Lactobacillus plantarum capable of efficiently producing acid and application of lactobacillus plantarum in corn silage

Abstract

The invention discloses lactobacillus plantarum capable of efficiently producing acid and application of the lactobacillus plantarum in corn silage, and relates to the technical field of microorganisms. The invention provides lactobacillus plantarum Mei1101.3, the preservation number of the lactobacillus plantarum Mei1101.3 is CGMCC No.30332, the lactobacillus plantarum Mei1101.3 has the characteristics of high acid production rate, low pH value and excellent growth performance, the lactobacillus plantarum can be used as a special strain for corn silage and is added into corn silage, the pH value of the corn silage is reduced to 3.02 30 days after silage, and the lactobacillus plantarum Mei1101.3 can be used as a special strain for corn silage. The sensory quality is close to the initial state of the raw materials, the energy consumption in the ensiling process is only 374.34 kcal. Kg <-1 >, the quantity of saccharomycetes is reduced, no mould or escherichia coli is detected, and the health and safety meet the national standard.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of plant lactobacillus that produces acid with high efficiency and application thereof in corn silage. Background Art

[0002] Silage refers to a method of preserving feed under sealed conditions, which can keep the feed's nutritional content and other characteristics relatively unchanged for a long time. During the silage process, lactic acid bacteria attached to the surface of the raw materials can produce a large amount of organic acids through a specific metabolic process under anaerobic conditions. Lactic acid is the main product. As lactic acid accumulates, the pH value of the silage will drop rapidly. Usually, when the pH value reaches the range of 3.5-4.2, it can effectively inhibit the growth of most harmful microorganisms. This is because the acidic environment of lactic acid is not conducive to the survival of other microorganisms. They cannot reproduce normally in such an environment, thereby achieving the purpose of extending the shelf life of the feed.

[0003] The key to influencing silage quality lies in the anaerobic fermentation process of lactic acid bacteria. Since lactic acid bacteria can cause the pH of silage raw materials to decrease and be preserved for a long time, the growth conditions and growth characteristics of lactic acid bacteria are considered to be important factors affecting the fermentation process and silage quality of silage, as well as prerequisites for screening lactic acid bacteria suitable for silage. Lactic acid bacteria not only play a key role in the storage of silage, but are also widely used in the food industry and agriculture because of their potential benefits to human and animal health. During the natural growth of plants, the number of lactic acid bacteria attached to their surface is low (<5 lgCFU / g), even below the detection level (<2 lgCFU / g). Lactic acid bacteria play a key role in the quality of silage. When the lactic acid bacteria content exceeds 10 5 CFU / g, it can significantly promote the fermentation process of feed, thereby effectively improving the quality and preservation effect of feed; however, when the number of lactic acid bacteria is less than 10 4 CFU / g may lead to a decrease in dry matter content and an increase in ammonia nitrogen content. Therefore, adding high-quality lactic acid bacteria to make up for the insufficient number of lactic acid bacteria in the raw materials and thus increase the number of silage microorganisms is an effective way to improve the fermentation process of silage.

[0004] During the ensilage process, lactic acid bacteria can fully metabolize and utilize the nutrients they need, accelerating the fermentation stage. This rapidly reduces the pH of the silage, reduces the growth and reproduction of harmful bacteria, reduces the natural loss of nutrients, and improves the quality of the silage. However, the type of raw material, the type of lactic acid bacteria, and the amount added all affect the role of lactic acid bacteria in the ensilage process. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art by providing a strain of Lactobacillus plantarum that efficiently produces acid and its use in corn silage. To further enhance the availability of highly acid-producing lactic acid bacteria, the present inventors conducted extensive screening and obtained a strain of Lactobacillus plantarum for corn silage that efficiently produces acid and consumes low energy for silage. This results in the technical solution of the present invention, achieving the objectives of this solution.

