Lactobacillus plantarum and leavening agent and application of lactobacillus plantarum and leavening agent in rotten vegetable leaf fermentation and feed

Abstract

The invention discloses lactobacillus plantarum, the Latin name of the lactobacillus plantarum is Lactiplantarum, the lactobacillus plantarum is named as BQ-3, the lactobacillus plantarum is preserved in Guangdong Microbial Culture Collection Center on January 15, 2025, and the preservation registration number of the lactobacillus plantarum is GDMCC No.65791; secondly, the invention further discloses a microbial fermentation agent, and the effective components of the microbial fermentation agent comprise the lactobacillus plantarum. In addition, the invention also discloses an application of the lactobacillus plantarum or the microbial fermentation agent in fermentation of the rotten vegetable leaves, and an application of the fermented rotten vegetable leaves prepared by the application of the lactobacillus plantarum or the microbial fermentation agent in fermentation of the rotten vegetable leaves in fattening feed. The lactobacillus plantarum strain disclosed by the invention has the beneficial effects that the lactobacillus plantarum strain is separated and screened from naturally-fermented rotten vegetable leaves, and has the characteristics of high lactic acid production speed and high yield; when applied to the fermentation of the rotten vegetable leaves, the strain can quickly produce acid, reduce the pH value, reduce the loss of dry matters, retain nutrients, inhibit the generation of ammoniacal nitrogen and putrefying acid, significantly improve the fermentation quality and prolong the storage life; the feed additive can promote fattening growth of livestock when applied to feeds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a lactobacillus plantarum and starter culture and application thereof in fermented tail and feed. BACKGROUND

[0002] Lactobacillus (such as Lactobacillus plantarum) plays an important role in the fermentation of tail and feed production, mainly in the following aspects: first, lactobacillus can significantly improve the content of crude protein and reduce the content of cellulose and nitrate through fermentation; second, lactic acid produced during fermentation can reduce the pH value, inhibit the growth of mold, bacillus and other spoilage microorganisms, prolong the shelf life of silage; in addition, metabolites (such as lactic acid, propionic acid) produced after fermentation can reduce the exudate produced by butyric acid bacteria activity, reduce the peculiar smell of silage and improve the palatability; in addition, by mixing different waste (such as high-moisture and low-moisture raw materials) for fermentation, sewage production can be reduced and treatment cost can be reduced.

[0003] For example, in document CN117511780A, a Lactobacillus plantarum with fast growth speed and strong acid-producing ability is provided, which is Lactobacillus plantarum DCF89, Lactobacillus plantarum DCF112 or Lactobacillus plantarum DCF120; the preservation number of Lactobacillus plantarum DCF89 is CCTCC NO: M20221420; the preservation number of Lactobacillus plantarum DCF112 is CCTCC NO: M20221421; the preservation number of Lactobacillus plantarum DCF120 is CCTCC NO: M20221422. The present application provides a microbial agent, wherein the effective component of the microbial agent includes one or more of Lactobacillus plantarum DCF89, Lactobacillus plantarum DCF112 and Lactobacillus plantarum DCF120 in the above technical solution. Preferably, the viable bacterial count of the microbial agent is ≥5×10 6 CFU / g. The present application provides a preparation method of the microbial agent in the above technical solution, which includes the following steps: culturing Lactobacillus plantarum in MRS liquid medium to obtain the microbial agent; the Lactobacillus plantarum is one or more of Lactobacillus plantarum DCF89, Lactobacillus plantarum DCF112 and Lactobacillus plantarum DCF120 in the above technical solution. Preferably, the culture temperature is 15-35℃, and the pH value is 3.5-7.0.

[0004] The Lactiplantibacillus plantarum provided in the scheme can reduce the number of molds, aerobic bacteria and yeasts in high-moisture broccoli stalk silage by producing acid and reducing pH value, and can improve the number of lactic acid bacteria and has good silage potential. However, it is sensitive to high-moisture silage raw materials, and when the moisture content of the silage raw materials is high, it may affect the reproduction speed of the strain, thereby indirectly leading to insufficient acid production and difficulty in quickly reducing the pH value. SUMMARY

[0005] The purpose of the present application is to provide a Lactiplantibacillus plantarum and a starter that can quickly produce acid and quickly reduce the pH value when treating high-moisture silage raw materials, and the application of the Lactiplantibacillus plantarum and the starter in stalk fermentation and feed.

[0006] In order to achieve the above-mentioned purpose, the basic scheme of the present application provides a Lactiplantibacillus plantarum, the Latin name of which is Lactiplantibacillus plantarum, named BQ-3, and preserved in the Guangdong Microbial Culture Collection Center on January 15, 2025, with the preservation registration number GDMCC No. 65791.

[0007] In the second aspect, the present application provides a microbial starter, and the effective component includes the Lactiplantibacillus plantarum described in the above-mentioned basic scheme.

[0008] In the third aspect, the present application provides the application of the Lactiplantibacillus plantarum or the microbial starter described above in stalk fermentation.

[0009] Preferably, the moisture content of the stalk treated in the stalk fermentation is 90-95% by mass percentage.

[0010] Preferably, the stalk fermentation process includes the following steps,

[0011] R1. Mixing stalks with wheat bran as raw materials;

[0012] R2. Spraying the bacterial solution of the Lactiplantibacillus plantarum in the raw materials prepared in R1 to seal and ferment to prepare fermented stalks;

[0013] The number of viable bacteria of the Lactiplantibacillus plantarum in the raw materials is ≥1.0×10 6 CFU / g, based on the wet weight of the raw materials.

[0014] Preferably, the stalk fermentation process includes the following steps,

[0015] S1. Adding the Lactiplantibacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae after activation to prepare a bacterial solution with a concentration ≥1×10 8 CFU / mL;

[0016] S2. Mixing stalks with wheat bran as raw materials, adding brown sugar and the bacterial solution prepared in S1 to seal and ferment to prepare fermented stalks.

[0017] In a fourth aspect, the present application provides the use of the above-mentioned Lactobacillus plantarum or microbial starter in the fermentation of the tail meal, and the use of the fermented tail meal in the fattening feed.

[0018] The present application has the following beneficial effects:

[0019] 1. The Lactobacillus plantarum strain of the present application is isolated from naturally fermented tail meal, has the characteristics of fast lactic acid production and high yield, and the lactic acid yield reaches more than 30 g / L after 12 h of growth, and the lactic acid content reaches 34.75 g / L after 18 h; secondly, the strain has resistance to clindamycin, neomycin, gentamicin, vancomycin, ofloxacin and polymyxin B, and excellent tolerance to acid, salt and bile salt, and also has strong nitrite degradation ability; in addition, the strain has high biological safety, no hemolytic effect and no adverse effects proved by animal acute toxicity test.

