Lactobacillus brevis strain and application thereof

Abstract

The invention provides a lactobacillus brevis strain HBUR51234 as well as a screening method and an application method of the lactobacillus brevis strain HBUR51234. The lactobacillus brevis strain HBUR51234 is specifically the lactobacillus brevis HBUR51234, and the preservation number of the lactobacillus brevis strain HBUR51234 is GDMCC (Graphics Database Microorganism Center) No.65258. The lactobacillus brevis strain HBUR51234 is a lactobacillus brevis strain HBUR51234. The lactobacillus brevis strain HBUR51234 is a lactobacillus brevis strain HBUR51234. The strain can improve the quality and nutritional ingredients of the alfalfa silage, effectively optimizes the storage conditions of the silage, and has the effects of increasing the number of lactic acid bacteria and inhibiting growth of mould, saccharomycetes and some other pathogenic bacteria. According to the method, the agricultural measure requirement that alfalfa is harvested in a field and is directly subjected to silage production can be met, the variety of alfalfa silage is increased, the problems of preparation of high-moisture alfalfa silage and seasonal forage gaps of herbivores in winter and spring are effectively solved, and meanwhile, the resource recycling channel of low-grade and inferior golden-silk jujubes is expanded.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a short-growing Lactobacillus strain and its screening and application. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is a perennial, high-quality forage grass belonging to the legume family. Its high nutritional value makes it widely used as a key protein source in ruminant feed systems, earning it the reputation of "King of Forage." However, during silage processing, alfalfa exhibits several characteristics that are detrimental to silage production: Firstly, alfalfa plants have high protein content and buffering capacity, meaning they are highly resistant to pH changes, which hinders the rapid establishment of an acidic environment in the early stages of silage fermentation. Secondly, alfalfa plants have low sugar content, providing relatively insufficient substrate for lactic acid bacteria fermentation, and the number of naturally attached lactic acid bacteria is small, making it difficult for them to quickly become the dominant flora and initiate the fermentation process under natural silage conditions. These factors make it difficult to produce high-quality silage when alfalfa is directly ensiled.

[0003] To address the challenges of alfalfa silage production, semi-dry (low moisture content, typically 40%–65%) silage technology is currently widely used. This technology involves drying and wilting the raw materials to reduce their moisture content to a suitable range, thereby minimizing the growth of harmful microorganisms and increasing the success rate of silage. However, while this semi-dry (low moisture) silage technology can produce high-quality silage to some extent, it still has significant drawbacks in practical application. For example, the drying and wilting process is highly dependent on weather conditions. Rainy weather can disrupt the drying process, and prolonged drying can lead to leaf drop and nutrient loss. Furthermore, rainwater can wash away nutrients during drying, further reducing its nutritional value and increasing silage costs and quality risks. In addition, existing technologies are ineffective at treating high-moisture (70%–80% moisture content) alfalfa during silage fermentation, failing to effectively inhibit the growth of harmful microorganisms and easily leading to mold and spoilage of the silage, affecting its palatability and nutritional value.

[0004] Therefore, developing a lactic acid bacteria additive that can be effectively applied to the fermentation of high-moisture alfalfa silage is of great practical significance and has an urgent market demand for solving the alfalfa silage problem, improving silage quality, reducing production costs, and improving preservation quality. Summary of the Invention

[0005] The purpose of this invention is to provide a short-growing Lactobacillus strain HBUR51234 and its application in the preparation of alfalfa silage, providing a lactic acid bacteria additive for the silage of high-moisture alfalfa.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A short-growing Lactobacillus strain, HBUR51234, specifically, is *Lactobacillus short-growing* (… Levilactobacillus brevis HBUR51234, deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 14, 2024, with accession number GDMCC No. 65258.

[0007] The above-mentioned short-growing Lactobacillus strains ( Levilactobacillus brevis The biological characteristics of HBUR51234 are as follows: The optimal growth temperature is 37±1℃. It is an anaerobic or facultative anaerobic culture. On MS medium, the colonies are milky white, opaque, round, thin at the edge and thick in the middle. The growth time is 48~72 hours, and the colony size is 3~5mm. The bacterial cells are rod-shaped, 3~5μm×4~5μm in size, Gram-positive, and stain purple with Gram stain.

[0008] The present invention also provides the application of the above-mentioned short-growing Lactobacillus strain HBUR51234 in the preparation of alfalfa silage.

[0009] Furthermore, the raw materials for the alfalfa silage also contain jujube powder, preferably at a content of 4%.

[0010] Furthermore, the application includes the following steps: The short-growing Lactobacillus strain HBUR51234 was prepared into a bacterial suspension, sprayed onto the silage raw material, vacuum-packed, and stored at room temperature for silage fermentation.

