A strain of Lactobacillus plantarum R6 and its application

Abstract

The present invention belongs to the field of microbial fermentation technology, and specifically relates to a strain of Lactobacillus plantarum R6 and its application. The Lactobacillus plantarum R6 has been deposited in the General Microbiological Center of the China Microbiological Strain Collection Management Committee on March 22, 2024, with the address of No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30096. The strain can produce linoleic acid isomerase, which can convert linoleic acid into c9, t11-CLA in MRS culture medium, and the fermentation level is as high as 1 mg / mL. At the same time, the strain can also produce high phytase, which can effectively decompose phytic acid. Therefore, the present invention can also use the strain to ferment silage, and use its characteristics of colonization in the rumen to increase the abundance of Lactobacillus plantarum R6 in the rumen, increase the CLA content in the rumen by biotransformation, and significantly increase the CLA content in fresh milk through metabolic transport, so as to obtain fresh milk rich in c9, t11-CLA, and meet the needs of consumers for nutritional health.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a strain of Lactobacillus plantarum R6 and application thereof. Background Art

[0002] Silage is made by chopping, sealing and fermenting plant straw and green forage. It is mainly used to feed ruminants. It is more durable than fresh feed and has better nutritional content than dry feed. Phytic acid, also known as inositol hexaphosphate, is a phosphorus-containing compound in plant tissues. For example, its content in corn straw is as high as 2%. Phytic acid has anti-nutritional effects such as reducing the absorption of mineral elements and the digestion of protein. At the same time, high-phosphorus feces aggravate the pollution of the environment. In order to reduce the side effects of phytic acid, phytase is often added to the feed. In addition, if the fermentation conditions are not well controlled during the fermentation process of silage, it often results in poor flavor, poor palatability, and even corruption of silage.

[0003] Conjugated linoleic acid (CLA) is a conjugated isomer of linoleic acid, and there are as many as 16 isomers, such as c9, t11-CLA and t10, c12-CLA. A large number of studies have found that CLA has physiological activities such as anti-cancer, weight loss and anti-diabetes. More and more studies have shown that its physiological activity is isomer-specific. It is generally believed that c9, t11-CLA isomers have anti-breast cancer, colon cancer and gastric cancer activities. In nature, CLA is mainly found in the fat in the milk and meat of ruminants such as cattle and sheep, and is mainly composed of isomers such as c9, t11-CLA and t10, c11-CLA, but its content is usually less than 10 mg / g fat, which cannot meet the development and application for health care and medical purposes. Summary of the invention

[0004] The object of the present invention is to overcome the deficiencies in the prior art and provide a strain of Lactobacillus plantarum R6 and an application thereof, specifically adopting the following technical scheme:

[0005] In a first aspect of the present invention, a strain of Lactobacillus plantarum R6 is provided, wherein the Lactobacillus plantarum ( Lactobacillus plants ) R6 was deposited in the General Microbiological Center of China Microbiological Strain Collection Administration on March 22, 2024, with the address at No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30096. The gene sequence of 16S rDNA of Lactobacillus plantarum R6 is shown in SEQ ID No: 1.

[0006] SEQ ID No: 1:

[0007]

[0008] In the present invention, Lactobacillus plantarum ( Lactobacillus plantarum ) R6 is isolated from the rumen of sheep. This strain can produce linoleic acid isomerase, which can convert linoleic acid into c9, t11-CLA in MRS culture medium, and the fermentation level is as high as 1 mg / mL. At the same time, this strain can not only produce high phytase and effectively decompose phytic acid, but also improve the digestion and absorption rate of mineral elements in straw. Therefore, the present invention ferments silage through this strain, utilizes its ability to colonize in the rumen, increases the abundance of Lactobacillus plantarum R6 in the rumen, increases the CLA content in the rumen through biotransformation, and significantly increases the CLA content in fresh milk through metabolic transport, thereby obtaining fresh milk rich in c9, t11-CLA and meeting consumers' demand for nutrition and health.

[0009] The second aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum R6 in preparing silage.

[0010] The third aspect of the present invention further provides a bacterial agent, the active ingredient of the bacterial agent includes the above-mentioned Lactobacillus plantarum R6.

[0011] The fourth aspect of the present invention further provides a silage additive, wherein the active ingredient of the silage additive includes the above-mentioned Lactobacillus plantarum R6.

[0012] The fifth aspect of the present invention further provides a silage, wherein the active ingredient of the silage additive includes the above-mentioned Lactobacillus plantarum R6.

