Lactobacillus buchneri and application thereof

Abstract

The invention relates to the technical field of silage microbial inoculants, and discloses a strain of Lactobacillus buchneri and application thereof, the Lactobacillus buchneri is Lactobacillus buchneri LB92, is preserved in the General Microbiological Center of China Committee for Culture Collection of Microorganisms on April 10, 2024, and has a preservation number of CGMCC NO.30296. The invention also discloses a method for preparing the lactobacillus buchneri. The lactobacillus buchneri LB92 provided by the invention can tolerate a high-temperature and high-acid environment during silage fermentation, can inhibit the reproduction of mould, reduce the pH value of oat silage and increase the number of lactic acid bacteria of the oat silage, and is an excellent strain capable of inhibiting the growth of mould in a high-temperature environment; the strain has a huge potential of being developed into a lactic acid bacterium agent in southern hot and humid areas.

Description

Technical Field

[0001] The invention relates to the technical field of silage microbial inoculants, and in particular to a strain of Lactobacillus buchneri and application thereof in oat silage. Background Art

[0002] Oats (Avena sativa L.) are an important forage variety for the development of my country's agriculture from a dual structure to a tri-structure and for grain-to-feed conversion. They are also one of the dominant crops planted in winter fallow fields in southern China. Silage is a method of preserving the green and juicy state of forage crops. It uses fresh green feed as raw material and lactic acid bacteria naturally attached to the surface of plants. Under closed conditions, through anaerobic fermentation, the soluble carbohydrates of plants are converted into organic acids such as lactic acid and acetic acid, resulting in a decrease in the pH value of the feed, thereby inhibiting the growth and reproduction of corrupt microbial flora, thereby achieving the purpose of maintaining the nutritional characteristics of crops. Silage has the advantages of sour and fragrant smell, soft and juicy, yellow-green color, and good palatability. Therefore, silage can better preserve the freshness of forage, store for a long time, and reduce nutrient loss. It is the main way to process and utilize oats in southern China.

[0003] Adding additives during the process of making silage can significantly improve the silage effect. Lactic acid bacteria are Gram-positive bacteria, resistant to catalase, anaerobic, non-spore, and non-motile. Adding lactic acid bacteria during the silage process can effectively inhibit the reproduction of harmful microorganisms, reduce the pH in silage, promote lactic acid fermentation, improve the quality of silage fermentation, and reduce nutritional losses. When the silage pit is sealed, anaerobic microorganisms can reproduce rapidly. If the conditions are suitable, lactic acid bacteria will quickly acidify the environment. When the pH drops to a certain level, harmful microorganisms competing with them will not survive, so that stable silage is obtained. On the contrary, bad microorganisms grow rapidly, causing silage to spoil or deteriorate in quality. The key to lactic acid fermentation is lactic acid bacteria. If the number of lactic acid bacteria attached to the material itself is small, or the number of lactic acid bacteria is large, but there is a lack of lactic acid bacteria that can withstand special environments (such as high temperature), it is difficult to obtain high-quality silage under the corresponding conditions. For example, it is difficult to prepare high-quality silage in a high-temperature silage environment without high-temperature resistant lactic acid bacteria.

[0004] Due to global warming, extreme weather occurs frequently, and oats are prone to lodging, especially in the southern region with abundant water and heat conditions. After lodging, oats will still be harvested and used to make silage, but the photosynthesis of lodging oats will be weakened, and the growth of mold will increase, increasing the risk of mycotoxins infecting silage. Under high temperature silage conditions, the risk of oat silage being infected by mold is further increased.

[0005] Therefore, exploring lactic acid bacteria resources that can inhibit mold growth in high temperature environments is of great significance to improving the quality of oat silage in the humid and hot southern regions. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a strain of Lactobacillus buchneri LB92 which can inhibit the growth of mold in a high temperature environment and its application.

[0007] The Lactobacillus buchneri LB92 provided by the present invention is isolated and screened from corn silage, and is determined to be a strain of Lactobacillus buchneri of the genus Lactobacillus through physiological and biochemical characteristics, molecular biological identification, etc. It has been deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on April 10, 2024, and its deposit number is CGMCC NO.30296.

[0008] The Lactobacillus buchneri LB92 provided by the present invention can inhibit the growth of molds in a high temperature environment, especially has a good inhibitory effect on Aspergillus fumigatus and Fusarium verticillioides, and can therefore be used to inhibit the growth of molds in a lactic acid bacteria fermentation environment. Further, the molds include Aspergillus fumigatus and Fusarium verticillioides.