[0006] The technical solutions of the present invention are as follows: The first aspect of the present invention provides a strain of Lactobacillus plantarum that produces acid efficiently. Lactiplantibacillus plantarum ) Mei1101.3, the depository is the General Microbiology Center of China Culture Collection Administration, the deposit number is CGMCC No. 30332, the deposit date is April 15, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Optionally, the Lactobacillus plantarum is isolated from kimchi.

[0008] Optionally, the Lactobacillus plantarum has at least one of the following properties: (1) It is acid-resistant and survives well at pH 3.0; (2) It is resistant to high temperatures and can survive well in an environment of 50°C.

[0009] The second aspect of the present invention provides a bacterial agent containing the aforementioned Lactobacillus plantarum.

[0010] Optionally, the bacterial agent is in the form of powder, pills or liquid preparation.

[0011] The third aspect of the present invention provides a method for preparing the bacterial agent, comprising the following steps: Under sterile conditions, the Lactobacillus plantarum is inoculated into an MRS liquid culture medium and cultured to obtain a fermentation liquid; The fermentation liquid is mixed evenly with physiological saline to prepare a bacterial agent.

[0012] Optionally, the culture conditions include: culturing at 37° C. and 160 rpm for 24 h to obtain a fermentation broth.

[0013] The fourth aspect of the present invention provides the use of the Lactobacillus plantarum or the bacterial agent in the preparation of corn silage.

[0014] Optionally, the specific method of using the plant lactobacillus or the bacterial agent lactobacillus in preparing corn silage comprises the following steps: The plant lactobacillus or the bacterial agent is added into corn kernels, and the kernels are ensiled for 30 days with the exclusion of air to prepare corn silage.

[0015] A fifth aspect of the present invention provides corn silage, which is prepared by adding the plant lactobacillus or the bacterial agent to corn kernels and silaging them for 30 days after removing air.

[0016] The strain of the present invention is Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Mei1101.3, deposited with CGMCC No. 30332, has the characteristics of rapid acid production, low pH, and excellent growth performance. The present invention's Lactobacillus plantarum can be used as a strain specifically for corn silage and added to corn silage. The pH of corn silage grown with the Lactobacillus plantarum Mei1101.3 strain of the present invention dropped to 3.02 after 30 days of ensiling, and the sensory quality was close to that of the original raw material. The energy consumption during the ensiling process was only 374.34 kcal·kg -1 The yeast count was reduced, and no mold or E. coli was detected, and the sanitation and safety met the national standards. Therefore, the plant lactobacillus of the present invention can well retain the nutritional components of corn silage, reduce energy consumption during the ensiling process, and inhibit the growth of harmful microorganisms, and has good application value.

[0017] The present invention has at least one of the following beneficial effects: 1. The present invention provides plant lactobacillus ( Lactiplantibacillus plantarum Mei1101.3 not only has a rapid acid production rate, but also exhibits excellent growth performance and is resistant to acid and temperature. Specifically, the Lactobacillus plantarum Mei1101.3 strain of the present invention drops to a pH of 3.44 after 24 hours of culture in MRS medium, enters the logarithmic phase around 3 hours, and enters the stable phase around 22 hours, capable of tolerating a pH of 3.0 and a temperature of 50°C.

[0018] 2. The plant lactobacillus provided by the present invention can be added to corn silage to effectively retain the nutritional components of corn, reduce energy consumption during the ensiling process, improve the fermentation quality of corn silage, and ensure its quality and safety. Specifically, the crude ash and crude fiber content are lower than those of untreated corn silage, while the crude protein and crude fat content are higher than those of untreated corn silage. Energy consumption is low, the pH drops to 3.55, the fermentation quality is excellent, and harmful microorganisms such as Escherichia coli and mold are absent. The product meets national health indicators and is a corn silage of excellent overall quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 3 is the acid production curve of Lactobacillus plantarum Mei1101.3 in the embodiment of the present invention.