[0013] A sixth aspect of the present invention further provides a method for preparing the above-mentioned silage, comprising the following steps:

[0014] The Lactobacillus plantarum R6, Saccharomyces cerevisiae and Bacillus subtilis cultivated separately are mixed in a volume ratio of 1:1:1 to prepare a multi-bacteria synergistic fermentation agent;

[0015] The soybean meal is evenly sprinkled into the corn stalks, and a multi-bacteria synergistic fermentation agent is sprayed at the same time, mixed evenly, and then fermented until the pH is less than 4.2, so as to obtain silage.

[0016] Among them, the above-mentioned brewer's yeast and Bacillus subtilis were purchased from the China General Microbiological Culture Collection Management Center. Saccharomyces cerevisiae ) was deposited in China General Microbiological Culture Collection Center on January 1, 1952, with the deposit number CGMCC 2.126; Bacillus subtilis ( Bacillus subtilis ) was deposited in the China General Microbiological Culture Collection Center on March 1, 1972, with the number CGMCC 1.821.

[0017] In the above method, the inventors have improved the wine flavor of silage by adding brewer's yeast through multi-strain synergistic fermentation, and the palatability is further improved. Adding Bacillus subtilis can quickly reduce the air in the gap between feeds in the early stage of fermentation, which is conducive to the rapid formation of an anaerobic environment, promote anaerobic fermentation, and avoid abnormal fermentation and corruption. In addition, these multi-strains are all probiotics, which can maintain the balance of intestinal flora of dairy cows, improve the immunity of dairy cows, and promote feed digestion and nutrient absorption.

[0018] As a further preferred embodiment, the bacterial concentrations of the above-mentioned Lactobacillus plantarum R6, Saccharomyces cerevisiae and Bacillus subtilis are all 1×10 8 CFU / mL.

[0019] As a further preferred embodiment, the usage ratio of the corn stalk, soybean powder and multi-bacteria synergistic fermentation agent is 1:1%-5%:0.1%-1.0%. More preferably, the usage ratio of the corn stalk, soybean powder and multi-bacteria synergistic fermentation agent is 1:2%:0.2%.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum ) R6, the plant lactobacillus comes from the sheep rumen and can produce high levels of linoleic acid isomerase and phytase. And the strain can be used to ferment silage. The data results of the embodiments of the present invention show that the strain can significantly reduce the content of phytic acid in silage by more than 50%. By utilizing its ability to colonize in the rumen, the abundance of plant lactobacillus R6 in the rumen is increased, the CLA content in the rumen is increased through biotransformation, and the CLA content in fresh milk is significantly increased to more than 25 mg / g milk fat through metabolic transport, and the content of c9,t11-CLA is>75%, obtaining fresh milk rich in c9,t11-CLA, with high CLA production, high purity of c9,t11-CLA isomers, simple production method and low cost.

[0022] (2) The present invention also provides a method for preparing silage by synergistic fermentation of multiple strains. Under the action of brewer's yeast, the method improves the wine flavor and palatability of the silage. Under the action of aerobic Bacillus subtilis, the air in the gap between feeds in the early stage of fermentation is rapidly reduced, an anaerobic environment is quickly formed, anaerobic fermentation is promoted, and abnormal fermentation and corruption are effectively avoided. The three selected strains of Lactobacillus plantarum, brewer's yeast and Bacillus subtilis are all probiotics that can maintain the balance of intestinal flora, improve the immunity of dairy cows, and promote feed digestion and nutrient absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is Lactobacillus plantarum ( Lactobacillus plantarum ) Horizontal graph of R6 synthetic CLA;

[0024] Figure 2 Shown is Lactobacillus plantarum ( Lactobacillus plantarum ) Composition analysis diagram of R6 synthetic CLA isomers;

[0025] Figure 3 Shown is the effect of multi-strain synergistic fermentation on the phytic acid content in corn silage;

[0026] Figure 4 The figure shows the effect of multi-strain synergistic fermentation of corn silage on the CLA content in fresh milk fat. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0028] Example 1

[0029] Lactobacillus plantarum ( Lactobacillus plantarum ) Screening, cultivation and identification of R6

[0030] (1) A method for screening and isolating Lactobacillus plantarum R6, comprising the following steps:

[0031] Sample processing: Immediately after the goats are slaughtered, an appropriate amount of rumen fluid is drawn into a sterile, oxygen-free, carbon dioxide-filled sealed glass bottle prepared in advance using a sterile syringe, and then quickly brought back to the laboratory and filtered using sterile gauze in an ultra-clean workbench.