[0009] The above-mentioned Lactobacillus buchneri LB92 can reduce the pH of oat green juice fermentation liquid under high temperature conditions and increase the number of lactic acid bacteria in the oat green juice fermentation liquid. Therefore, the present invention also provides a silage feed additive, and the active ingredient of the silage feed additive includes the above-mentioned Lactobacillus buchneri LB92.

[0010] Furthermore, the present invention also provides a silage, wherein the silage contains the above-mentioned Lactobacillus buchneri LB92.

[0011] Furthermore, the preparation method of the silage is to mix the silage raw material to be fermented with the silage additive to prepare the silage, and the ratio of Lactobacillus buchneri LB92 to the silage raw material in the silage additive is 3×10 7 cfu:1kg.

[0012] Furthermore, the silage raw material is oats.

[0013] Furthermore, the oats include lodging oats.

[0014] Aiming at the problems existing in the silage of lodged oats, the present invention separates and screens lactic acid bacteria which can inhibit the growth of molds and improve the fermentation quality of silage under a high temperature environment. As a result, a strain of Lactobacillus buchneri LB92 is found, which has an inhibition rate of 36.15% and 56.94% against Aspergillus fumigatus and Fusarium verticillium at a culture temperature of 45°C. When the strain is inoculated into oat green juice fermentation liquid, the pH of the oat green juice fermentation liquid can be reduced under a high temperature condition, the number of lactic acid bacteria in the oat green juice fermentation liquid can be increased, and molds, yeasts and enterobacteria are not detected in the fermentation liquid.

[0015] The Lactobacillus buchneri LB92 provided by the present invention has the following microbiological characteristics: Gram-positive, rod-shaped, uses glucose to produce gas, is a heterofermentative lactic acid bacterium, has a suitable growth temperature of 37-50°C, and a growth pH of 3.5-7; some physiological and biochemical characteristics are shown in Tables 1 and 2 below.

[0016] Table 1. Microbiological characteristics of LB92

[0017] Note: W: OD600nm between 0.1 and 0.3 indicates weak growth of the strain; +: OD600nm between 0.3 and 0.5 indicates normal growth of the strain; ++: OD600nm>0.5 indicates that the strain grows well.

[0018] Table 2. Sugar fermentation characteristics of LB92

[0019] Note: + means that more than 90% of the strains can ferment the substrate; w means that a small amount of the substrate can be fermented; - means that 90% or more of the strains cannot ferment the substance.

[0020] The present invention has the following beneficial effects: The Lactobacillus buchneri LB92 provided by the present invention can tolerate the high temperature and high acid environment during silage fermentation, and can inhibit the reproduction of mold, reduce the pH value of oat silage, and increase the number of lactic acid bacteria in oat silage. It is an excellent strain that can inhibit the growth of mold in a high temperature environment and has great potential for being developed into a lactic acid bacteria agent in the hot and humid southern regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the phylogenetic tree diagram of the Lactobacillus buchneri LB92 strain in Example 1; Figure 2 The growth curve of Lactobacillus buchneri LB92 strain in Example 2; Figure 3 This is the acid production curve of Lactobacillus buchneri LB92 strain in Example 2. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples. It is understood that the specific examples and the accompanying drawings are only exemplary descriptions of the present invention and should not be regarded as the entirety of the present invention or as a limitation or restriction to the technical solution of the present invention. Unless otherwise specified, the raw materials used in the following examples and comparative examples can be obtained through conventional commercial channels.

[0023] Example 1: Isolation, screening, identification and preservation of Lactobacillus buchneri LB92 1. Test materials 831 strains of lactic acid bacteria tested (isolated and screened from silage); The indicator fungi were Aspergillus flavus (A. flavus), Aspergillus niger (A. niger), Aspergillus fumigatus (A. fumigatus), and Fusarium verticillioides (F. verticillioides). Aspergillus flavus (CGMCC3.3950) and Aspergillus niger (CGMCC3.3928) were purchased from the China General Microbiological Culture Collection Center, and Aspergillus fumigatus and Fusarium verticillioides were isolated and screened from lodging oats.

[0024] 2. Preparation of lactic acid bacteria liquid: Add the lactic acid bacteria strain in the cryopreserved tube into MRS broth culture medium at a 3% inoculation rate, place it in a 37°C constant temperature incubator for anaerobic culture for 48 h, activate it twice continuously, and prepare lactic acid bacteria liquid for use.

[0025] 3. Preparation of mold spore solution: Inoculate 4 mold strains into PDA medium and place in a 28℃ constant temperature incubator for 5-7 days. Rinse the surface of the culture dish with 2% Tween 80 and adjust the spore concentration to 10 with a hemocytometer. 4 cfu / mL, stored at 4°C until used.