[0020] 2. When the strain and the microbial starter prepared therefrom are applied to the fermentation of high-moisture tail meal, they can quickly produce acid and reduce the pH value, reduce the loss of dry matter, retain nutritional ingredients, inhibit the generation of ammonia nitrogen and putrefactive organic acid and other putrefactive substances, and significantly improve the storage time and the fermentation quality of the tail meal.

[0021] 3. When the feed added with the fermented tail meal of the strain is applied to livestock fattening, it can significantly improve the final weight and average daily weight gain of livestock, reduce the feed conversion ratio and save costs.

[0022] Biological preservation instructions:

[0023] The Lactobacillus plantarum BQ-3 strain, whose Latin name is Lactiplantibacillus plantarum, was preserved in the Guangdong Microbial Culture Collection Center on January 15, 2025, and the preservation registration number is GDMCC No.65791. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is the colony morphology (A) and gram staining diagram (B) of Lactobacillus plantarum BQ-3.

[0025] Figure 2 It is the phylogenetic tree of Lactobacillus plantarum BQ-3.

[0026] Figure 3 It is the growth, pH and lactic acid curve of Lactobacillus plantarum BQ-3.

[0027] Figure 4 It is the hemolysis test result of Lactobacillus plantarum BQ-3.

[0028] Figure 5 It is the effect of the strain on the body weight of mice in acute toxicity test.

[0029] Figure 6 For acute toxicity, mouse organ tissue sections (40x, 50 μm) were cut. DETAILED DESCRIPTION

[0030] In the present application, the specific experimental conditions and methods are not specified, which are selected according to the conventional methods and conditions in the technical field, or according to the product instructions.

[0031] The various materials and reagents used in the present application are materials and reagents commonly used in the art, and can be obtained through conventional commercial channels.

[0032] The "Classification and Identification of Lactic Acid Bacteria and Experimental Methods" mentioned in the present application, written by Ling Daiwen and Dong Xiuzhu, was published by China Light Industry Press in 1999.

[0033] The "Common Bacterial System Identification Manual" mentioned in the present application, written by Dong Xiuzhu and Cai Miaoying, was published by Science Press in 2001.

[0034] The "Berger Bacterial Identification Manual" mentioned in the present application, written by R·E·Buckanan and N·E·Gibbons, was published in Chinese version by Science Press in 1984.

[0035] The "Guidelines for the Management and Use of Laboratory Animals" mentioned in the present application, written by He Zhengming, Li Genping, Zhu Desheng and Lu Shengming, was published by Science Press in May 2016.

[0036] With the rapid development of China's vegetable industry, the annual output of vegetable processing tail vegetables (such as root, stem, leaf and other processing by-products) has exceeded 400 million tons. Such tail vegetables generally have the characteristics of high moisture (> 90%) and easy spoilage, and are rich in protein, dietary fiber and soluble sugar and other nutrients. If not properly handled, it will not only cause environmental pollution, but also lead to resource waste. At present, traditional composting, landfill or incineration is still the main way of tail vegetable resource processing, but these methods have low processing efficiency, high risk of secondary pollution and other problems, for example, traditional composting is prone to anaerobic fermentation due to high moisture content of tail vegetables, producing foul-smelling gas and leachate pollution; landfill will occupy land resources, and even cause soil salinization. Therefore, using microbial fermentation technology to convert tail vegetables into high-value-added products such as feed and fertilizer has become a current research hotspot.

[0037] In the field of microbial fermentation of silage, lactic acid bacteria have become the commonly used fermentation strains due to their advantages of producing lactic acid by metabolizing sugars, reducing the pH of the fermentation system, inhibiting the growth of harmful microorganisms, and improving the quality of fermentation products. However, the special nature of silage poses strict requirements on the lactic acid bacteria used for fermentation. Firstly, silage is high in moisture and perishable, requiring lactic acid bacteria to have the ability to grow and reproduce rapidly and produce acid efficiently to establish an acidic environment in a short time and avoid spoilage. Secondly, the pH changes during the fermentation process of silage, requiring lactic acid bacteria to have strong environmental adaptability. In addition, silage has diverse nutritional components, requiring lactic acid bacteria to fully utilize the carbon sources and other nutrients. However, the current commonly used lactic acid bacteria strains have obvious shortcomings. Most strains grow slowly and enter the logarithmic growth phase late, making it difficult to quickly form a dominant population. At the same time, the speed and total amount of lactic acid production are limited, which cannot produce enough lactic acid to quickly reduce the environmental pH value in a short time. For perishable silage, such performance cannot meet the demand of rapid and stable treatment, limiting the application effect of lactic acid bacteria in silage treatment and affecting the efficiency of silage resource utilization.

[0038] Therefore, the present application provides a specific embodiment of Lactiplantibacillus plantarum and starter culture capable of rapid acid production and rapid pH reduction when treating high-moisture silage material, as well as the application of the same in silage fermentation and feed.

[0039] The specific embodiments are further described in detail below:

[0040] The Lactiplantibacillus plantarum provided in the present embodiment has the Latin name Lactiplantibacillus plantarum and is named BQ-3. It was preserved in the Guangdong Microbial Culture Collection Center on January 15, 2025, and the preservation registration number is GDMCC No. 65791.

[0041] Secondly, a specific embodiment of a microbial starter culture is also provided, which includes the Lactiplantibacillus plantarum in the above-mentioned embodiments as an effective component.

[0042] In addition, a specific embodiment of the application of the Lactiplantibacillus plantarum or microbial starter culture in silage fermentation is also provided.

[0043] More preferably, the silage treated in the silage fermentation has a water content of 90-95% by mass.

[0044] More preferably, the silage fermentation process includes the following steps:

[0045] R1. Mixing silage and wheat bran as raw materials;

[0046] R2. Spraying the bacterial solution of Lactiplantibacillus plantarum on the raw materials prepared in R1 and sealing for fermentation to prepare fermented silage;

[0047] The viable cell number of the Lactobacillus plantarum in the raw material is ≥1.0×10 6 CFU / g.

[0048] As another preferred embodiment, the following step of fermenting offal is included,

[0049] S1. After being activated, the Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae are prepared into a bacterial solution with a concentration ≥1×10 8 CFU / mL;

[0050] S2. The offal is mixed with wheat bran as raw material, and then brown sugar and the bacterial solution prepared in S1 are added for fermentation to obtain fermented offal.