[0032] Enrichment culture: Use a pipette to pipette 0.5 mL of filtered rumen fluid into 5.0 mL of MRS culture medium and culture at 39°C for 48 h.

[0033] Initial screening by streaking on a flat plate: streak the bacterial sample in the MRS culture medium on the MRS solid culture medium with an inoculation loop in an ultra-clean workbench. After constant temperature incubation at 37°C for 48 h, pick out a single colony and incubate it in MRS culture medium at 37°C for 24 h. Then take it out and store it at 4°C for later use.

[0034] Rescreening: The preserved strain was activated and subcultured once with an inoculation volume of 5%. After culturing at 37°C for 24 h, 1.0 g / L of emulsified linoleic acid was added to the fermentation system. After fermentation at 37°C for 24 h, n-hexane was added for extraction. The extract was collected by centrifugation and the CLA content was determined. The specific determination method was carried out according to the method in (Xiaohua Liu, Yusheng Cao, Yan Chen. Separation of conjugated linoleic acid isomers by cyclodextrin-modified micellarelectrokinetic chromatography. Journal of Chromatography A, 2005, 1095:197-200).

[0035] (2) Cultivating Lactobacillus plantarum R6, comprising the following steps:

[0036] The R6 bacterial solution of Lactobacillus plantarum stored at 4°C was inoculated into MRS culture medium at a 5% (v / v) inoculation rate, cultured at 37°C for 24 h, and the bacterial solution was stored at 4°C for later use.

[0037] (3) Identification of Lactobacillus plantarum R6, comprising the following steps:

[0038] Morphological characteristics and Gram staining observation: Observe the colony size, shape, color, edge structure, surface smoothness, transparency and other morphological characteristics of the bacteria on MRS solid medium. Perform Gram staining and observation. The colony diameter is 1-2mm, milky white and translucent, with smooth edges and a slight bulge in the middle. The bacteria are Gram-positive and rod-shaped.

[0039] 16S rDNA gene sequencing analysis: According to the conventional operation method, the genome of the strain was extracted, the amplification primers were universal primers for bacterial identification, the genomic DNA of the strain was amplified by PCR, the PCR product was purified, and sequencing was performed. The 16S rDNA sequence obtained by sequencing was compared by BLAST in the NCBI database to compare its homology.

[0040] (4) Lactobacillus plantarum Lactobacillus plantarum ) Fermentation characteristics of R6

[0041] Skim milk powder was added with water to prepare 10% skim milk, 5 mL per test tube, sterilized at 85℃ for 30 min, and MRS culture medium was prepared at the same time, 5 mL per test tube, sterilized at 121℃ for 20 min, cooled to room temperature and inoculated with plant lactobacillus R6 strain, with an inoculation volume of 5%. Then 0.12% linoleic acid was added to each group of test tubes, mixed evenly, and placed in a constant temperature incubator at 37℃ for culture. 0.5 mL of samples were taken every 12 hours, and the CLA content was determined respectively. The specific determination method was carried out according to the method in (Xiaohua Liu, Yusheng Cao, Yan Chen. Separation of conjugated linoleic acid isomers bycyclodextrin-modified micellar electrokinetic chromatography. Journal of Chromatography A, 2005, 1095:197-200). The results of fermentation for 72 hours are as follows: Figure 1 As shown in the figure, the fermentation levels of CLA in the two culture media were comparable, and the CLA content reached 0.89 mg / mL after 24 h of fermentation. Subsequently, the CLA production entered a stable period, reaching a maximum of 1.11 mg / mL. Lactobacillus plantarum ) The isomer of CLA synthesized by R6 in skim milk and MRS medium was only one monomer, c9,t11-CLA. Figure 2 shown.

[0042] Example 2

[0043] A method for preparing silage for feeding dairy cows, which specifically comprises the following steps:

[0044] (1) Lactobacillus plantarum ( Lactobacillus plantarum R6) CGMCC 30096 strains were inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum culture medium; Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) CGMCC2.126 strain was inoculated into malt juice culture medium and cultured at 25℃ for 24 h to obtain brewer's yeast culture medium; Bacillus subtilis ( Bacillus subtilis ) CGMCC 1.821 strain was inoculated into nutrient broth and cultured at 30°C for 24 h to obtain Bacillus subtilis culture. The cell concentrations of the above three culture solutions were adjusted to 1×10 8 CFU / mL;

[0045] (2) mixing Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Bacillus subtilis liquid in a volume ratio of 1:1:1 to obtain a multi-strain synergistic fermentation agent;

[0046] (3) Cut the harvested whole corn into 2 cm long stalk segments, crush the soybeans and pass them through a 30-mesh sieve, evenly sprinkle 20 kg of soybean powder into 1000 kg of corn stalks, and spray 2 kg of a multi-strain synergistic fermentation agent to mix evenly. After packaging with a silage baler, naturally ferment for more than 15 days until the pH is less than 4.2, and finally obtain corn silage.