[0021] Figure 2 3 is the growth curve of Lactobacillus plantarum Mei1101.3 in the embodiment of the present invention.

[0022] Figure 3 This is the acid resistance test of Lactobacillus plantarum Mei1101.3 in the embodiment of the present invention; Figure 4 This is the temperature resistance test of Lactobacillus plantarum Mei1101.3 in the embodiment of the present invention; Figure 5 This is a physical picture of the corn silage prepared in the embodiment of the present invention. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The MRS liquid culture medium used in the present invention (Guangdong Zhongshan Baiwei Microbiology Technology Co., Ltd.) is composed of the following components: 10.0 g / L peptone, 5.0 g / L beef extract powder, 4.0 g / L yeast extract powder, 20.0 g / L glucose, 1.0 g / L Tween-80, 2.0 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 2.0 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and a pH of 6.2 ± 0.2. The preparation method is as follows: Weigh 54 g of the culture medium sample, add 1 L of distilled or deionized water, stir, heat, and boil until completely dissolved. Autoclave at 121°C for 15 minutes before use.

[0025] The MRS agar medium (Guangdong Zhongshan Baiwei Microbiology Technology Co., Ltd.) in the present invention is composed of the following components: 10.0 g / L peptone, 5.0 g / L beef extract powder, 4.0 g / L yeast extract powder, 20.0 g / L glucose, 1.0 g / L Tween-80, 2.0 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium acetate, 2.0 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 15.0 g / L agar, and a pH of 6.2±0.2. The preparation method is as follows: Weigh 64.3 g of the medium sample, add 1 L of distilled or deionized water, stir, heat, and boil until completely dissolved, and sterilize by autoclaving at 121°C for 15 minutes before use.

[0026] If specific experimental steps or conditions are not specified in the examples, the experiments were performed according to the conventional experimental procedures or conditions described in the literature in the field. All raw materials and instruments used are commercially available, including but not limited to those used in the examples of this application.

[0027] Example 1 Isolation and identification of Lactobacillus plantarum Mei1101.3 1. Strain Isolation Lactobacillus plantarum Mei1101.3 was screened from kimchi. The method for screening this strain is as follows: weigh 20 g of kimchi and add it to a conical flask containing 180 mL of sterile saline. Shake thoroughly. Under a sterile environment, use a 1 mL pipette to accurately measure 1 mL of kimchi fermentation liquid and add it to test tube No. 1 containing 9 mL of sterile water. Shake well to obtain 10 -1 Dilution; According to this method, after continuous dilution, 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Serial gradient dilution of the bacterial solution. Prepare MRS solid culture medium and sterilize it. After sterilization, pour the plates under a sterile environment and wait for the plates to cool and solidify. Pipette 1 mL of the dilution solution of different dilutions, spread it evenly on the plate, and culture it in an anaerobic incubator for 48 hours. After the colonies grow, observe the morphology and size of the colonies, select small colonies that are milky white and needle-shaped, and use an inoculation loop to pick up suspected colonies and streak them on the plate culture medium. Invert the plate, place it in an anaerobic incubator, and culture it for 48 hours. A total of 11 strains of bacteria were isolated and named Mei1101.1-Mei1101.11 respectively. The purified single colonies were stored on slant surfaces at 4°C and frozen in glycerol tubes at -80°C for later use.

