermentation was significantly lower than that of the control group, ranging from 3.62 to 4.81. Among them, S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 had a pH below 3.80 after 48 h of fermentation of Canadian goldenrod pollen, and were recorded as 6 initial screening strains.

[0047] 1.3 Growth curves and acid production performance determination of lactic acid bacteria strains

[0048] The six lactic acid bacteria strains initially screened in step 1.2 were activated. The activated strains were inoculated into MRS liquid medium at an inoculum rate of 1% (V / V), mixed thoroughly, and the OD of the fermentation broth was measured. 600 nm Initial values ​​and pH values ​​were set, and the culture was incubated at 37°C. The bacterial culture was shaken and the OD was measured every 2 hours over 32 hours. 600 nm pH. Fermentation broth for 36 h was filtered through a 0.22 μm aqueous filter membrane, and the types and contents of organic acids in the filtrate were analyzed using high-performance liquid chromatography (HPLC). S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 were designated as strain 1, strain 2, strain 3, strain 4, strain 5, and strain 6, respectively. The results are shown in Tables 2-4.

[0049] Table 2. OD values ​​of different strains at different times 600 nm value

[0050]

[0051] Table 3. pH values ​​of different strains at different times

[0052]

[0053] Table 4. Types and contents (mmol / L) of organic acids in the filtrates of different bacterial strains

[0054]

[0055] As shown in Table 2, strains S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 can begin to grow rapidly from 2 to 4 hours, reach their maximum growth rate from 4 to 12 hours, and then slow down and enter a stationary phase at approximately 14 hours. Subsequently, the OD of the bacterial culture... 600 nm The changes were minimal, and no cell growth decline was observed after 32 h. The OD values ​​of the bacterial broths from strains S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 after 12 h of fermentation were compared. 600 nm The values ​​were 1.96, 1.95, 1.98, 1.97, 1.98 and 1.98 respectively, showing similar growth rates.

[0056] As shown in Table 3, the acid production rate of the six strains was relatively fast from 2 to 8 h, gradually decreased from 8 to 12 h, and remained stable from 14 to 32 h, with the final pH remaining at approximately 3.40. The pH values ​​of strains S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 after 12 h of fermentation were 3.76, 3.74, 3.72, 3.72, 3.72, and 3.70, respectively, showing similar acid production rates.

[0057] As shown in Table 4, the six strains mainly produce lactic acid.

[0058] 1.4 Analysis of physiological and biochemical characteristics of lactic acid bacteria strains

[0059] Reference [Zhao J, Yin Characteristics, proteolysis and bacterial community of high-moisture corn stalk and stylo silage prepared with Bauhinia variegate flower. Bioresource Technology, 2020, 296: 122336. ] and [Muck RE, Nadeau EMG, Mc Allister TA, et al. Silage review: Recent advances and future uses of silage additives. Journal of Dairy Science, 2018, 101(5): 3980-4000. The methods described herein are used for physiological and biochemical characteristic analysis, as detailed below:

[0060] The six lactic acid bacteria strains initially screened in step 1.2 were activated. The activated strains were inoculated into MRS liquid medium at an inoculum rate of 1% (V / V) and incubated statically at 5℃, 15℃, 25℃, 35℃, and 45℃ for 24 h, respectively. The OD of the bacterial culture was measured. 600 nm The growth of the strains at different temperatures was compared.

[0061] The six lactic acid bacteria strains initially screened in step 1.2 were activated. The activated strains were inoculated at a rate of 1% (V / V) into MRS liquid medium with pH values ​​of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, and 2.5, respectively, and incubated at 37°C for 24 h. The OD of the bacterial culture was then measured. 600 nm The acid resistance of lactic acid bacteria was analyzed.

[0062] The six lactic acid bacteria strains initially screened in step 1.2 were activated. The activated strains were inoculated at a 1% inoculum into MRS liquid medium with NaCl concentrations of 3.0%, 6.5%, 10.0%, and 20.0% (w / v, g / 100mL), respectively. After incubation at 37℃ for 24 h, the OD of the bacterial culture was measured. 600 nm The salt tolerance of lactic acid bacteria was analyzed.