[0051] In addition, the application also provides an embodiment of the application of the Lactobacillus plantarum or microbial starter in the fermentation of offal and the application of the fermented offal in fattening feed.

[0052] Example 1

[0053] 1. Isolation, identification and screening of Lactobacillus plantarum BQ-3

[0054] 1.1 Experimental materials

[0055] The naturally fermented offal of preserved vegetable was collected from the Animal Nutrition Laboratory of Chongqing Institute of Animal Husbandry in Rongchang District, Chongqing.

[0056] The Lactobacillus plantarum BQ-3 (LPB-3 for short) was derived from the naturally fermented offal of preserved vegetable collected from the Animal Nutrition Laboratory of Chongqing Institute of Animal Husbandry in Rongchang District, Chongqing, and was preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No. 65791.

[0057] 1.2 Culture medium

[0058] The composition of the MRS liquid culture medium is as follows: 10.0 g of proteose peptone, 10.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 2.0 g of potassium phosphate dibasic, 0.05 g of manganese sulfate, 0.1 g of magnesium sulfate, 1.0 mL of Tween-80, and distilled water to 1 L, with a pH of 6.2±0.2.

[0059] The MRS solid culture medium is prepared by adding 1.5%-2% (w / v) agar powder to the above-mentioned MRS liquid culture medium.

[0060] The CaCO3-MRS solid culture medium is prepared by adding 10.0 g of CaCO3 to the above-mentioned MRS solid culture medium.

[0061] 1.3 Isolation and purification of strains

[0062] Take 20 g of naturally fermented dregs sample, add 180 mL of sterile normal saline, and shake at 37°C, 220 r / min for 30 min to prepare a suspension. Take 1 mL of the suspension and dilute it 10 times with sterile normal saline. Take 100 μL of each gradient and evenly spread it on CaCO3-MRS solid medium. Each gradient is done in triplicate, and the plates are incubated at 37°C for 48 h. After observing the colony morphology, use a loop to pick colonies that are clearly dissolved in calcium carbonate and are greater than 5 mm in diameter. The suspected lactic acid bacteria are then inoculated on MRS solid medium and incubated at 37°C. The pure strain is then suspended in 25% glycerol solution and stored at -80°C for future use.

[0063] 1.4 Screening of high-yield lactic acid bacterial strains

[0064] The purified single colonies are inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 24 h to obtain the primary seed liquid. The primary seed liquid is inoculated into new MRS liquid medium at a 1% (v / v) inoculation amount and incubated at 37°C for another 24 h. The pH value of the culture solution is measured. Strains with a pH value less than 4.0 are preliminarily screened out as 56 acid-producing strains for subsequent quantitative analysis of lactic acid production.

[0065] The primary seed liquid of the above 56 strains is inoculated into 25 mL of MRS liquid medium at a 1% inoculation amount. After incubation at 37°C for 24 h, the supernatant is obtained by centrifugation at 12000 r / min for 10 min. The lactic acid production of each strain is quantitatively detected by GC-MS (gas chromatography-mass spectrometry). After comprehensive evaluation, the optimal high-yield lactic acid strain is determined to be Lactobacillus BQ-3.

[0066] 1.5 Preliminary identification of the strain

[0067] 1.5.1 Morphological observation

[0068] Strain BQ-3 is inoculated into MRS liquid medium for observation of growth. The colony surface is smooth and moist, with a neat edge, and is round, opaque, and milky white with a raised center (see Figure 1 -A). Under a light microscope, BQ-3 strain is stained purple, and the bacterial cells are short rods, mostly single, paired, or in chains, and do not produce spores (see Figure 1 -B).

[0069] 1.5.2 Physiological and biochemical identification

[0070] According to the "Lactic Acid Bacteria Classification and Identification and Experimental Methods", the screened strain BQ-3 was cultured in MRS liquid medium at 37°C for 24h, inoculated into the corresponding bacterial micro-reaction tube, and continued to be cultured at 37°C for 24-48h. The color change of the reaction tube was observed and recorded, and the identification was carried out according to the "Common Bacterial System Identification Manual".

[0071] The physiological and biochemical identification results showed that the BQ-3 strain could utilize glucose, sucrose, maltose, inulin, raffinose, mannitol, cellobiose, fructose, rhamnose, mushroom sugar, lactose, galactose, D-ribose, salicylic acid, and escin, but could not utilize starch, xylose, sorbose, sorbitol, and the hydrogen sulfide test, protein hydrolysate water test, gelatin liquefaction test, and nitrate reduction test were all negative.

[0072] According to the colony morphological characteristics, gram staining results and physiological and biochemical identification results, the BQ-3 strain was preliminarily judged as Lactobacillus according to the "Berger Bacterium Identification Manual".

[0073] 1.6 Molecular identification of the strain

[0074] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit, and PCR amplification was performed using universal primers. The amplification product was subjected to 1% gel electrophoresis and gel recovery and purification, and then sent to Shanghai Sunway Biological Engineering Co., Ltd. for sequencing to obtain the complete 16S rRNA sequence. The sequencing results were analyzed by homology comparison analysis through the BLAST program in NCBI, and then the Neighbor-Joining method in MEGA7 software was used to construct a phylogenetic tree.

[0075] The results showed that the homology of the BQ-3 strain and Lactiplantibacillus plantarum (NR_115605.1) reached 99%( Figure 2 ). Therefore, it is determined that the plant lactobacillus used in the present application is Lactiplantibacillus plantarum BQ-3 (hereinafter referred to as LPB-3), which has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) located at No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, China, with a postal code of 510070. The preservation number is GDMCC NO: 65791, and the preservation date is January 15, 2025.

[0076] 2. Growth characteristics and acid-producing capacity of Lactiplantibacillus BQ-3

[0077] 2.1 Test method

[0078] 2.1.1 Strain activation

[0079] Dip an inoculation loop into a -80℃ glycerol tube containing frozen bacterial culture and streak it onto MRS solid medium. Incubate at 37℃ for 24–48 hours. Once colonies have grown, select the best-growing single colony and inoculate it into 5 mL of MRS liquid medium. Incubate overnight at 37℃ to obtain activated bacterial culture.

[0080] 2.1.2 Determination of growth curve and acid production curve

[0081] The activated bacterial culture was inoculated into MRS liquid medium at a rate of 1% (v / v) (three replicates) and incubated at 37°C. Samples were taken every 2 hours during the incubation period for the following tests:

[0082] (1) Determination of bacterial concentration: Take 1 mL of bacterial solution, centrifuge at 5000 r / min for 10 min, discard the supernatant, wash the precipitate twice with physiological saline, and measure the OD using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Values ​​were used to plot the growth curve of the strain; at the same time, 0.1 mL of bacterial solution was serially diluted 10-fold, and three suitable dilutions were selected and plated on MRS solid medium. After incubation at 37℃ for 48 h, the number of colonies was counted (using plates with 30-300 CFU / plate).