[0028] 2. Strain Screening Pipette 200 μL of the preserved strain from the glycerol storage tube into 50 mL of MRS liquid medium and incubate at 37°C and 160 rpm until the culture solution becomes turbid. Activate twice. Inoculate the activated strain into MRS liquid medium and incubate at 37°C and 160 rpm for 24 hours. Samples are taken at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours, and the pH of the fermentation broth is measured. Acid production varies among strains; see Table 1 below for details. Strain pH value time / h 0 2 4 6 8 10 12 14 16 18 20 22 24 Mei1101.1 5.45 5.2 4.61 4.13 3.89 3.76 3.7 3.61 3.61 3.6 3.53 3.58 3.41 Mei1101.2 5.47 5.34 4.49 4.26 4.04 3.9 3.8 3.73 3.64 3.63 3.57 3.59 3.52 Mei1101.3 5.44 5.16 4.62 4.18 3.93 3.75 3.71 3.61 3.55 3.53 3.5 3.49 3.44 Mei1101.4 5.44 5.23 4.66 4.15 3.87 3.77 3.72 3.66 3.6 3.57 3.5 3.53 3.46 Mei1101.5 5.62 5.5 5.2 4.65 4.08 3.87 3.76 3.65 3.59 3.56 3.54 3.5 3.49 Mei1101.6 5.64 5.58 5.43 4.95 4.28 3.99 3.88 3.73 3.73 3.64 3.63 3.61 3.6 Mei1101.7 5.63 5.49 5 4.41 4.03 3.86 3.82 3.71 3.71 3.64 3.61 3.61 3.59 Mei1101.8 5.69 5.5 5.08 4.51 4.03 3.83 3.79 3.73 3.63 3.6 3.58 3.56 3.56 Mei1101.9 5.74 5.7 5.66 5.62 5.56 5.27 5.3 5.03 4.34 4.16 4.08 3.98 3.91 Mei1101.10 5.72 5.69 5.65 5.63 5.61 5.5 5.57 5.45 5.33 5.22 4.88 4.74 4.36 Mei1101.11 5.65 5.55 5.3 4.72 4.16 3.93 3.78 3.67 3.61 3.57 3.57 3.52 3.54 Among them, the plant lactobacillus Mei1101.3 with strong acid production ability was screened out, and its acid production curve is shown in FIG. Figure 1 The pH value of the fermentation liquid of the Lactobacillus plantarum Mei1101.3 of the present invention drops sharply after the initial cultivation and reaches a stable state after about 16 hours. The pH value of the fermentation liquid is finally stabilized at about 3.44.

[0029] 3. Strain Identification The strain Mei1101.3 was extracted using a kit and amplified by PCR. The DNA was then sent to Shanghai Paisonno Biotechnology Co., Ltd. for 16S rDNA gene sequencing. The sequencing results were compared with the homologous sequences in NCBI using the BLAST tool to clarify the species relationship of the strain. The strain was confirmed to be Lactobacillus plantarum ( Lactiplantibacillus plantarum Therefore, the plant lactobacillus ( Lactiplantibacillus plantarum ) Mei1101.03 bacteria were deposited in the China General Microbiology Center (CGMCC) under the China Culture Collection Administration, with the deposit number CGMCC No. 30332 and the deposit date April 15, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0030] The 16S rDNA sequence of this strain is shown in SEQ ID NO.1:

[0031] Example 2 Study on the biological characteristics of Lactobacillus plantarum Mei1101.3 1. Determination of the Growth Curve of Lactobacillus plantarum Mei1101.3 200 μL of the preserved bacteria were drawn from the glycerol storage tube and placed in 50 mL of MRS liquid medium. The culture was incubated at 37°C and 160 rpm until the culture solution became turbid. The activated Lactobacillus plantarum Mei1101.3 was inoculated into MRS liquid medium and incubated at 37°C and 160 rpm for 48 h. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 38, 40, 42, 44, 46, and 48 h, and the OD value of the fermentation liquid was measured. 600 Value. See the result Figure 2 The Lactobacillus plantarum Mei1101.3 of the present invention enters the logarithmic phase at around 3 hours and enters the stable phase at around 18 hours.

[0032] 2. Determination of acid resistance of Lactobacillus plantarum Mei1101.3 After activation twice, Lactobacillus plantarum Mei1101.3 was inoculated into MRS liquid medium at pH 3.0 at a concentration of 10% and cultured at 37°C for 3 h. The number of viable bacteria at 0 h in culture medium at pH 3.0 was used as the control group. Figure 3 The survival rate was calculated according to the following formula: survival rate = number of viable cells after acid treatment / original number of viable cells. The calculated survival rate was 104.26%. The plantarum Lactobacillus Mei1101.3 of the present invention can still grow in an environment of pH 3.0, indicating that it has a certain acid resistance.