[0047] In this embodiment, a control group is set up, in which no multi-strain cooperative fermentation agent is added during the preparation process, so as to obtain corn silage without adding a fermentation agent.

[0048] The effects of two fermentation methods (corn silage and corn silage without fermentation agent) on the phytic acid content in corn silage were determined respectively. The specific detection method was based on the method described in (Luo Kun et al., Effects of high-phytase lactic acid bacteria fermentation on protein quality and baking characteristics of black bean bread, Food Science, 2021, 42(06): 111-117). The detection results are shown in the figure. Figure 3 shown by Figure 3 It can be seen that after 15 days of multi-strain synergistic fermentation, the phytic acid content in corn silage decreased by more than 50%, while the control group was less than 15%. At the same time, the corn silage fermented by multi-strain synergistic fermentation had a stronger aroma and no abnormal fermentation occurred.

[0049] The above corn silage and corn silage without fermentation agent were used for dairy cow feeding. The daily diet of dairy cows consisted of 10 kg of concentrate, 3 kg of alfalfa hay, 4 kg of straw and 10 kg of silage. The concentrate was 45% corn, 20% wheat bran, 17% soybean meal, 12% cotton cake, 2% baking soda, 1% salt, 1% shellfish powder and 2% bone meal. The control group was corn silage without fermentation agent. It was found that corn silage fermented by multiple strains had better palatability and significantly reduced the incidence of gastrointestinal digestive diseases in dairy cows. The effects of the two corn silages on the CLA content in fresh milk were determined respectively. The specific determination method was to determine the CLA content according to the method in (Xiaohua Liu, Yusheng Cao, Yan Chen. Separation of conjugated linoleic acidisomers by cyclodextrin-modified micellar electrokinetic chromatography. Journal of Chromatography A, 2005, 1095:197-200). The results are shown as follows: Figure 4 shown by Figure 4It can be seen that after 6 days of feeding with corn silage fermented by multiple strains, the CLA content in fresh milk increased to as high as 25.4 mg / g milk fat, while the CLA content in fresh milk of cows fed with traditional silage was 8.3 mg / g milk fat. And the c9,t11-CLA isomer content of the former was 76.1%, while that of the latter was only 52.2%. At the same time, it was found that if soybean powder was not added during the fermentation of corn silage, the CLA content in fresh milk did not increase significantly, usually <10 mg / g milk fat. By feeding dairy cows with corn silage fermented by multiple strains of the present invention, fresh milk rich in c9,t11-CLA can be obtained, with high CLA yield, high purity of c9,t11-CLA isomers, simple production method and low cost.

[0050] Example 3

[0051] A method for preparing silage for feeding dairy cows, which specifically comprises the following steps:

[0052] (1) Lactobacillus plantarum ( Lactobacillus plantarum R6) CGMCC 30096 strains were inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum culture medium; Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) CGMCC2.126 strain was inoculated into malt juice culture medium and cultured at 25℃ for 24 h to obtain brewer's yeast culture medium; Bacillus subtilis ( Bacillus subtilis ) CGMCC 1.821 strain was inoculated into nutrient broth and cultured at 30°C for 24 h to obtain Bacillus subtilis culture. The cell concentrations of the above three culture solutions were adjusted to 1×10 8 CFU / mL;

[0053] (2) mixing Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Bacillus subtilis liquid in a volume ratio of 1:1:1 to obtain a multi-strain synergistic fermentation agent;

[0054] (3) Harvested whole corn plants were cut into 2 cm long stalk segments, alfalfa was cut into 5 cm long segments, soybeans were crushed and passed through a 30-mesh sieve, 600 kg of corn stalks and 200 kg of alfalfa were mixed in a mass ratio of 3:1, and 16 kg of soybean powder was evenly sprinkled on. At the same time, 1.6 kg of multi-strain synergistic fermentation agent was sprayed on the mixture and mixed evenly. After packaging with a silage baler, the mixture was naturally fermented for more than 15 days until the pH was less than 4.2. Finally, corn stalk and alfalfa silage were obtained. It was found that the silage fermented by multi-strain synergistic fermentation had a stronger aroma and no abnormal fermentation occurred.