[0083] (2) pH value determination: Take 3 mL of bacterial solution and measure the pH value of the bacterial solution directly using a calibrated pH meter (accuracy ±0.01).

[0084] (3) Quantitative analysis of lactic acid:

[0085] Sample preparation: Centrifuge the bacterial culture at 8000 r / min for 10 min, take 2 mL of the supernatant into a 10 mL colorimetric tube, heat in a 100℃ water bath for 10 min, cool, add 0.2 mL of 50% (volume fraction) H2SO4 and mix well, then add 1 mL of methanol, heat in a 58℃ water bath for 30 min, then add 1 mL of ultrapure water, add 2 mL of chloroform, shake vigorously for 3 min to fully extract, centrifuge at 1000 r / min for 2 min to separate the layers, take the lower chloroform layer, filter through a 0.22 μm organic filter membrane and transfer to a brown sample vial for analysis.

[0086] Preparation of standard curve: D-lactic acid standard was dissolved in 1 mL of ultrapure water to prepare a 10000 mg / L stock solution, which was then serially diluted to prepare a series of standard solutions of 5000, 2500, 1250, 625, 312.5, and 156.25 mg / L. 2 mL of each standard solution was taken and processed according to the sample preparation steps described above, with each concentration injected three times. A standard curve was plotted with the standard solution concentration (X, mg / L) on the x-axis and the GC-MS peak area (Y) on the y-axis. The linear regression equation was Y = 28.74X - 9889.45 (R² = 0.9987), with a linear range of 156.25–5000 mg / L.

[0087] 2.2 Test results

[0088] Figure 3 The results showed that Lactobacillus plantarum BQ-3 grew fast and entered the logarithmic phase after 2h of inoculation, and the cell density increased exponentially, and entered the stationary phase after 12h (the number of colonies reached 6 x 10 9 CFU / mL), and entered the exponential growth phase earlier than similar strains. The pH value decreased continuously with the growth of the cells, and decreased from the initial 5.39 to 3.76 in the logarithmic phase (2-12h), and stabilized at about 3.65 after 14h, indicating that the strain produced acid fast. The dynamic display of lactic acid production showed that the lactic acid content reached a peak of 34.75g / L at 18h, which was significantly higher than the lactic acid production level of most reported Lactobacillus plantarum (usually 20-30g / L), and then fell slightly due to substrate consumption and metabolic product accumulation. The viable cell count of the strain was stable at more than 6 x 10 9 CFU / mL within 12-18h, and had the characteristics of high-density growth and high-acid production, and had the potential for industrial fermentation production.

[0089] 3. Antibiotic sensitivity determination of Lactobacillus plantarum BQ-3

[0090] 3.1 Test method

[0091] The activated bacterial solution was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v), and incubated at 37°C until the end of the logarithmic growth phase. 10 8 CFU / mL of the bacterial suspension was uniformly coated on the MRS solid plate, and after the water on the surface of the plate was completely absorbed by the agar, sterile forceps were used to tightly adhere the various drug-sensitive paper sheets to the agar surface, and incubated at 37°C for 24h. The inhibition zone diameter was observed and measured, and the drug sensitivity was determined according to the inhibition zone size. The determination standard was established according to the US NCCLS document.

[0092] 3.2 Test results

[0093] As shown in Table 1, Lactobacillus plantarum BQ-3 was sensitive to ampicillin, kanamycin, tetracycline, chloramphenicol and cefuroxime, moderately sensitive to erythromycin, and not sensitive to clindamycin, neomycin, gentamicin, vancomycin, ofloxacin and polymyxin B, i.e. resistant.

[0094] Table 1 Results of antibiotic sensitivity experiment

[0095]

[0096] Note: "-" represents no inhibition zone.

[0097] 4. Determination of the acid tolerance, bile salt tolerance and NaCl tolerance of Lactobacillus plantarum BQ-3

[0098] 4.1 Test Methods

[0099] The activated test strain was inoculated into MRS liquid medium at a rate of 1% (v / v) and cultured in a constant temperature incubator at 37℃ until the end of the logarithmic growth phase. 1 mL of bacterial suspension was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with PBS. The cells were then resuspended in an equal volume of PBS. The bacterial suspension was then inoculated into 10 mL of MRS liquid medium with pH values ​​of 2, 2.5, and 3, bile salt concentrations of 0.1%, 0.2%, and 0.3%, and NaCl concentrations of 2%, 4%, 6%, 8%, and 10%, respectively. Unadjusted MRS medium was used as a blank control. Three replicates were set up for each group. Viable cell counts were measured after 3 h and 6 h of incubation at 37℃. The survival rates for acid tolerance, bile salt tolerance, and NaCl tolerance were calculated using the following formulas:

[0100] Survival rate = (lg CFU N1 / lg CFU N0) × 100%

[0101] In the formula: N1 represents the number of viable bacteria after treatment; N0 represents the number of viable bacteria in the blank control group.

[0102] 4.2 Test Results

[0103] Table 2 shows that *Lactobacillus plantarum* BQ-3 can stably survive in acidic environments (pH 2–3), bile salt concentrations (0.1%–0.3%), and NaCl concentrations (2%–10%). Specifically, the survival rate of this strain reached 40.15% after 6 hours of treatment with MRS culture medium at pH 2, 87.47% after 6 hours of treatment with MRS culture medium containing 10% NaCl, and 71.97% after 6 hours of treatment with MRS culture medium containing 0.3% bile salt. These results indicate that *Lactobacillus plantarum* BQ-3 has strong tolerance to acidic, high-salt, and bile salt environments. This characteristic gives it excellent adaptability to fermentation environments and makes it highly efficient for use in the fermentation of vegetable waste.

[0104] Table 2. Tolerance of Lactobacillus plantarum BQ-3 to acid, bile salts, and NaCl (mean ± standard error, %)

[0105]

[0106] 5. Determination of the ability of Lactobacillus plantarum BQ-3 to reduce nitrite

[0107] 5.1 Test Methods

[0108] The activated test strain was inoculated at 2% (v / v) into MRS medium containing sodium nitrite (200 mg / L) and cultured at 37°C. MRS medium without sodium nitrite was used as a blank control. Samples were taken at 12, 24, 36, and 48 hours, and the supernatant was collected after centrifugation at 8000 rpm for 10 min. The nitrite content in the supernatant was determined using the naphthylethylenediamine hydrochloride method (micro-method), and the degradation rate was calculated. The formula for calculating the nitrite degradation rate is as follows:

[0109] Nitrite degradation rate (%) = (N2 - N1) / N2 * 100

[0110] In the formula: N1 represents the nitrite content in the culture medium inoculated with the strain; N2 represents the nitrite content in the culture medium inoculated with the strain not inoculated.