[0063] The results are shown in Table 5.

[0064] Table 5 Physiological and biochemical characteristics of lactic acid bacteria

[0065]

[0066] Note: "—" represents OD 600 nm < 0.05 indicates no growth; "+" means 0.05 ≤ OD 600 nm < 0.1 indicates weak growth; "++" means 0.1 ≤ OD 600 nm < 0.5 indicates normal growth; "++" indicates OD 600 nm A value ≥ 0.5 indicates good growth.

[0067] As shown in Table 5, strains S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14 are all Gram-positive and catalase-negative, all of which are bacilli. They did not produce gas in the glucose fermentation gas production experiment and are homofermentative lactic acid bacteria. All six lactic acid bacteria can grow well (+++) in MRS liquid medium with a NaCl concentration of 3.0%, a temperature range of 15–35°C, and a pH range of 3.5–7.0. All six lactic acid bacteria can grow moderately (++) in MRS medium with a NaCl concentration of 6.5% and a pH of 3.0; grow weakly (+) in MRS medium with a NaCl concentration of 10.0% and a temperature range of 5°C and 45°C; and cannot grow (-) in MRS liquid medium with a NaCl concentration of 20.0% and a pH of 2.5.

[0068] 1.5 16S rRNA sequence analysis of lactic acid bacteria strains

[0069] Genomic DNA was extracted from the six lactic acid bacteria strains initially screened in step 1.2. PCR amplification was performed using universal prokaryotic 16S rRNA primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3'). The amplified products were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The obtained 16S rRNA sequences were submitted to NSI Genbank (http: / / blast.NSi.nlm.nih.gov / Blast) for BLAST homology analysis to determine the genus and species information of the strains.

[0070] The 16S rRNA sequence of strain S_LAB_ColdC is shown in SEQ ID NO.3, and is as follows:

[0071]

[0072] The 16S rRNA sequence of strain S_LAB_Y03 is shown in SEQ ID NO.4, and is as follows:

[0073]

[0074] Table 6. NCBI alignment results based on 16S rRNA sequences.

[0075]

[0076] When the similarity of 16S rRNA is greater than 97.5%, the two taxonomic units belong to the same genus. As shown in Table 6, the similarity between two lactic acid bacteria and *Lactobacillus plantarum* is greater than 97.5%, and six strains were identified as *Lactobacillus plantarum*. S_LAB_ColdC and S_LAB_Y03 were named *Lactobacillus plantarum* ColdC and *Lactobacillus plantarum* YLAB_03, respectively, and were deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC NO: M2026974 and CCTCC NO: M2026975, respectively.

[0077] Example 2

[0078] Canadian goldenrod was harvested, chopped into 2-3 cm pieces, and wilted overnight. The raw material had a dry matter content of 22.08%, soluble carbohydrates of 14.28%, starch of 4.58%, crude protein of 17.21%, neutral detergent fiber of 37.59%, acid detergent fiber of 22.97%, and hemicellulose of 14.62%. The numbers of lactic acid bacteria, aerobic bacteria, yeast, and mold in the Canadian goldenrod raw material, expressed as colony forming units (CFU / g), were 4.98, 7.97, 5.02, and 3.45 log, respectively. 10 CFU / g.

[0079] Different lactic acid bacteria inoculants (the six lactic acid bacteria inoculants initially screened in step 1.2 of Example 1) were added to *Solidago canadensis* at a concentration of 1.0 × 10⁻⁶. 9 The concentration of CFU / kg Canadian goldenrod was determined by spraying the chopped goldenrod evenly onto the powder using a spray bottle. For the control group, the corresponding volume of sterile water (30 mL) was added. After thorough mixing, 300 g of the mixture was weighed and placed into a 40 cm × 30 cm polyethylene bag, which was then vacuum-sealed. Three replicates were prepared for each treatment. After 60 days of fermentation at room temperature in the dark, samples were taken for analysis of fermentation nutritional quality, fermentation quality, and counting of fermentation microorganisms.