[0033] 3. Determination of the heat resistance of Lactobacillus plantarum Mei1101.3 After activation twice, Lactobacillus plantarum Mei1101.3 was inoculated into MRS liquid culture medium at a 10% inoculum volume. The culture was then placed in a constant temperature water bath at 37°C, 50°C, 60°C, and 70°C for 20 minutes, and then placed in a shaking incubator at 37°C for 12 hours. The number of viable bacteria cultured at 37°C was used as the control group. Figure 4 The survival rate was calculated according to the following formula: survival rate = number of viable cells after heat treatment / original number of viable cells. The calculated survival rate was 100.57% under 50°C environmental conditions. The Lactobacillus plantarum Mei1101.3 of the present invention still had a certain level of activity in a 50°C environment, indicating that it had a certain tolerance to a 50°C environment.

[0034] Example 3 Preparation of Lactobacillus plantarum Mei1101.3 starter culture This embodiment provides a method for preparing a starter culture using Lactobacillus plantarum Mei1101.3, comprising the following steps: Under sterile conditions, Lactobacillus plantarum Mei1101.3 was inoculated into MRS liquid medium (commercially available) and cultured at 37°C and 160 rpm for 24 h. The fermentation broth was mixed evenly with physiological saline to prepare a bacterial solution with a concentration of 10 8 CFU / mL fermentation broth, ready to use.

[0035] Example 4 Application of Lactobacillus plantarum Mei1101.3 in the preparation of corn silage 1. Preparation of Corn Silage with Lactobacillus plantarum Mei1101.3 Weigh 200 g of fresh corn kernels, add 1 mL of liquid starter (Lactobacillus plantarum Mei1101.3 starter in Example 3), mix thoroughly, place in a breathing bag (size: 20 × 24 cm), seal and remove air, and silage at room temperature for 30 days.

[0036] 2. Treatment of corn silage samples with Lactobacillus plantarum Mei1101.3 Take 10 g of fresh samples after ensiling for 30 days and put them into a 250 mL stoppered Erlenmeyer flask. Add 90 mL of ultrapure water and extract at room temperature for 30 min. Centrifuge at 4800 r / min for 15 min, retain the supernatant, and store it in a refrigerator at 4°C. The extract is used for the determination of pH, organic acid, and microbial count; the remaining sample is dried in an oven at 105°C, and the dried sample is crushed for the determination of crude ash, crude fat, crude protein, crude fiber, etc.

[0037] 3. Effects of Lactobacillus plantarum Mei1101.3 on the sensory quality of corn silage The evaluation is mainly based on the color and smell of the silage samples. Figure 5 Its smell has no butyric smell, but has aromatic fruity or obvious bread aroma; its color is similar to the raw material, bright yellow, indicating excellent sensory quality.

[0038] 4. Effects of Lactobacillus plantarum Mei1101.3 on the nutritional quality of corn silage (1) Determination of moisture Weigh about 5 g of sample into a drying dish, place it in a 105°C oven, and dry it to constant weight. Take out the drying dish and place it in a desiccator to cool to room temperature, and then weigh it.

[0039] (2) Determination of crude ash Weigh about 5 g of sample into a crucible, place it on an electric furnace and carefully heat it until carbonized, then transfer it to a muffle furnace and calcine it at 550°C for 6 h. Take it out and place it in a desiccator, cool it to room temperature and weigh it quickly.