[0111] 5.2 Test Results

[0112] Table 3 shows that the nitrite degradation capacity of *Lactobacillus plantarum* BQ-3 increases with prolonged culture time. The nitrite degradation rate reached 98.64% after 12 hours of culture at 37℃, and as high as 99.05% after 48 hours, indicating that it can efficiently degrade nitrite in the environment through metabolic activity and has the potential to reduce nitrite accumulation during the fermentation of vegetable waste.

[0113] Table 3. Degradation rate of nitrite by Lactobacillus plantarum BQ-3 (mean ± standard error, %)

[0114]

[0115] 6. Safety evaluation of Lactobacillus plantarum BQ-3

[0116] 6.1 Hemolytic test

[0117] 6.1.1 Test Methods

[0118] Using a sterile inoculation loop, take a suspension of *Lactobacillus plantarum* BQ-3 and inoculate it into a blood agar plate. Incubate at 37°C for 48 hours. Use *Staphylococcus aureus* as a positive control and observe whether there is hemolysis around the colonies. If there is no obvious clear zone around the colony (γ hemolysis), it is considered that there is no hemolytic reaction; if a grass-green ring appears around the colony (α hemolysis), it is an incomplete hemolytic reaction; if a completely clear hemolytic ring of 2–4 mm appears (β hemolysis), it is considered that the strain can produce hemolysin and a complete hemolytic reaction exists.

[0119] 6.1.2 Test Results

[0120] Hemolysis test results as follows Figure 4 As shown, after 48 hours of culture, a β-hemolytic zone appeared around Staphylococcus aureus, while no hemolytic zone appeared around Lactobacillus plantarum BQ-3, indicating that Lactobacillus plantarum BQ-3 does not have hemolytic function.

[0121] 6.2 Animal acute oral toxicity test

[0122] 6.2.1 Test method

[0123] 6.2.1.1 Preparation of test bacterial solution

[0124] The activated Lactobacillus plantarum BQ-3 bacterial solution was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v), and then incubated at 37°C for 12 h. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the same volume of sterile normal saline was added for washing. The centrifugation and washing were repeated twice, and the bacterial concentration was adjusted to 10 11 CFU / mL with sterile normal saline to prepare a bacterial solution for gavage.

[0125] 6.2.1.2 Animals and treatment

[0126] Twenty healthy SPF Kunming mice with a body weight of 18-20 g were selected, and after one week of adaptive feeding, they were randomly divided into two groups (n=10) with half males and half females, namely a control group and a Lactobacillus plantarum (LP) group. The control group was gavaged with sterile normal saline, and the LP group was gavaged with a BQ-3 bacterial suspension (1×10 11 CFU / mL of live bacteria), with a gavage dose of 0.4 mL / 20 g of body weight, once a day for 14 consecutive days. After the end of the test, all mice were subjected to orbital plexus blood collection, and then were sacrificed in a clean environment. The heart, liver, spleen, lung, and kidney were dissected and weighed, and the spleen, kidney, and ileum were collected for examination.

[0127] All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Chongqing Institute of Animal Science. The experimental mouse room was maintained at a temperature of (25±1) °C, a relative humidity of 56±5%, and a light / dark cycle of 12h / 12h. The mice were fed with a pure diet and had free access to food and water. During the experiment, the survival, mental state, and fecal characteristics of the mice were observed and recorded every day. The body weight and food intake were recorded every 3 days.

[0128] 6.2.2 Detection index

[0129] (1) Isolation of plasma and serum for blood routine and serum biochemical index detection;

[0130] (2) Collection of ileum, liver, and kidney tissues fixed in 4% paraformaldehyde for HE tissue sectioning for pathological morphological analysis.

[0131] 6.2.3 Test results

[0132] 6.2.3.1 General physical observation

[0133] During the experiment, all mice grew well and were in good mental state, and their food intake, water drinking, respiration and defecation were normal, and there was no death or poisoning. The body weight changes of mice in each group were as shown in Table 2, and the body weight of mice in LP group at each stage of the experiment had no significant difference compared with the control group (P>0.05). Figure 5

[0134] 6.2.3.2 Organ index

[0135] After the experiment, the mice were dissected, and macroscopic observation showed that there were no organic lesions in the heart, liver, spleen, lung, kidney, intestine and other internal organs. The organ index results are shown in Table 4. The heart / body ratio, liver / body ratio, spleen / body ratio and kidney / body ratio of mice in the LP group had no significant difference compared with the control group (P>0.05). This indicates that Lactobacillus plantarum BQ-3 has no obvious adverse effects on the development of the main organs of mice.

[0136] Table 4 Organ index of mice in each group of acute toxicity experiment (mean ± standard error)

[0137]

[0138] 6.2.3.3 Blood routine and serum biochemical parameters

[0139] The blood routine test results (Table 5) showed that the white blood cell count, red blood cell count, hemoglobin content, platelet count and lymphocyte ratio of mice in the LP group were within the normal physiological range and had no significant difference compared with the control group (P>0.05), indicating that the intragastric administration of Lactobacillus plantarum BQ-3 did not interfere with the hematopoietic function and immune cell distribution of mice. The blood biochemical analysis results (Table 5) showed that the liver function indicators (glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase), kidney function indicators (creatinine, urea nitrogen) and metabolism related indicators (total protein, triglyceride, cholesterol) of the two groups of mice had no significant difference (P>0.05), indicating that Lactobacillus plantarum BQ-3 had no obvious adverse effects on the metabolic function of important organs such as liver and kidney of mice.

[0140] Table 5 Blood routine and serum biochemical parameters of mice in each group of acute toxicity experiment (mean ± standard error)

[0141]

[0142] 6.2.3.4 Pathomorphological analysis

[0143] Microscopic examination of HE-stained sections of liver, kidney and ileum tissues found that Figure 6 ​), the liver lobular structure of each group of mice was complete, the liver cells were arranged in order, and there was no degeneration, necrosis or inflammatory cell infiltration; the glomerular and tubular structure was clear, and no cast or interstitial damage was observed; the ileum mucosa epithelium was complete, and the villi were arranged in order. No necrosis, fibrosis or normal structure destruction was observed at the tissue level, further confirming that Lactobacillus plantarum BQ-3 had no acute damage effect on the important organs of mice.