[0011] Furthermore, the bacterial suspension is prepared by the following method: Activated Lactobacillus short-lived growth factor HBUR51234 was inoculated into MRS liquid medium and cultured in a shaker at 37±1℃ and 200±50 r / min for 45~48h to obtain the bacterial suspension.

[0012] Furthermore, the effective viable count of the bacterial suspension is ≥5×10⁻⁶. 8 CFU / mL.

[0013] Furthermore, the inoculation viable count of the short-growing Lactobacillus strain HBUR51234 was 5 × 10⁻¹. 7 cfu / g silage raw materials.

[0014] The present invention also provides a lactic acid bacteria additive, the effective ingredient of which includes the above-mentioned short-growing Lactobacillus strain HBUR51234.

[0015] The beneficial effects of this invention are as follows: This invention provides a short-growing Lactobacillus strain HBUR51234, which is particularly suitable as a lactic acid bacteria agent for the preparation of alfalfa silage with high moisture content (70-80%), and its effect is even better when a small amount of jujube powder is added to the alfalfa raw material.

[0016] Specifically, the short-growing Lactobacillus strain HBUR51234 described in this invention can improve the quality and nutritional composition of alfalfa silage, effectively improve the storage conditions of silage, such as increasing the lactic acid and acetic acid content of alfalfa silage to obtain a better lactic acid / acetic acid ratio; reducing the cellulose content and loss of alfalfa dry matter in alfalfa silage, retaining more nutrients, increasing palatability for feed animals, etc., and has the effect of increasing the number of lactic acid bacteria and inhibiting the growth of molds, yeasts and some other pathogens.

[0017] This invention will meet the agricultural demand for direct silage production from alfalfa harvested in the field. It not only increases the variety of alfalfa silage feed, effectively solves the problems of high-moisture alfalfa silage preparation and seasonal forage shortage for herbivores in winter and spring, but also expands the reuse channels for low-grade and inferior jujubes. Attached Figure Description

[0018] Figure 1 This is a colony diagram of the growth of some lactic acid bacteria strains isolated and purified in Example 1 on MRS solid medium.

[0019] Figure 2 The growth curves of the top 10 fastest-growing lactic acid bacteria strains in MRS liquid medium are shown.

[0020] Figure 3 The graph shows the acid production rate curves of the top 10 lactic acid bacteria strains.

[0021] Figure 4 This is a salt tolerance gradient diagram of the top three lactic acid bacteria strains with the best growth performance.

[0022] Figure 5 The image shows the antibacterial effect of the short-growing Lactobacillus strain HBUR51234 against Serratia marcescens and holly powdery mildew. Figure 5 A represents the inhibitory effect of the short-growing Lactobacillus strain HBUR51234 on Serratia marcescens; Figure 5 B represents the application of *Lactobacillus subtilis* strain HBUR51234 before the application of *Lactobacillus subtilis* bacterial solution; Figure 5 C represents the application of *Lactobacillus subtilis* bacterial solution (HBUR51234) after treatment with holly white powder.

[0023] Figure 6Phylogenetic tree of 16S rRNA genes of the short-growing Lactobacillus strain HBUR51234 and its reference type strain.

[0024] Figure 7 The images show the bacterial cells (left) and colony morphology (right) of the short-growing Lactobacillus strain HBHBUR51234. Detailed Implementation

[0025] The following embodiments further illustrate the present invention in detail, but do not limit the invention in any way.

[0026] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0027] The screening of the short-growing Lactobacillus strain HBUR51234 of this invention includes primary screening, secondary screening, and strain identification of lactic acid bacteria strains.

[0028] Example 1: Initial screening of lactic acid bacteria strains 1. Preparation of silage samples Alfalfa planted in the vicinity of the 27th Brigade, Third Division, Nandagang District, Huanghua City, Cangzhou City, Hebei Province, was harvested when it reached the initial budding stage. Treatments were carried out with and without jujube powder, with the jujube powder treatment involving the addition of 4% dried jujube powder. The silage was vacuum-packed and made into alfalfa silage. Samples were taken at 0, 3, 5, 15, 30, 45, and 60 days of silage fermentation to obtain silage samples.

[0029] 2. Isolation of lactic acid bacteria (a) Weigh 10g of silage sample and quickly place it into an Erlenmeyer flask containing 90ml of sterile water and glass beads, then seal the flask. Place the flask on a shaker and shake for 20 minutes to prepare a 10-fold diluted bacterial suspension. Subsequent serial dilutions are then performed to prepare 10-fold diluted suspensions. -2 ~10 -9 A series of diluted bacterial suspensions; (b) Plate spreading isolation: Pipette 0.2 mL of bacterial suspension from each dilution tube onto an MRS solid medium plate, spread evenly using a sterile spreader, repeating 3 times per group, and label each plate. Place the petri dishes on a horizontal surface and let them stand for about 20 minutes to allow the bacterial suspension to be fully absorbed; then, according to the growth requirements of the strain, invert the medium and incubate it in each incubator. After 48 hours, observe the growth of the colonies.