[0080] Take 150.0 g of Canadian goldenrod raw material or fermentation sample, blanch at 105℃ for 30 min, dry at 65℃ to constant weight, and determine the dry matter (DM) content. The determination method is described in [Zhang Jing, Yu Zhu, Shao Tao. Effects of propionic acid and lactic acid bacteria additives on the fermentation quality of ryegrass. Acta Grasslandica Sinica, 2009, 17(2): 162-165.]. After pulverizing through a 0.38 mm sieve, the crude protein (CP) content was determined by distillation using a FOSS 8400 fully automatic Kjeldahl nitrogen analyzer. The determination of water-soluble carbohydrates (WSC) and starch content was performed according to the method described in [Yoshida S. Laboratory manual for physiological studies of rice. IRRI, Los Baios, Philippines, 1976, 43.]. The Van Soest method was used to determine the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF), and the hemicellulose content was calculated as follows: hemicellulose (%) = NDF (%) - ADF (%). The method referenced is [Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition[J]. Journal of Dairy Science, 1991, 74: 3583-3597.].

[0081] Take 20.0 g of Canadian goldenrod or its fermented sample, place it in an Erlenmeyer flask, add 180.0 mL of distilled water, mix thoroughly, and let stand at 4℃ for 24 h. After filtering through four layers of gauze, obtain the filtrate. The pH value of the filtrate is measured using a Mettler Toledo pH meter. After filtering the filtrate through a 0.22 μm aqueous filter membrane, the contents of lactic acid (LA), acetic acid (AA), and isobutyric acid (ISOBA) in the filtrate are analyzed according to the high performance liquid chromatography (HPLC) detection conditions described in the literature [Zhao J, Dong Z, Li J, et al. Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. Bioresource Technology, 2018, 266: 158-165.]. The method described in the reference [Salawu MB, Acamovic T, Stewart CS, et al. The use of tannins as silage additives: Effects on silage composition and mobile bag disappearance of dry matter and protein. Animal Feed Science and Technology, 1999, 82(3 / 4): 243-259.] was used to determine the ammonia nitrogen (NH3-N) content in the filtrate using the phenol-sodium hypochlorite colorimetric method.

[0082] Take 10.0 g of Canadian goldenrod or its fermentation sample, place it in a sealed Erlenmeyer flask containing 90.0 mL of sterile physiological saline (0.9%), shake at 120 rpm at room temperature for 1 h, filter with a single layer of sterile gauze, and obtain 10 -1 The gradient filtrate was then subjected to gradient dilution to obtain 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7Serial dilutions were prepared according to the method described in [He LW, Wang C, Xing YQ, et al. Ensiling characteristics, proteolysis and bacterial community of high-moisture corn stalk and stylo silage prepared with Bauhinia variegate flower. Bioresource Technology, 2020, 296: 122336.]. Aerobic bacteria, lactic acid bacteria, yeast, and mold were counted using the plate count method. 100 μL of each of the different serial dilutions was plated onto nutrient agar, deMan, Rogosa, and Sharpe (MRS) agar, and Bengal red agar, respectively [Zhao J, Yin X, Wang S, et al. Separating the effects of chemical and microbial factors onfermentation quality and bacterial community of napier grass silage by using gamma-ray irradiation and epiphytic microbiota transplantation. Animal Feed Science and Technology, 2021, 280: 115082.]. Aerobic bacteria were counted on nutrient agar medium after incubation at 37°C for 1 day, lactic acid bacteria were counted on MRS agar medium after incubation at 37°C for 2 days, and yeast were counted on Bengal red agar medium after incubation at 28°C for 1 day and molds after incubation for 3 days.

[0083] The results are shown in Tables 7 and 8.

[0084] Table 7. Effects of different strains on the fermentation quality of Canadian goldenrod.

[0085]

[0086] Table 8. Effects of different bacterial strains on the quality of fermented Canadian goldenrod.