[0144] In summary, under the conditions of the present test, Lactobacillus plantarum BQ-3 has no acute toxicity by oral gavage in mice, and has good biological safety.

[0145] Example 2

[0146] 1. Effect of Lactobacillus plantarum BQ-3 on fermentation of lettuce tail

[0147] 1.1 Materials and methods

[0148] 1.1.1 Fermentation raw materials and strains

[0149] Fresh lettuce tail: The main part of the fresh lettuce tail is long and slender needle-shaped leaves, which are crisp and tender in texture, have a very high water content (such as 90-95%), and rot quickly. The tail needs to be processed in a timely manner.

[0150] Fresh lettuce tail was collected from a local vegetable planting base and cut into small pieces of 3-4 cm after removing the rotten tissue by screening; wheat bran was a commercially available food-grade raw material. The main nutritional components of the fermentation raw materials are shown in Table 6.

[0151] The test strain was Lactobacillus plantarum BQ-3, which was isolated and preserved in the laboratory. After activation, the bacterial liquid was inoculated in MRS liquid medium and incubated at 37°C for 12 h, and the bacterial liquid concentration was adjusted to 1×10 6 CFU / mL, which was used as a fermentation inoculum.

[0152] Table 6 Nutritional components of fermentation raw materials (dry matter basis%)

[0153]

[0154] 1.1.2 Test design

[0155] The lettuce tail and wheat bran were mixed at a mass ratio of 304:196 (total mass 500 g), so that the dry matter content of the material reached 350 g / kg fresh weight. The mixture was divided into two groups, one of which was sprayed with Lactobacillus plantarum BQ-3 bacterial liquid (final concentration of 1×10 6LP group; another group was sprayed with the same volume of sterile water, recorded as the blank control (CON) group. The two groups of materials were respectively packed into 20 cm x 30 cm breathable type of respiration bag, sealed by vacuum sealing machine, and placed at room temperature for fermentation. On the 0th, 7th, 15th, and 30th days of fermentation, 3 bags of samples were randomly selected from each group for sampling and index determination.

[0156] 1.1.3 Determination index and method

[0157] 1.1.3.1 Determination of general nutritional components

[0158] The collected fermentation samples were placed in a 65℃ air-drying oven for constant temperature drying for 48h, and the dry matter (DM) content was determined; the crude protein (CP) content was determined by Kjeldahl nitrogen determination method; the soluble protein (SP) content was determined by Coomassie brilliant blue G-250 colorimetric method; the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined by automatic fiber instrument; the soluble sugar (WSC) content was determined by anthrone-sulfuric acid colorimetric method.

[0159] 1.1.3.2 Determination of fermentation quality index

[0160] 10g of fermentation sample was taken, mixed with 90mL of sterile distilled water, sealed and placed in a 4℃ refrigerator for extraction for 24h, filtered with 4 layers of gauze, and then filtered with a 0.22μm filter membrane to determine the relevant indexes. The pH value of the filtrate was directly determined using a portable pH meter; the ammonia nitrogen (NH3-N) content was determined using phenol-sodium hypochlorite colorimetric method. The organic acid (lactic acid, acetic acid, propionic acid, butyric acid) content was determined by GC-MS instrument.

[0161] 1.1.4 Statistical analysis

[0162] The general linear model in SPSS 27.0 software was used for two-way ANOVA analysis to analyze the main effects of adding Lactobacillus plantarum BQ-3 (T) and time (D) and their interaction. Tukey method was used for multiple comparisons. When P<0.05, it was considered to have significant difference.

[0163] 1.2 Test results

[0164] 1.2.1 Effect of Lactobacillus plantarum BQ-3 on fermentation nutritional quality of oil lettuce tail

[0165] As shown in Table 7, only the T*D interaction of WSC content was significant (P<0.05). The DM content of the LP group was significantly higher than that of the CON group on the 15th and 30th day of fermentation (P<0.05), indicating that the addition of Lactobacillus plantarum can reduce the loss of dry matter during fermentation. The CP and SP contents of the two groups showed a trend of first increasing and then decreasing with the fermentation time, but there was no significant difference (P>0.05). The NDF content of the two groups gradually decreased with the extension of fermentation time. The ADF content of the LP group was significantly lower than that of the CON group at the beginning of fermentation (P<0.05), while the WSC content at each period was significantly higher than that of the CON group (P<0.05), with a retention rate of 41.02% higher than that of the CON group at 30d, indicating that the strain can slow down the excessive consumption of sugar.

[0166] Table 7 Effect of Lactobacillus plantarum BQ-3 on the fermentation nutritional quality of leaf mustard tail

[0167]

[0168] Note: The same row data is marked with different capital letters, indicating that there is a significant difference (P<0.05) between different treatments at the same fermentation time. The same column data is marked with different lowercase letters, indicating that there is a significant difference (P<0.05) between different fermentation times under the same treatment. T represents strain addition treatment; D represents fermentation time; T*D represents the interaction of treatment and fermentation time; SEM represents standard error. The following table is the same.

[0169] 1.2.2 Effect of Lactobacillus plantarum BQ-3 on the fermentation characteristics of leaf mustard tail

[0170] As shown in Table 8, only the T*D interaction of lactic acid content was not significant (P>0.05). With the progress of fermentation, the ammonia nitrogen and lactic acid contents of the two groups increased, while the contents of acetic acid, propionic acid and butyric acid decreased. The ammonia nitrogen content of the LP group was significantly lower than that of the CON group on the 7th, 15th and 30th day of fermentation (P<0.05), and was only 40.3% of that of the CON group at 30d. The pH of the LP group decreased to 3.71 on the 7th day of fermentation, which was significantly lower than that of the CON group (P<0.05). The LP group continuously accumulated lactic acid, reaching 75.06g / kg DM at 30d, which was 19.4% higher than that of the CON group (P<0.05); the contents of acetic acid, propionic acid and butyric acid were significantly lower than those of the CON group at 0-7d (P<0.05), and the butyric acid content was only 0.13g / kg at 30d, indicating that Lactobacillus plantarum can effectively inhibit the generation of spoilage organic acid butyric acid.

[0171] Table 8 Effect of Lactobacillus plantarum BQ-3 on the fermentation characteristics of leaf mustard tail

[0172]

[0173] 1.3 Summary

[0174] Lactobacillus plantarum BQ-3 can improve the nutrient retention of leaf mustard tail by reducing dry matter loss and retaining soluble sugars; at the same time, it can promote lactic acid production to rapidly reduce the pH value of the system, reduce the accumulation of ammonia nitrogen and spoilage organic acids (butyric acid), and significantly improve the fermentation quality.