[0026] 4. Initial screening of bacterial strains The 96-well plate method was used for primary screening. 10 μL of mold spore liquid was added to a sterile 96-well plate containing 190 μL of lactic acid bacteria liquid (CFS) using a pipette, and three replicates were set for each strain. After culturing in a 37°C constant temperature incubator for 48 h, OD580nm was measured using an enzyme reader (Thermo Fisher Varioskan lux). 10 μL of mold spore liquid and 190 μL of MRS broth medium were inoculated in the control.

[0027] The antibacterial rate was calculated according to the following formula: ; Where: ODLAB represents the OD value of the mold after being cultured in lactic acid bacteria CFS for 48 h, and ODCK represents the OD value of the mold after being cultured in MRS broth medium for 48 h.

[0028] After initial screening, strains with an inhibition rate of more than 30% were selected.

[0029] 5. Rescreening of strains The lactic acid bacteria with an inhibition rate of >30% were further screened using the double-layer plate method. The lactic acid bacteria liquid was dipped with a disposable inoculation loop to draw two lines with an interval of 1.5 cm and a length of 3 cm on the MRS solid medium. The culture was then carried out at different culture temperatures (37°C, 45°C) for 48 h. 6 cfu / mL spore solution, 10 mL of PDA semisolid medium (1% agar) was poured into the upper layer of MRS and cultured at 28 °C for 48 h. The inhibition zone was determined with a vernier caliper.

[0030] After repeated screening, a lactic acid bacteria strain (No. 92) was selected that is resistant to high temperature and can simultaneously inhibit the growth of Aspergillus fumigatus and Fusarium verticillium. The inhibitory effect is shown in Table 3 below.

[0031] Table 3. Inhibitory effect of strain 92 on Aspergillus fumigatus and Fusarium verticillium

[0032] 6. Physiological and biochemical characteristics of strain No. 92 According to the lactic acid bacteria identification method reported by Cai et al., the sugar fermentation property test and catalase test of strain 92 were performed (Luqiao Technology Co., Ltd., Beijing, China).

[0033] Sugar fermentation characteristic test: Use a sterile inoculation loop to pick a loop of strain No. 92 and place it in ampoules containing sucrose, lactose, inulin, raffinose, sorbitol, salicin, mannitol, maltose, cellobiose, esculin, glucose, rhamnose, arabinose, fructose, galactose, mesidic acid, and mannose.

[0034] Catalase test: Culture strain 92 on solid MRS medium at 37°C for 48 h. Use an inoculation stick to pick a ring on a glass slide and add 2-3 drops of 3% H2O2 (aq). + (positive): bubbles are generated; - (negative): no bubbles are generated.

[0035] The temperature resistance, acid resistance and salt resistance of strain 92 were determined according to the following test methods.

[0036] (1) Different culture temperatures The inoculum was inoculated into MRS broth medium at a 3% inoculum volume (v:v) and cultured at 10℃, 20℃, 37℃, 40℃, 45℃ and 50℃ for 48 h, with 3 replicates at each temperature. The OD value was measured to determine the appropriate growth temperature of the strain.

[0037] (2) Different pH The inoculum was inoculated at a 3% inoculum (v:v) into MRS broth culture media with pH values ​​of 2.5, 3, 3.5, 4, 5, 6, and 7, respectively, and cultured in a constant temperature incubator at 37°C under anaerobic conditions for 48 h. Three replicates were performed for each pH value, and the OD value was measured to determine the suitable pH value for bacterial growth.

[0038] (3) Salt tolerance The inoculum size was 3% (v:v) and the cultures were inoculated into MRS broth containing 3% NaCl and 6.5% NaCl, respectively. The cultures were placed in a constant temperature incubator at 37 °C under anaerobic conditions for 48 h. Three replicates were set at different salt concentrations, and the OD values ​​were measured to determine the salt tolerance of the strains.

[0039] The test results are shown in Tables 1 and 2 above.

[0040] 7. Identification and preservation of strain No. 92 After strain 92 was cultured in a 37°C incubator for 24 h, it was centrifuged at 12,000 rpm for 5 min at 4°C, and the remaining operations were performed according to the instructions of the TIANamp bacterial DNA kit (DP302-02, Tiangen, Beijing, China) to extract the DNA genome of strain 92.

[0041] The 16S rDNA gene fragment was amplified by PCR from the extracted DNA genome, and the PCR amplification products were assembled and sequenced (sequencing was entrusted to Shanghai Bioengineering Co., Ltd., primer sequences: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACG ACTT-3'). The sequencing results were compared with the GenBank by Blast analysis, and it was found that the homology between strain 92 and Lactobacillus buchneri reached 99%. Combined with the physiological and biochemical characteristics of the strain, strain 92 was finally determined to be Lactobacillus buchneri.