[0040] (3) Determination of crude protein Determination was performed using a fully automated Kjeldahl nitrogen analyzer. Approximately 0.5 g of sample was weighed, wrapped in weighing paper, and carefully placed in a digestion tube. An appropriate amount of Kjeldahl nitrogen determination tablets and 10 mL of concentrated sulfuric acid were added. Digestion was performed using a graphite digester. After the digested sample cooled to room temperature, its crude protein content was determined using a fully automated Kjeldahl nitrogen analyzer.

[0041] (4) Determination of crude fat The fat content is determined using a fat analyzer. Weigh approximately 2 g of sample, wrap it tightly in filter paper, and place it in the sample holder. Attach the holder to the instrument using a magnet. After drying to a constant weight, add 50 mL of petroleum ether to the extraction cup. Place the extraction cup on a hot plate, ensuring the filter paper tube is completely immersed in the solvent. Begin the measurement using the instrument according to the instructions.

[0042] (5) Determination of crude fiber Weigh approximately 1 g of sample into an Erlenmeyer flask and digest with acid. Add approximately 150 mL of sulfuric acid solution, bring to a boil as quickly as possible, and maintain boiling for approximately 30 minutes. During boiling, use a condenser reflux device to maintain a constant volume. If foaming occurs during boiling, add a few drops of antifoaming agent. First, perform the first filtration. Pre-pour a layer of diatomaceous earth into the filter crucible. When digestion is complete, slowly pour the liquid into the crucible and filter using a vacuum pump. Wash the residue with approximately 10 mL of hot water. Repeat this washing five times. Stop vacuuming and add an appropriate amount of acetone to just cover the residue. Let it sit for approximately 5 minutes before vacuuming. Degrease with 30 mL of petroleum ether. Repeat this degreasing process three times. Transfer the residue from the first filtration to the same Erlenmeyer flask used for acid digestion. Add approximately 150 mL of potassium hydroxide solution, bring to a boil as quickly as possible, and maintain boiling for approximately 30 minutes. During boiling, use a condenser reflux device to maintain a constant volume. If foaming occurs during boiling, add a few drops of antifoaming agent. Begin the second filtration, following the same procedures as for the first. Finally, place the filter crucible in an ashing dish. Dry the ashing dish and its contents in a 105°C oven, cool in a desiccator, and weigh. Place the filter crucible and ashing dish in a muffle furnace and ash at 500°C for 6 hours. Remove and cool in a desiccator, then weigh. Perform a blank measurement as for the sample, but without adding sample.

[0043] (6) Determination of soluble carbohydrates Place 1 mL of each glucose standard solution in a 10 mL centrifuge tube, add 5 mL of the anthrone solution, and carefully and rapidly shake the tube to mix thoroughly. Place in a boiling water bath for 10 minutes, then remove and cool. Measure the absorbance at 620 nm and plot a standard curve with glucose content as the abscissa and absorbance as the ordinate. Place 1 mL of the sample extract in a 10 mL centrifuge tube and repeat the same procedure as for the standard solution three times. Calculate the soluble carbohydrate content in the sample based on the glucose standard curve.

[0044] (7) Determination of total energy Calculation method of total energy: Organic matter (%) = dry matter (%) - crude ash (%); Nitrogen-free extract (%) = organic matter (%) - crude protein (%) - crude fiber (%) - crude fat (%); Total energy (kcal / kg) = 5.6 × crude protein (g / kg) + 4.2 × crude fiber (g / kg) + 9.4 × crude fat (g / kg) + 4.2 × nitrogen-free extract (g / kg).

[0045] (8) Results The results of nutritional quality indicators are shown in Table 2. After 30 days of silage, the crude fat and crude protein contents of corn silage incubated with Lactobacillus plantarum Mei1101.3 increased, accounting for 2.66% and 4.20% of the fresh sample, respectively. The crude fiber content decreased, accounting for 0.88% of the fresh sample. The total energy content decreased, with the energy consumption being only 59.26 kcal·kg -1 Crude fiber, crude fat, and crude protein are the most basic indicators for evaluating feed quality. Low fiber can improve feed palatability, ease digestion, and improve feed quality. Only by providing livestock and poultry with sufficient crude fat, crude protein, and energy can the basic nutritional needs of livestock and poultry be met. Adding Lactobacillus plantarum will consume energy in the feed. The crude fat content, crude protein content, and crude fiber degradation capacity of the natural corn silage fermentation used as the control group were lower than those of the corn silage of the present invention, and its energy consumption was close to that of the corn silage of the present invention.