[0175] 2. Rapid pH-lowering characteristics of Lactobacillus plantarum BQ-3 in high-moisture preserved vegetable tail

[0176] 2.1 Test method

[0177] Fresh preserved vegetable tail (moisture content 90%-95%) was selected, which was not easy to store. After the preserved vegetable tail was crushed, 100 g was accurately weighed and placed in a 250 mL triangular flask. The test was divided into 3 groups: the LPB-3 group was inoculated with Lactobacillus plantarum BQ-3 bacterial liquid (viable bacterial concentration 1×10 8 CFU / mL) at an inoculation amount of 1%, the standard strain group was inoculated with the same amount of Lactobacillus plantarum standard strain CGMCC1.577 bacterial liquid, and the blank control (CON) group was inoculated with the same amount of sterile water. After mixing well, the three groups were fermented at 25°C. The pH value of the system was measured at 0, 4, 8, 18 and 20 h, and each time point was repeated 3 times to evaluate the acid production efficiency and advantage of the strain in a high-moisture environment.

[0178] 2.2 Test results

[0179] As shown in Table 9, there was no significant difference in the initial pH of each group (P>0.05), and they were all in a neutral to acidic state. From 4 to 8 h of fermentation, the pH values of the LPB-3 group and the standard strain group were not significantly different (P>0.05), but were significantly lower than that of the CON group (P<0.05). From 8 to 20 h of fermentation, the pH-lowering rate of the LPB-3 group significantly accelerated. At 18 h, the pH value of the LPB-3 group rapidly decreased from 6.56 to 4.28, which was significantly lower than that of the standard strain group and the CON group (P<0.05); at 20 h, the pH value of the LPB-3 group continued to decrease to 4.13, which was significantly lower than that of the standard strain group by 12.68% and that of the CON group by 26.64% (P<0.05).

[0180] Table 9 Effect of Lactobacillus plantarum BQ-3 on the pH value of preserved vegetable tail fermentation (mean ± standard error)

[0181]

[0182] Note: The same column data marked with different lowercase letters indicates significant difference (P<0.05) between different treatments at the same time point.

[0183] 2.3 Summary

[0184] Lactobacillus plantarum BQ-3 could significantly reduce the pH of pickled mustard tail (pH 4.13 after 20 h fermentation). During the fermentation period (0-20 h), the strain could effectively overcome the potential restriction of high-moisture substrate on microbial metabolism, and rapidly reduce the pH of the system by efficiently starting and accelerating the acid production process, which was suitable for efficient fermentation of pickled mustard tail.

[0185] 3. Optimization of fermentation process of pickled mustard tail based on Lactobacillus plantarum BQ-3

[0186] 3.1 Materials and methods

[0187] 3.1.1 Test materials

[0188] Pickled mustard tail (moisture content 90%-95%, not easy to store) was collected from the pickled mustard processing base in Fuling, Chongqing, and was fresh and mold-free. It was mechanically pulverized to a particle size of 2-3 mm for use. Wheat bran was food-grade, and brown sugar was commercially available first-grade product (sucrose content ≥ 99%).

[0189] Test strains: Lactobacillus plantarum BQ-3 (GDMCC No. 65791, isolated and preserved by the laboratory, viable cell count 1×10 9 CFU / mL), Bacillus subtilis, and Saccharomyces cerevisiae, which were activated and prepared into bacterial suspensions (concentration 1×10 8 CFU / mL).

[0190] 3.1.2 Test design

[0191] L25(56) orthogonal test design was adopted. Lactobacillus plantarum BQ-3 addition amount (A, volume ratio), Bacillus subtilis addition amount (B, volume ratio), Saccharomyces cerevisiae addition amount (C, volume ratio), ratio of pickled mustard tail to wheat bran (mass ratio, D), brown sugar addition amount (E, mass ratio), and fermentation time (F) were used as observation factors. Each factor had 5 levels (Table 10). The pH, lactic acid content, short-chain fatty acid content, and ammonia nitrogen in the fermented feed were detected. The membership values of each group were obtained by using membership function analysis for range and variance analysis of the orthogonal test, and the optimal fermentation process was screened.

[0192] Table 10 Factors and levels of orthogonal experiment

[0193]

[0194] 3.1.3 Fermentation and determination methods

[0195] The tail of preserved szechuan pickled mustard and wheat bran were weighed according to orthogonal design, and the corresponding amount of brown sugar and bacterial liquid (total addition amount was calculated based on the fresh weight of the material) was added. After mixing thoroughly, they were put into sterile and breathable fermentation bags (20 cm x 30 cm) and vacuum packaged for constant temperature fermentation at 25 DEG C. The total material amount of each group was 500 g, and each group was repeated 3 times. The indicators were measured by sampling at the preset time.

[0196] The determination methods of pH value, lactic acid content, short-chain fatty acid content and ammonia nitrogen were the same as those in the above examples.

[0197] The fermentation quality of fermented feed was comprehensively evaluated by using membership function analysis. An index (lactic acid content) positively correlated with the fermentation quality of feed was designed, and its membership value was calculated by using formula (1). An index (ammonia nitrogen content, pH, and acetic acid content) negatively correlated with the fermentation quality of feed was designed, and its membership value was calculated by using formula (2). Finally, the average value of the membership values of each fermentation index was calculated as the membership value of the group.

[0198] Fij+= (Xij-Xmin) / (Xmax-Xmin); (1)

[0199] Fij-=1-(Xij-Xmin) / (Xmax-Xmin). (2)

[0200] In the formula, Fij is the membership value of the jth index of the ith treatment; Xij is the measured value of the jth index of the ith treatment; Xmax is the maximum measured value of the jth single index in each test treatment; and Xmin is the minimum measured value of the jth single index in each test treatment.

[0201] 3.1.4 Data processing

[0202] Range analysis and variance analysis were performed by using SPSS 27.0 software to determine the optimal process for the index. In the range analysis, the order of factors was sorted according to the range R (the larger the R, the more significant the influence).

[0203] 3.2 Test results

[0204] 3.2.1 Orthogonal test results and range analysis

[0205] The orthogonal test results and comprehensive scores are shown in Table 11. By comparing the range sizes in Table 11, the size order of the factors affecting the membership value (Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, wheat bran ratio, brown sugar and time are represented by A, B, C, D, E and F respectively) is D>A>E>C>B>F, and the optimal combination is A5B4C5D2E2F5.