[0042] The 16S rDNA sequence of strain 92 is shown in the sequence table. The 16S rDNA sequence of strain 92 was compared with the known sequences in GenBank by Blast, and the 16S rDNA of related species was obtained from the database. The phylogenetic tree was constructed using Mega6.0 software ( Figure 1 ),from Figure 1The phylogenetic tree shows that strain 92 and Lactobacillus buchner LC094429 are clustered in the same branch, with a similarity of 100%. The strain was determined to belong to the genus Lactobacillus through the physiological and biochemical characteristics of the strain and experimental data such as 16S rDNA sequence, and was finally determined to be Lactobacillus buchneri.

[0043] The strain No. 92 (LB92) obtained above was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on April 10, 2024, with the deposit number CGMCC NO.30296.

[0044] Example 2: Acid production capacity of Lactobacillus buchneri LB92 The lactic acid bacteria liquid was inoculated into MRS broth medium at an inoculum rate of 3% (v:v), and cultured in a constant temperature incubator at 37 ℃ under anaerobic conditions for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, and 48 h. The OD600nm value and pH value of the bacterial liquid were measured, and three replicates were set for each strain. The growth curve and acid production curve were drawn. The results are shown in Table 1. Figure 2 and Figure 3 shown.

[0045] See also Figure 2 After 4 h of cultivation, 9 strains of lactic acid bacteria entered the logarithmic phase. Among them, the growth rates of the 9 strains of lactic acid bacteria were the fastest during 6-8 h. The growth rates of strains 828 and 92 were significantly higher than those of other strains. After 16 h of cultivation, the growth of the strains entered the stable phase. The OD600nm values ​​of strains 522, 19, 11, 823, 821, 36, 42d, 828, and 92 after 16 h of fermentation were 1.765, 1.830, 1.821, 1.831, 1.728, 1.849, 1.780, 1.800, and 1.779, respectively.

[0046] See also Figure 3The pH of all lactic acid bacteria was around 5.6-5.7 when the culture time was 2-4 h. The pH of all lactic acid bacteria showed a downward trend when the culture time was 4-16 h. Among them, the pH of strains 828 and 92 was significantly lower than that of other lactic acid bacteria. The acid production rates of strains 828 and 92 reached the maximum when the culture time was 6-8 h. After 8 h of culture, the acid production rates of each strain gradually decreased. After 12 h of culture, the pH values ​​of strains 36, 828, and 92 all dropped to below 4.0. After 16 h of culture, the pH stabilized at around 3.9.

[0047] In summary, strain 92 (LB92) has excellent acid production ability.

[0048] Example 3: Effect of Lactobacillus buchneri LB92 on oat green juice fermentation broth Preparation of oat green juice fermentation liquid: take 50g of fresh whole oat plant, add 10ml of sterilized distilled water, stir 5ml in a blender, and filter with 4 layers of sterilized gauze to make oat green juice fermentation liquid.

[0049] Mold and lactic acid bacteria LB92 were inoculated into oat green juice fermentation liquid according to the inoculation amount of the following groups to obtain bacterial liquids with different inoculation amounts of bacterial species. The bacterial liquid of each group was cultured at different culture temperatures (25°C, 35°C, 45°C) under anaerobic conditions for 6h, 12h, 24h, and 36h. The pH value of the bacterial liquid was measured respectively, and the bacterial liquid after 36h of culture was subjected to microbial detection. The test results are shown in Table 4 below.

[0050] Group AF2: 1 mL of Aspergillus fumigatus spore suspension was inoculated into every 10 mL of oat green juice fermentation liquid; FV group: 1 mL of Fusarium verticillium spore suspension was inoculated into every 10 mL of oat green juice fermentation liquid; Group M: 0.5 mL of Aspergillus fumigatus and Fusarium verticillium spore suspension were inoculated into every 10 mL of oat green juice fermentation liquid; JAF2 group: 0.5 mL of Aspergillus fumigatus spore suspension and 0.5 mL of LB92 bacterial solution were inoculated into every 10 mL of oat green juice fermentation liquid; JFV group: 0.5 mL of Fusarium spore suspension and 0.5 mL of LB92 bacterial suspension were inoculated into every 10 mL of oat green juice fermentation liquid; JM group: Every 10 mL of oat green juice fermentation liquid was inoculated with 0.25 mL of Aspergillus fumigatus spore suspension, 0.25 mL of Fusarium verticillium spore suspension, and 0.5 mL of LB92 bacterial liquid.