[0087]

[0088] As shown in Tables 7 and 8, compared with the control group, all six tested lactic acid bacteria strains improved the fermentation quality of *Solidago canadensis*. Among them, strain S_LAB_Y03 significantly increased the dry matter (DM) content of the fermentation product (P < 0.05). All six lactic acid bacteria strains significantly increased the water-soluble carbohydrate (WSC) content and lactic acid bacteria count in the fermentation product (P < 0.05), while significantly decreasing the pH value, isobutyric acid content, ammonia nitrogen content, aerobic bacteria count, and yeast count (P < 0.05). None of the strains had a significant effect on the neutral detergent fiber (NDF) content, acid detergent fiber (ADF) content, or mold count (P > 0.05). Strains S_LAB_ColdC and S_LAB... S_4, S_LAB_Y03, S_LAB_12, and S_LAB_14 all significantly increased the crude protein (CP) content, lactic acid content, and lactic acid / acetic acid ratio of the fermentation products (P < 0.05). Compared with the control group, strains S_LAB_ColdC, S_LAB_4, S_LAB_Y03, and S_LAB_12 significantly increased the starch content of the fermentation products (P < 0.05) and significantly decreased the acetic acid (AA) content of the fermentation products (P < 0.05).

[0089] Example 3

[0090] In this embodiment, the harvested Canadian goldenrod was chopped to a length of 2-3 cm and subjected to three withering treatments of 0h (W0), 2.5h (W1), and 5h (W2). The dry matter content of the raw materials after withering was 19.87%, 23.42%, and 26.96%, respectively.

[0091] Four lactic acid bacteria strains obtained from screening in Example 1 were selected, namely S_LAB_ColdC, S_LAB_4, S_LAB_Y03, and S_LAB_12, and numbered A, B, C, and D respectively; the experiment was first set up with 5×10 5 cfu / g, 1×10 6 cfu / g, 5×10 6 Three bacterial cultures with concentrations of CFU / g were added, numbered 1, 2, and 3 respectively. Different concentrations of the strains were added to Canadian goldenrod that had undergone a 5-hour wilting (W2) treatment for fermentation. The optimal concentration of the strains was then determined. Subsequently, the optimal concentration was 5 × 10⁻⁶. 6 A strain of CFU / g was added to raw materials that had undergone different wilting treatments (0 h, 2.5 h, and 5 h) to verify the regulatory effect of the strain on the fermentation effect of Canadian goldenrod at different moisture contents. Finally, using a commercially available inoculant as a control, 5 × 10⁻⁶ CFU / g of the strain was added to the Canadian goldenrod raw material that had undergone 5 h of wilting. 6The fermentation effects of the tested strain and the commercial inoculant were compared using cfu / g lactic acid bacteria. The commercial inoculant used in this experiment was a mixed feed additive-type straw fermentation agent, prepared from a compound of *Lactobacillus acidophilus* and *Saccharomyces cerevisiae*, purchased from the Nanhua Qianmu flagship store on Taobao, with an addition amount of 0.1 mg / g. The fermentation sample preparation, pH, lactic acid content, acetic acid content, ammonia nitrogen content, number of lactic acid bacteria, number of aerobic bacteria, and other fermentation quality indicators, as well as the determination methods for nutrient indicators such as DM, WSC, starch, CP, NDF, and ADF, were all consistent with those in Example 2. The results are shown in Tables 9-14.

[0092] As shown in Tables 9 and 10, the lactic acid and acetic acid contents in the fermentation material inoculated with the strains were significantly higher than those in the blank control group (P < 0.05), with the highest contents in both categories in the strain C treatment group. There was no significant difference in the lactic acid / acetic acid ratio among the different strain treatment groups (P > 0.05), and the difference was also not significant compared with the blank control group (P > 0.05). The ammonia nitrogen content in the fermentation material inoculated with strains A and B was significantly lower than that in the control group and the other strain treatment groups (P < 0.05).