[0206] Table 11 Orthogonal range analysis of different factors for fermented preserved szechuan pickled mustard tail

[0207]

[0208] 3.2.2 Analysis of variance

[0209] As shown in Table 12, the correction model of membership value has a significant influence on the test results, and the orthogonal test results have credibility. A, B, C, D, E, F all significantly affect the fermentation quality of preserved vegetable tail. According to the F value, the size of the factors affecting membership value is A>D>E>C>B>F.

[0210] Table 12 Orthogonal analysis of variance of different factors for fermenting preserved vegetable tail

[0211]

[0212] 3.3 Summary

[0213] The optimal fermentation process of preserved vegetable tail is: 2% of Lactobacillus plantarum BQ-3, 1.5% of Bacillus subtilis, 2% of Saccharomyces cerevisiae, 0.5% of brown sugar, and the rest is a mixture of 7:3 of preserved vegetable tail. The fermentation time is 28d. The fermentation under this process can provide technical support for the efficient resource utilization of preserved vegetable tail. Lactobacillus plantarum BQ-3 as the core functional strain, through the rapid acid production and metabolic synergy, significantly improves the fermentation efficiency and product quality of preserved vegetable tail, and provides technical support for its resource utilization.

[0214] Example 3 Effect of Lactobacillus plantarum BQ-3 fermented tail meal on the growth performance of Fuling black pigs

[0215] 1. Test method

[0216] 96 Fuling black pigs with similar body weight and good body condition (initial body weight 48.34±0.38 kg) were selected and randomly divided into 2 groups (control group and test group), 4 replicates in each group, 12 pigs in each replicate, and the stocking density was 0.5 pigs / m 2 , the temperature range was 19-24℃, and the humidity range was 60%-75%.

[0217] Control group: fed with basic diet (refer to NY / T65-2004 meat and fat type pig nutrition requirement preparation);

[0218] Test group: added with 32% of the fermented feed prepared by the optimal process of Example 2 (not dried feed, calculated as 18% of the dried basic feed), and mixed with 68% of the basic diet for feeding. Before feeding every day, the fermented tail meal and the basic diet (equivalent to corn and soybean meal) were mixed, and then water was added to mix uniformly at a ratio of 1:5 for liquid feeding. The test pigs were allowed to freely eat, and the daily feed intake was recorded. The test lasted for 65 days, and the pigs were weighed on an empty stomach at the beginning and end of the test to calculate the growth performance index.

[0219] 2. Test results

[0220] As shown in Table 13, after 65 days of feeding, the final body weight and average daily gain of the test group of Fuling black pigs were significantly higher than those of the control group, increasing by 4.35% and 8.18% respectively (P<0.05); the feed conversion ratio of the test group was significantly lower than that of the control group by 10.11% (P<0.05); and the average daily feed intake of the two groups was not significantly different (P>0.05).

[0221] Table 13 Effect of Lactobacillus plantarum BQ-3 fermented silage of over-wintered Chinese cabbage on growth performance of Fuling black pigs (mean ± standard error)

[0222]

[0223] Note: The same column data marked with different lowercase letters indicates significant difference between groups (P<0.05).

[0224] 3. Summary

[0225] The addition of Lactobacillus plantarum BQ-3 fermented silage of over-wintered Chinese cabbage in the daily diet of Fuling black pigs can significantly increase the final body weight and average daily gain, and reduce the feed conversion ratio. This shows that the fermented silage of over-wintered Chinese cabbage can be effectively used for Fuling black pig breeding, improving growth performance, and reflecting the practical value of Lactobacillus plantarum BQ-3 in the application of silage of over-wintered Chinese cabbage.

[0226] Example 4

[0227] In December 2024, Lactobacillus plantarum BQ-3 was used to ferment preserved vegetable silage (using the optimal process of Example 2), which was stored in a sealed fermentation barrel at room temperature and away from light. In May-July 2025, the fermented preserved vegetable silage was transported to the pig farm, and was mixed with 68% basic daily diet to feed the pigs, with one preparation of 3 days of feed (the same as the preparation method of Example 3 test group). After 3 days of preparation, the prepared feed was detected, as shown in Table 14, the pH value of the feed was basically the same as the initial value, the number of microorganisms did not exceed the standard, and the feed was not spoiled.

[0228] The control group and the test group were set up in the same way as in Example 3.

[0229] Table 14 Effect of Lactobacillus plantarum BQ-3 fermented preserved vegetable silage on shelf life

[0230]

[0231] The above-mentioned are only embodiments of the present application, and the common knowledge of specific structures and characteristics in the scheme is not described in detail. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent.

Claims

1. A Lactobacillus plantarum, whose Latin name is Lactiplantibacillus plantarum , named BQ-3, preserved in Guangdong Microbial Culture Collection Center on January 15, 2025, with a preservation registration number of GDMCC No. 65791.

2. A microbial starter characterized in that the active ingredient comprises the Lactiplantibacillus plantarum of claim 1.

3. Use of the Lactiplantibacillus plantarum or microbial starter of claim 1 or 2 in the fermentation of pig manure.

4. Use of Lactobacillus plantarum or a microbial starter according to claim 3 for the fermentation of pomace, characterized in that: The pig manure treated in the fermentation of pig manure contains 90-95% of water by mass percentage.

5. Use of Lactobacillus plantarum or a microbial starter according to claim 3 or 4 for the fermentation of pomace, characterized in that: The fermentation of pig manure comprises the following steps, R1. Mixing pig manure with wheat bran as raw materials; R2. Preparing fermented pig manure by sealing fermentation after spraying the bacterial solution of the Lactiplantibacillus plantarum on the raw materials prepared in R1. The viable cell number of the Lactobacillus plantarum in the raw material is ≥1.0×10 6 CFU / g, based on the wet weight of the raw material.

6. Use of Lactobacillus plantarum or a microbial starter according to claim 3 or 4 for the fermentation of pomace, characterized in that: The fermentation of pig manure comprises the following steps, S1. After the Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae are activated, prepare a bacterial solution with a concentration of ≥1×10 8 CFU / mL. S2. Add the bacterial solution to the fermentation tank, and then add the raw materials to the fermentation tank to start fermentation. S2. Preparing fermented pig manure by sealing fermentation after mixing pig manure with wheat bran as raw materials, adding brown sugar and the bacterial solution prepared in S1.

7. Use of the fermented pig manure prepared by the Lactiplantibacillus plantarum or microbial starter of any one of claims 3-6 in the fermentation of pig manure in fattening feed.

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