[0055] The corn stalks and alfalfa silage prepared above were used for dairy cow feeding. The daily diet of dairy cows consisted of 10 kg of concentrate, 2 kg of alfalfa hay, 5 kg of straw, and 10 kg of corn stalks and alfalfa silage. The concentrate was 45% corn, 20% wheat bran, 17% soybean meal, 12% cotton cake, 2% baking soda, 1% salt, 1% shell powder, and 2% bone meal. The control group was corn silage without the addition of a fermentation agent. It was observed that the corn silage fermented by multiple strains had better palatability and significantly reduced the incidence of gastrointestinal digestive diseases in dairy cows. After 6 days of feeding with silage fermented by multiple strains, the content of CLA in fresh milk increased to 26.1 mg / g milk fat, while the content of CLA in fresh milk of dairy cows fed with traditional silage was 9.2 mg / g milk fat. And the c9,t11-CLA isomer content of the former was 76.5%, while that of the latter was only 51.4%. By feeding dairy cows with corn and alfalfa silage synergistically fermented by multiple strains of the present invention, fresh milk rich in c9, t11-CLA can be obtained, the yield of CLA is high, the purity of c9, t11-CLA isomers is high, the production method is simple and the cost is low.

[0056] Example 4

[0057] A method for preparing silage for feeding beef cattle, which specifically comprises the following steps:

[0058] (1) Lactobacillus plantarum ( Lactobacillus plantarum R6) CGMCC 30096 strains were inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum culture medium; Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) CGMCC2.126 strain was inoculated into malt juice culture medium and cultured at 25℃ for 24 h to obtain brewer's yeast culture medium; Bacillus subtilis ( Bacillus subtilis ) CGMCC 1.821 strain was inoculated into nutrient broth and cultured at 30°C for 24 h to obtain Bacillus subtilis culture. The cell concentrations of the above three culture solutions were adjusted to 1×10 8 CFU / mL;

[0059] (2) mixing Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Bacillus subtilis liquid in a volume ratio of 1:1:1 to obtain a multi-strain synergistic fermentation agent;

[0060] (3) Cut the harvested whole corn into 2 cm long stalk segments, crush the soybeans and pass them through a 30-mesh sieve, evenly sprinkle 20 kg of soybean powder into 1000 kg of corn stalks, and spray 2 kg of a multi-strain synergistic fermentation agent to mix evenly. After packaging with a silage baler, naturally ferment for more than 15 days until the pH is less than 4.2, and finally obtain corn stalk silage.

[0061] The corn straw silage obtained above was used for beef cattle feeding. The daily diet of beef cattle consisted of 5 kg of concentrate, 1 kg of alfalfa hay, 2 kg of straw, and 4 kg of silage. The concentrate was 60% corn, 11% distiller's grains, 15% soybean meal, 8% rapeseed meal, 2% baking soda, 1% salt, 1% shellfish powder, and 2% bone meal. It was observed that the corn silage fermented by multiple strains had better palatability and significantly reduced the incidence of gastrointestinal digestive diseases in beef cattle. After 30 days of feeding with corn silage fermented by multiple strains, the CLA content in beef fat increased to as high as 20.5 mg / g fat, while the CLA content in beef fat fed with traditional silage was 7.8 mg / g fat. The c9,t11-CLA isomer content of the former was 75.2%, while that of the latter was only 51.5%. At the same time, it was found that if soybean meal was not added during the fermentation of corn silage, the CLA content in beef did not increase significantly, usually <10 mg / g milk fat. By feeding beef cattle with corn silage using multiple strains of the present invention, marbled beef rich in c9, t11-CLA can also be obtained, with high CLA yield, high purity of c9, t11-CLA isomers, simple production method and low cost.