[0093] Significant differences were observed in pH, lactic acid content, acetic acid content, and lactic acid / acetic acid ratio among different bacterial suspension concentrations (P < 0.05), while no significant difference was found in ammonia nitrogen content (P > 0.05). The pH of suspension concentrations 2 and 3 was significantly lower than that of suspension 1 (P < 0.05); the lactic acid content of suspensions 1 and 2 was significantly lower than that of suspension 3 (P < 0.05); the acetic acid content of suspension 1 was significantly lower than that of suspensions 2 and 3 (P < 0.05), while there was no significant difference between suspensions 2 and 3 (P > 0.05); the lactic acid / acetic acid ratio was highest in suspension concentration 2, significantly higher than that of suspensions 3 and 1 (P < 0.05), and this ratio was higher in suspension 3 than in suspension 1. The interaction between bacterial strain and suspension concentration had a highly significant effect on the lactic acid / acetic acid ratio, the number of lactic acid bacteria, and the number of aerobic bacteria (P < 0.01), but no significant effect on the other indicators (P > 0.05). The pH of all treatment groups was below 4.5. Among them, the lactic acid content of strain C and bacterial concentration 2 treatment group was the highest, at 9.15 g / kg·DM.

[0094] Table 9. Effects of different strains and dosages on the fermentation quality of Canadian goldenrod after 5 hours of wilting.

[0095]

[0096] Note: Strains A, B, C, and D are S_LAB_12, S_LAB_4, S_LAB_Y03, and S_LAB_ColdC, respectively; bacterial concentrations 1, 2, and 3 are 5 × 10⁻⁶. 5 cfu / g, 1×106 cfu / g, 5×10 6 cfu / g. The same applies to Table 10 below.

[0097] The number of lactic acid bacteria was highest in the strain A treatment group, followed by the strain B treatment group, both of which were significantly higher than the other strain treatment groups and the blank control group (P < 0.05). In terms of the number of aerobic bacteria, there was no significant difference between the strain A and strain B treatment groups (P > 0.05), both of which were significantly higher than the other treatment groups (P < 0.05), and there was no significant difference between the blank control group, the strain C treatment group and the strain D treatment group (P > 0.05).

[0098] Table 10. Effects of different strains and dosages on the feed quality of fermented Canadian goldenrod after 5 hours of wilting.

[0099]

[0100] The effects of different bacterial strains on the nutrient composition of fermentation materials showed that the dry matter (DM) content of fermentation materials inoculated with strains B and D was significantly higher than that of the blank control group and the other strain treatment groups (P < 0.05), while there were no significant differences among the other strain treatment groups (P > 0.05). The water-soluble carbohydrate (WSC) content was highest in the strain B treatment group, followed by the strain A treatment group, both significantly higher than the blank control group and the other strain treatment groups. The WSC content of the blank control group was higher than that of the strain C and strain D treatment groups. The starch content and non-structural carbohydrate (NSC) content were highest in the strain C treatment group, followed by the strain D treatment group, both significantly higher than... For the other treatment groups (P < 0.05), there was no significant difference between the blank control group and the strain A treatment group (P > 0.05), but both were significantly higher than the strain B treatment group (P < 0.05). In terms of crude protein (CP) content, all strain treatment groups were higher than the control group (P < 0.05), with strain B treatment group having the highest content, followed by strain C and strain D treatment groups. The content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was highest in strain C and strain D treatment groups, both of which were significantly higher than the other treatment groups (P < 0.05), while the content of strain B treatment group was the lowest. There was no significant difference between the strain A treatment group and the blank control group (P > 0.05).

[0101] There were no significant differences in the contents of DM, NDF and CP among different bacterial suspension concentrations (P>0.05), and the NSC content in the bacterial suspension concentration 1 treatment group was higher than that in the concentration 2 treatment group.

[0102] Based on the analysis of fermentation quality and feed quality of fermented materials treated with different concentrations and strains, all tested strains showed good regulatory effects on fermentation, with strains S_LAB_ColdC and S_LAB_Y03 showing better results. The optimal concentration of the bacterial solution was 5 × 10⁻⁶. 6 cfu / g.

[0103] Table 11 Effects of different strains on the fermentation quality of different raw materials

[0104]

[0105] Note: Wilting treatments W0, W1, and W2 represent 0 hours, 2.5 hours, and 5 hours of wilt adjustment, respectively. The same applies below. CK is the blank treatment, with sterile water added.