[0030] The MRS solid culture medium formula is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g K2HPO4, 2g diammonium hydrogen citrate, 5g sodium acetate, 1mL Tween 80, 0.58g MgSO4·7H2O, 0.25g MnSO4·4H2O, 18g agar powder, and 1000mL distilled water (adjusted to pH 6.2~6.6, sterilized at 115℃ for 20 min).

[0031] 3. Purification and preservation of bacterial strains Typical single colonies exhibiting differences in colony morphology were selected and purified using either the streak plate method or the dilution method to obtain single colonies. The purified single colonies were inoculated into short and long agar slant media and cultured until the bacterial growth was complete. The bacterial growth was then washed with 20% glycerol liquid medium to prepare glycerol tubes. The agar slant cultures were stored at 4°C. Store the glycerin tubes in a -80°C freezer for later use.

[0032] Through the above isolation and purification process, a total of 90 strains were initially screened. The lactic acid bacteria colonies were milky white or grayish white, with smooth surfaces, regular edges, and varied in size. Figure 1 The image shown is a colony image of some lactic acid bacteria strains grown on MRS solid medium.

[0033] Example 2: Secondary screening of lactic acid bacteria The strains obtained from the initial screening were subjected to tests on growth curves, acid production rates, salt tolerance, acid and alkali tolerance, and antibacterial activity. Based on these indicators, the top 3-10 strains were selected.

[0034] Growth curve determination of lactic acid bacteria strains (1) Activation of strains: Take the lactic acid bacteria glycerol tube strain obtained from the initial screening, transfer it to a plate medium, and incubate it upside down in an incubator at 37℃±1℃; after 48 hours of incubation, pick the colonies growing on the plate and inoculate them onto the slant medium by streak, and continue to incubate them upside down in an incubator at 37℃±1℃; after 48 hours of slant culture, observe the growth of the bacterial growth, and use it for use if it is qualified. (2) Preparation of seed fermentation broth: One loop of bacteria was picked from the slant of lactic acid bacteria obtained from the initial screening and inoculated into 100 ml of MRS liquid medium and cultured for 24 h. (3) Determination of the growth curve of lactic acid bacteria: The seed fermentation broth of lactic acid bacteria was inoculated into MRS liquid medium at an inoculation rate of 5% and cultured at 37℃±1℃. During the culture period, 5 mL of fermentation broth of different strains was taken every 2 hours. With MRS liquid medium as a blank control, the absorbance of the fermentation broth at each time point was measured using a UV spectrophotometer at a wavelength of 600 nm. The growth curve of lactic acid bacteria was plotted with different time points as the abscissa and the absorbance value at the corresponding time point as the ordinate.

[0035] The growth curves of the top 10 fastest-growing lactic acid bacteria in MRS liquid medium are shown below. Figure 2 As shown, most strains grow rapidly after the start of fermentation, entering the logarithmic phase from 2 hours onwards, and entering the stationary phase after 12 hours of fermentation. Thereafter, the growth rate slows down, and the OD... 600 The numerical change was small, even after 22 hours of fermentation, OD 600 The value decreases. Some strains grow slowly after fermentation begins, with a relatively long lag phase, but after a period of cultivation, they will enter the logarithmic growth phase at different times, and then the growth rate will slow down and enter the stationary phase.

[0036] Determination of acid production rate of lactic acid bacteria strains The fermentation broth of lactic acid bacteria obtained from the initial screening was inoculated into MRS liquid medium at an inoculum volume of 5%. The inoculated medium was then incubated at 37℃±1℃ for 24 hours. During the incubation period, 5 mL of fermentation broth from different strains was taken every 2 hours to determine the pH value. The acid production rate curve of the lactic acid bacteria was plotted with different time points as the x-axis and the corresponding pH value at the y-axis.

[0037] The initial pH of the MRS liquid medium used to culture different lactic acid bacteria strains was 6.4. After 24 hours of culture, the pH decrease trend of the strains was generally consistent. The pH change curves of the top 10 lactic acid bacteria strains with the highest acid production rates are shown in the figure. Figure 3 It can be seen that the acid production rate of different lactic acid bacteria increased from 0 to 6 hours, and the pH value decreased rapidly. From 6 to 14 hours, the pH value continued to decrease and eventually stabilized. Among them, 10 lactic acid bacteria strains had a pH value below 4.00 and had a good acid production rate.