[0046] Table 2 Nutritional quality indicators of corn silage

[0047] Table 3 Fermentation quality indicators of corn silage 5. Effect of Lactobacillus plantarum Mei1101.3 on the fermentation quality of corn silage (1) Determination of pH value Take the sample extract and immediately measure its pH value using a pH meter.

[0048] (2) Determination of ammonia nitrogen The phenol-sodium hypochlorite method was used for determination. For the blank group, 50 μL of standard ammonium solutions of varying concentrations were placed in a centrifuge tube. For the blank group, 50 μL of distilled water was added. 2.5 mL of phenol reagent was added to each centrifuge tube and shaken thoroughly. Then, 2 mL of sodium hypochlorite reagent was added and mixed thoroughly. The mixture was heated in a 95°C water bath for 5 minutes to develop a color reaction. After cooling, the absorbance was measured at 630 nm. A standard curve was plotted with the standard ammonium content as the abscissa and the absorbance as the ordinate. For the sample group, 50 μL of the extract was placed in a test tube and the same procedure as for the standard solution was repeated three times. The ammoniacal nitrogen content in the samples was calculated based on the standard curve.

[0049] (3) Determination of organic acids The sample extract was filtered through an organic filter (0.22 μm) and the contents of lactic acid, acetic acid, propionic acid, and butyric acid were determined using high-performance liquid chromatography (HPLC). The determination conditions were: an HPX-87 column, 45°C column temperature, 210 nm wavelength, a flow rate of 0.5 mL / min, and a 5.52 mmol / L H₂SO₄ solution as the mobile phase.

[0050] (4) Results Table 3 shows the fermentation quality indicators. After 30 days of corn silage incubation with Lactobacillus plantarum Mei1101.3, the pH dropped to 3.02, the ammoniacal nitrogen / total nitrogen ratio decreased to 2.37, the lactic acid content increased to 1.10%, the acetic acid content decreased to 0.12%, and the lactic acid / acetic acid ratio increased to 9.38. The pH decreases during silage. When the pH drops to a certain level, microbial growth is inhibited, resulting in long-term nutrient preservation. The ammoniacal nitrogen / total nitrogen ratio reflects the degree of crude protein degradation in the silage. Furthermore, because ammoniacal nitrogen has a foul odor, a high ammoniacal nitrogen content can affect animal feed intake. A higher ammoniacal nitrogen / total nitrogen ratio indicates a lower nutritional value, significantly impacting fermentation quality and, consequently, lower silage quality. As the primary organic acid in silage, a higher lactic acid content indicates a more vigorous fermentation process and a stronger inhibitory effect on harmful microorganisms. High-quality silage contains more lactic acid and less acetic acid. Compared with the natural corn silage as the control group, the pH value, ammonia nitrogen / total nitrogen and acetic acid content of the corn silage of the present invention are lower than those of the control group, and the lactic acid content and lactic acid / acetic acid ratio are higher than those of the control group, indicating that the fermentation quality of the corn silage of the present invention is better.

[0051] Table 3 Fermentation quality indicators of corn silage

[0052] 6. Effect of Lactobacillus plantarum Mei1101.3 on the microbial population in corn silage (1) Determination of the number of microorganisms in corn silage with Lactobacillus plantarum Mei1101.3 The sample extract was diluted gradiently, and appropriate dilution concentrations were spread on potato dextrose agar plates, crystal violet neutral bile salt agar plates, MRS plates, potato dextrose agar plates, and nutrient agar plates for the count of mold, Escherichia coli, lactic acid bacteria, yeast, and aerobic bacteria, respectively. The plates were inverted in a constant temperature incubator and incubated for 24 h on crystal violet neutral bile salt agar plates, 72 h on MRS plates, and 96 h on potato dextrose agar plates and nutrient agar plates. The plates were observed and counted, and the number of colonies on each dilution was the average of the two plates.