[0051] The concentration of the Aspergillus fumigatus spore suspension is 10 6 cfu / mL, the concentration of Fusarium spore suspension was 10 6 cfu / mL, the number of viable bacteria in LB92 bacterial solution is 10 6cfu / mL.

[0052] According to the test results of the pH value of the bacterial liquid, it can be found that when the culture temperature is 25℃, 35℃, and 45℃, the pH of the treatment of inoculation of mold + lactic acid bacteria drops faster than that of inoculation of mold. When the culture temperature is 45℃, the pH value of the oat green juice fermentation liquid containing mold and inoculated with lactic acid bacteria LB92 can drop to below 4.2 after fermentation for 12 hours.

[0053] Table 4. Microbial composition of oat green juice fermentation broth

[0054] Note: The bacterial liquid groups are classified as follows: AF2: Aspergillus fumigatus, FV: Fusarium verticillium, M: Aspergillus fumigatus + Fusarium verticillium mixed at a ratio of 1:1; JAF2: Aspergillus fumigatus + 92, JFV: Fusarium verticillium + 92, JM: Aspergillus fumigatus + Fusarium verticillium mixed at a ratio of 1:1 + strain 92; Different lowercase letters indicate significant differences between different treatments at the same culture temperature (P<0.05), and different uppercase letters indicate significant differences between different temperatures at the same treatment (P<0.05). ND means not detected.

[0055] As can be seen from Table 4, at 25 ℃, the number of lactic acid bacteria in JAF2, JFV and JM treatments was lower than that in AF2, FV and M treatments, respectively; at 35 ℃ and 45 ℃, the number of lactic acid bacteria in JAF2, JFV and JM treatments was higher than that in AF1, AF2, FV and M treatments, respectively. No yeast, enterobacterium and mold were found in all treatments. The interaction between temperature and whether lactic acid bacteria were inoculated had a significant effect on the microbial composition in green juice fermentation broth.

[0056] Example 4: Application of Lactobacillus buchneri LB92 in oat silage Experimental group: The oats that were not rotten or spoiled were cut into 2-3 cm segments and used as silage materials. 3×10 strains of LB92 were added per kg of silage materials. 7 The oat feed with LB92 strain added was put into silage bags and divided into two treatments, one of which was placed at 45℃ for 30 days of anaerobic fermentation and the other was placed at 25℃ for 30 days of anaerobic fermentation.

[0057] Control group 1: An equal amount of sterile saline was used instead of LB92 strain as a control; Control group 2: An equal amount of commercially available lactic acid bacteria additive (Yaxin Bio Silage Additive) was used to replace the LB92 strain as a control.

[0058] After silage is completed, observe the quality of silage, and test the content of various nutrients (dry matter, soluble carbohydrates, starch, crude protein, neutral and acidic detergent fiber, ammonia nitrogen), organic acid content, mold, yeast content and aerobic stability in silage. The test results are shown in Table 6 below.

[0059] Table 5. Chemical and microbial composition of forage before ensilage

[0060] Note: DM: dry matter; FM: fresh matter; lg: data is logarithmic to the base 10; cfu / g: colony forming unit.

[0061] Table 6. Effect of Lactobacillus buchneri LB92 on the fermentation quality of oat silage

[0062] Note: ND means not detected.

[0063] It can be seen from Tables 5 and 6 that compared with the control group, the test group with the addition of Lactobacillus buchneri LB95 significantly improved the fermentation quality of oat silage, whether in a high-temperature fermentation environment of 45°C or a normal-temperature fermentation environment of 25°C.

[0064] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A strain of Lactobacillus buchneri LB92 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 10, 2024, and its deposit number is CGMCC NO.30296.

2. Application of Lactobacillus buchneri LB92 according to claim 1 in inhibiting mold growth in a lactic acid bacteria fermentation environment.

3. The use according to claim 2, Features: The molds include Aspergillus fumigatus and Fusarium verticillium.

4. A silage additive, Features: The active ingredient of the silage additive includes the Lactobacillus buchneri LB92 according to claim 1.

5. A silage, Features: The silage contains the Lactobacillus buchneri LB92 according to claim 1.

6. The silage according to claim 5, Features: The silage raw material to be fermented is mixed with the silage feed additive according to claim 4 to prepare the silage feed, wherein the ratio of Lactobacillus buchneri LB92 to the silage raw material in the silage feed additive is 3×10 7 cfu:1kg.

7. The silage according to claim 6, Features: The silage raw material is oats.

8. The silage according to claim 7, Features: The oats include lodging oats.

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