[0062] Example 5

[0063] A method for preparing silage for feeding dairy cows, which specifically comprises the following steps:

[0064] (1) Lactobacillus plantarum ( Lactobacillus plantarum R6) CGMCC 30096 strains were inoculated into MRS culture medium and cultured at 37℃ for 24 h to obtain Lactobacillus plantarum culture medium; Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) CGMCC2.126 strain was inoculated into malt juice culture medium and cultured at 25℃ for 24 h to obtain brewer's yeast culture medium; Bacillus subtilis ( Bacillus subtilis ) CGMCC 1.821 strain was inoculated into nutrient broth and cultured at 30°C for 24 h to obtain Bacillus subtilis culture. The cell concentrations of the above three culture solutions were adjusted to 1×10 8 CFU / mL;

[0065] (2) mixing Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Bacillus subtilis liquid in a volume ratio of 1:1:1 to obtain a multi-strain synergistic fermentation agent;

[0066] (3) Cut the harvested whole corn into 2 cm long stalk segments, crush the soybeans and pass them through a 30-mesh sieve, evenly sprinkle 20 kg of soybean powder into 1000 kg of corn stalks, and spray 2 kg of multi-strain synergistic fermentation agent to mix evenly. After packaging with a silage baler, naturally ferment for more than 15 days until the pH is less than 4.20, and finally obtain corn silage.

[0067] In this embodiment, two control groups were set up, in which only Lactobacillus plantarum liquid or a mixed liquid of Saccharomyces cerevisiae and Bacillus subtilis was added during the preparation process, respectively, to obtain corn silage with two different starter cultures.

[0068] The effects of three fermentation methods on the phytic acid content in corn silage were measured separately. After 15 days of fermentation with a mixture of three strains or a single strain of Lactobacillus plantarum, the phytic acid content in corn silage decreased by more than 50%, while the phytic acid content in the mixed fermentation group of Saccharomyces cerevisiae and Bacillus subtilis decreased by less than 15%. At the same time, the aroma of corn silage fermented by multiple strains was stronger, and no abnormal fermentation occurred. The wine aroma of fermentation with a single strain of Lactobacillus plantarum was insufficient.

[0069] The three groups of corn silage were used for dairy cow feeding. The daily diet of dairy cows consisted of 10 kg of concentrate, 3 kg of alfalfa hay, 4 kg of straw, and 10 kg of silage. The concentrate was 45% corn, 20% wheat bran, 17% soybean meal, 12% cotton cake, 2% baking soda, 1% salt, 1% shellfish powder, and 2% bone meal. It was observed that the palatability of corn silage fermented by multiple strains was better, and the incidence of gastrointestinal digestive diseases in dairy cows was significantly reduced. The palatability of corn silage fermented by a single strain of Lactobacillus plantarum was slightly worse. After feeding with corn silage fermented by multiple strains for 6 days, the CLA content in fresh milk increased to 25.4 mg / g milk fat. The CLA content in fresh milk of dairy cows fed only with silage fermented by Lactobacillus plantarum was 23.8 mg / g milk fat, while the CLA content in fresh milk of dairy cows fed with silage fermented by a mixture of Saccharomyces cerevisiae and Bacillus subtilis was 8.7 mg / g milk fat.

[0070] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A strain of Lactobacillus plantarum R6, characterized in that: The Lactobacillus plantarum ( Lactobacillus plantarum ) R6 was deposited on March 22, 2024 at the General Microbiology Center of the China Microbiological Collection Administration, located at No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.30096.

2. Use of Lactobacillus plantarum R6 according to claim 1 in preparing silage.

3. A bacterial agent, characterized in that: The active ingredient of the bacterial agent includes the Lactobacillus plantarum R6 described in claim 1.

4. A silage additive, characterized in that: The active ingredient of the silage additive includes the Lactobacillus plantarum R6 according to claim 1.

5. A silage, characterized in that: The silage contains the silage additive according to claim 4.

6. The method for preparing silage according to claim 5, characterized in that: The following steps are involved: The Lactobacillus plantarum R6, Saccharomyces cerevisiae and Bacillus subtilis cultivated separately are mixed in a volume ratio of 1:1:1 to prepare a multi-bacteria synergistic fermentation agent; The soybean meal is evenly sprinkled into the corn stalks, and a multi-bacteria synergistic fermentation agent is sprayed at the same time, mixed evenly, and then fermented until the pH is less than 4.2, so as to obtain silage.

7. The preparation method according to claim 6, characterized in that: The bacterial liquid concentrations of Lactobacillus plantarum R6, Saccharomyces cerevisiae and Bacillus subtilis were all 1×10 8 CFU / mL.

8. The preparation method according to claim 6, characterized in that: The usage ratio of the corn stalk, soybean powder and multi-bacteria synergistic fermentation agent is 1:1%-5%:0.1%-1.0%.

9. The preparation method according to claim 8, characterized in that: The usage ratio of the corn stalk, soybean powder and multi-bacteria synergistic fermentation agent is 1:2%:0.2%.

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