[0053] (2) Results The results of the microbial counts of corn silage with Lactobacillus plantarum Mei1101.3 are shown in Table 4. After 30 days of corn silage with Lactobacillus plantarum Mei1101.3, the number of lactic acid bacteria increased to 7.71 lgCFU·g -1 , mold and E. coli were not detected. The national standard GB 13078-2017 "Feed Hygiene Standard" stipulates that the total number of molds in feed should be less than 10 4 CFU / g, the number of E. coli should be less than 10 3 The lactic acid bacteria in the corn silage of the present invention were higher than those in the control group, and no mold and E. coli were detected, which met the national standards. This shows that the quality of the corn silage of the present invention with Lactobacillus plantarum Mei1101.3 is better than that of the control group in terms of microbial quantity.

[0054] Table 4 Microbial counts in corn silage

[0055] 7. Effects of Lactobacillus plantarum Mei1101.3 on the hygienic and safety indicators of corn silage The determination methods of aflatoxin B1, zearalenone and vomitoxin content shall be carried out according to the instructions of the test card.

[0056] The test results of aflatoxin B1 content, zearalenone content and vomitoxin content in the corn silage of the Lactobacillus plantarum Mei1101.3 of the present invention are all negative, indicating that the detection limit is not exceeded.

[0057] The national standard GB 13078-2017, "Feed Hygiene Standard," specifies limits for various toxins in corn. Among these, the limit for aflatoxin is 30 μg / kg, the limit for zearalenone is 0.5 mg / kg, and the limit for vomitoxin is 5 mg / kg. Therefore, the Lactobacillus plantarum Mei1101.3 corn silage of the present invention is safe for feeding.

[0058] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum that is highly efficient in producing acid, characterized in that: The plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum ) Mei1101.3, the depository is the General Microbiology Center of China Culture Collection Administration, the deposit number is CGMCC No. 30332, the deposit date is April 15, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Plant Lactobacillus according to claim 1, characterized in that The plant lactobacillus is isolated from kimchi.

3. Plant Lactobacillus according to claim 1, characterized in that The plant lactobacillus has at least one of the following properties: (1) It is acid-resistant and survives well at pH 3.0; (2) It is resistant to high temperatures and can survive well in an environment of 50°C.

4. A bacterial agent, characterized in that Contains the Lactobacillus plantarum according to any one of claims 1 to 3.

5. The microbial agent according to claim 4, characterized in that The bacterial agent is in the form of powder, pills or liquid preparation.

6. The method for preparing the microbial agent according to claim 4, characterized in that: The following steps are involved: Under sterile conditions, the Lactobacillus plantarum is inoculated into an MRS liquid culture medium and cultured to obtain a fermentation liquid; The fermentation liquid is mixed evenly with physiological saline to prepare a bacterial agent.

7. The preparation method according to claim 6, characterized in that The culture conditions include: culturing at 37° C. and 160 r / min for 24 h to obtain a fermentation liquid.

8. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to claim 4 in preparing corn silage.

9. The use according to claim 8, characterized in that The specific method of using the plant lactobacillus or the bacterial agent lactobacillus in preparing corn silage comprises the following steps: The plant lactobacillus or the bacterial agent is added into corn kernels, and the kernels are ensiled for 30 days with the exclusion of air to prepare corn silage.

10. A corn silage, characterized in that: The ensilage is prepared by adding the plant lactobacillus according to claim 1 or the bacterial agent according to claim 2 to corn kernels and removing air and silaging for 30 days.

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