[0106] Table 12 Effects of different bacterial strains on the quality of different materials after fermentation

[0107]

[0108] As shown in Tables 11 and 12, the changes in the number of lactic acid bacteria in the fermented material of *Solidago canadensis* after inoculation with different strains for different wilting times were as follows: the strain A, strain B, and strain C treatment groups were all significantly higher than the control group (P < 0.05), with strain A treatment group having the highest number of lactic acid bacteria. There was no significant difference between strain B and strain C treatment groups (P > 0.05), while the number of lactic acid bacteria in strain D treatment group was lower than the control group. The number of aerobic bacteria in all four strain treatment groups was significantly lower than the control group (P < 0.05), with strain D treatment group having the lowest number of aerobic bacteria, followed by strains B and C. The lactic acid content was highest in strain D treatment group, followed by strain B treatment group, both significantly higher than strain A treatment group, strain C treatment group, and the control group (P < 0.05). Regarding ammonia nitrogen content, there was no significant difference between strain D treatment group and the blank control group (P > 0.05), while the other strain treatment groups were significantly higher than the control group (P < 0.05).

[0109] The results of different withering time treatments showed that the pH was highest in the 0h withering time treatment group (4.77), which was significantly higher than that in the 2.5h and 5h withering time treatment groups (P < 0.05); the lactic acid to acetic acid ratio was lowest in the 0h withering time treatment group, while there was no significant difference between the 2.5h and 5h withering time treatment groups (P > 0.05); the ammonia nitrogen content was highest in the 0h withering time treatment group, which was significantly higher than that in the other treatment groups (P < 0.01), followed by the 2.5h withering time treatment group, and the lowest in the 5h withering time treatment group.

[0110] The effects of different bacterial strains on the nutrient composition of fermentation showed that: after different strain treatments, there was no significant difference in the dry matter (DM) content of the fermentation material (P>0.05); regarding the non-structural carbohydrate (NSC) content, there was no significant difference between the strain B treatment group and the blank control group (P>0.05), and both were significantly lower than the other strain treatment groups (P<0.05), with the strain C treatment group having the highest NSC content, followed by the strain A treatment group. Regarding crude protein (CP) content, the strain B and strain C treatment groups were significantly higher than the control group and the other strain treatment groups (P<0.05); regarding neutral detergent fiber (NDF) content, all strain treatment groups were significantly higher than the blank control group (P<0.05), with the strain C and strain D treatment groups having significantly higher NDF content than the other treatment groups (P<0.05); regarding acid detergent fiber (ADF) content, the strain D treatment group was significantly higher than the other treatment groups (P<0.05), followed by the strain C and strain A treatment groups. Regarding the pruning time, the DM, NSC, and CP indicators of the 5h pruning time treatment were significantly better than those of other pruning time treatments (P<0.05).

[0111] In summary, strains S_LAB_12, S_LAB_4, S_LAB_Y03, and S_LAB_ColdC all showed significant improvement effects on the fermentation of high-moisture materials with varying moisture content after different wilting treatments.

[0112] Table 13 Comparison of fermentation quality between screened strains and commercial strains

[0113]

[0114] Note: Strains A, B, C, and D are S_LAB_12, S_LAB_4, S_LAB_Y03, and S_LAB_ColdC, respectively, as shown in Table 14 below.

[0115] Table 14 Comparison of feed quality effects between screened strains and commercial strains

[0116]

[0117] As shown in Tables 13 and 14, when comparing the fermentation effects of the four strains isolated in this study with those of commercial strains, significant differences in pH were observed among the different treatment groups (P < 0.05), but the pH of all treatment groups was below 4.5. Regarding lactic acid and acetic acid content, the treatment groups of the four tested strains were significantly higher than those of the commercial strain treatment group (P < 0.05). The lactic acid to acetic acid ratio was significantly higher in strain A treatment group than in all other treatment groups (P < 0.05), while there were no significant differences among the other treatment groups (P > 0.05). The ammonia nitrogen content was also significantly higher in strain A treatment group than in the other treatment groups. The number of lactic acid bacteria was lowest in treatment group B, and significantly lower than that in the commercial strain treatment group and strain A treatment group (P < 0.05). There were no significant differences between the other tested strain treatment groups and the commercial strain treatment group (P > 0.05). The number of lactic acid bacteria was highest in treatment group B, followed by the commercial strain treatment group, then strain A and strain C treatment groups, and lowest in treatment group D. Regarding the number of aerobic bacteria, the number of aerobic bacteria in treatment groups B, C, and D was significantly lower than that in the commercial strain treatment group (P < 0.05), with the lowest number of aerobic bacteria in treatment group C. Nutritional analysis showed that the dry matter (DM) content of the fermentation materials from the four tested strains was significantly higher than that of the commercial strains (P < 0.05). Regarding non-structural carbohydrate (NSC) content, except for strain B which was significantly lower than the commercial strain (P < 0.05), the NSC content of the other tested strains was significantly higher than that of the commercial strains (P < 0.05), with strain C having the highest NSC content. In terms of crude protein (CP) content, strains B and C were significantly higher than the commercial strains (P < 0.05). Regarding neutral detergent fiber (NDF) and acid detergent fiber (ADF) content, strains C and D were significantly higher than the commercial strains (P < 0.05).

[0118] Based on the fermentation quality and feed quality indicators of the fermented materials after fermentation, all four tested strains in this study showed good fermentation promotion performance. Among them, strains C and D, namely S_LAB_Y03 and S_LAB_ColdC, showed better application effects.

[0119] In summary, this invention isolated and screened six lactic acid bacteria strains from fermented samples of Canadian goldenrod, namely S_LAB_ColdC, S_LAB_4, S_LAB_6, S_LAB_Y03, S_LAB_12, and S_LAB_14. These strains were identified as *Lactobacillus plantarum*. Among them, *Lactobacillus plantarum* S_LAB_Y03 and S_LAB_ColdC exhibit rapid fermentation, strong acid production capacity, excellent acid resistance, and a wide range of carbohydrate utilization. Inoculation with these strains can reduce the number of molds, aerobic bacteria, and yeasts in fermented Canadian goldenrod, while increasing the abundance of lactic acid bacteria. Simultaneously, it effectively improves the fermentation quality and nutritional quality of the fermented product, demonstrating good potential for fermentation applications. This invention provides excellent strain resources for the fermentation and preparation of high-moisture materials.

[0120] It should be noted that the above embodiments are only some exemplary implementations of the present invention, and not all implementations. Those skilled in the art can obtain other implementations based on these embodiments without creative effort, and all such implementations fall within the protection scope of the present invention.

Claims

1. A type of lactic acid bacteria, characterized in that, The lactic acid bacteria are *Lactobacillus plantarum* DCFColdC or *Lactobacillus plantarum* DCFY03; the preservation number of *Lactobacillus plantarum* DCFColdC is CCTCC NO: M2026974; the preservation number of *Lactobacillus plantarum* DCFY03 is CCTCC NO: M2026975.

2. A microbial inoculant, characterized in that, The effective components of the microbial agent include Lactobacillus plantarum DCFColdC and / or Lactobacillus plantarum DCFY03 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of the microbial agent is ≥5×10⁻⁶. 6 CFU / g.

4. The method for preparing the microbial inoculant according to any one of claims 2 to 3, characterized in that, The process includes the following steps: culturing *Lactobacillus plantarum* in MRS liquid medium to obtain the microbial agent.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 15~35℃, and the pH value is 3.5~7.

0.

6. The application of the lactic acid bacteria of claim 1, or the microbial agent of claim 2 or 3, or the microbial agent prepared by the preparation method of claim 4 or 5, in the fermentation of Canadian goldenrod.

7. The application according to claim 6, characterized in that, The fermentation raw material for Canadian goldenrod has a moisture content of 75% to 83%.

8. The application according to claim 7, characterized in that, The fermentation raw materials include the stems and leaves of Canadian goldenrod.

9. A method for improving the fermentation quality of Canadian goldenrod, characterized in that, Includes the following steps: The microbial agent described in claim 2 or 3 is mixed with the stems and leaves of Canadian goldenrod and fermented.

10. The method according to claim 9, characterized in that, Based on the wet weight of the fermentation raw materials, the ratio of the microbial inoculant to the stems and leaves of *Solidago canadensis* is ≥5.0 × 10⁻⁶. 6 CFU: 1kg.

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