[0038] Determination of stress resistance of lactic acid bacteria strains (1) Salt tolerance test MRS liquid culture media containing different NaCl mass fractions (1%, 3%, 5%, 7%, 9%) were prepared. Seed fermentation broth of lactic acid bacteria was inoculated into each MRS liquid culture medium at an inoculation rate of 5%. The media were incubated at 37℃±1℃ for 24 h, and the absorbance was measured using a UV spectrophotometer at a wavelength of 600 nm.

[0039] Salt tolerance tests were conducted on the lactic acid bacteria strains obtained from the initial screening. The OD values ​​of the lactic acid bacteria under different NaCl concentrations were measured. 600 The salt tolerance value is used to test its salt resistance. Figure 4 It can be seen that the growth trend of each strain gradually weakens with the continuous increase of NaCl concentration. When the NaCl concentration reaches above 7%, the growth of the strains becomes very slow; when the NaCl concentration reaches 9%, they cannot grow at all. In comparison, strains HBU51234, HBU51284, and HBU51285 have higher salt tolerance than other strains, with strain HBU51234 showing the best performance, surviving at a NaCl concentration of 9%.

[0040] (2) Acid and alkali resistance test MRS liquid culture media with different pH values ​​(3, 4, 5, 6, 8, 9, 10) were prepared. The seed fermentation broth of lactic acid bacteria was inoculated into each MRS liquid culture medium at a 5% inoculum. The cultures were incubated at 37℃±1℃ for 24 h, and the absorbance was measured using a UV spectrophotometer at a wavelength of 600 nm.

[0041] Acid and alkali resistance tests revealed that most strains were relatively acid-resistant, reaching a pH of 3.0, but had poor tolerance to alkalis, failing to grow at a pH of 8.0.

[0042] Detection of antibacterial activity of lactic acid bacteria strains (1) Indicator bacteria: Fusarium graminearum ( Fusarium graminearum ), Botrytis cinerea ( Botrytis cinerea ) and Serratia marcescens ( Serratia marcescens ); (2) Preparation of lactic acid bacteria metabolites The strains obtained from the initial screening were inoculated onto MRS solid medium for activation. Single colonies were picked and inoculated onto MRS solid slant medium and incubated statically at 37℃±1℃ for 2–5 days. One loopful of bacterial growth was picked and transferred to MRS liquid medium and incubated at 37℃±1℃ and 200 rpm for 24 h. 1 mL of the above liquid culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 10 min. The supernatant was aspirated with a sterile syringe and filtered through a 0.22 μm bacterial filter to obtain sterile filtrate, which was stored at 4℃ for later use. (3) Preparation of indicator bacteria and antibacterial test Using a sterilized scalpel, cut approximately 0.5 cm diameter cubes of *Fusarium graminearum* and *Botrytis cinerea* colonies. Place the side with mycelium on a solid PDA agar plate and incubate at 25°C for 3-5 days. Use a sterile bamboo skewer to pick up a small amount of mycelium into a sterile test tube, add a small amount of sterile water, stir to release the spores, and filter through sterile absorbent cotton into another sterile test tube to prepare a spore suspension. Adjust the OD... 600 Adjust the concentration to 0.6-0.8. Add the spore suspension to the unconsolidated PDA solid medium at a ratio of 1:50, mix well, and then pour the mixture onto a plate. PDA medium: 200g potato, 20g sucrose (or glucose), 15-20g agar, 1000ml water, adjust pH to 6.2-6.6, sterilize at 115℃ for 20 min.

[0043] (4) Preparation of bacterial suspension Serratia marcescens was transferred to NA solid medium and incubated at 37℃±1℃ for 1-2 days; then inoculated into 10mL NA liquid medium and incubated at 37℃±1℃ and 200rpm for 24h. The bacterial suspension was then adjusted to OD using 10mL of uninoculated NA liquid medium. 600 Adjust the concentration to 0.6-0.8. Add the bacterial suspension to the unconsolidated NA solid medium at a ratio of 1:50, mix well, and then pour the mixture onto a plate. NA liquid culture medium: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, pH 7.2~7.4, sterilized at 115℃ for 20 min.

[0044] (5) Antimicrobial test of lactic acid bacteria metabolites Perforated plates were made in PDA medium containing spore suspension or NA medium containing bacterial suspension, with MRS liquid medium as a blank control. 100 μL of fermentation supernatant of the lactic acid bacteria strain obtained from the initial screening was added to each well. The plates were incubated at 25℃ (fungi) and 37℃±1℃ (bacteria) respectively, repeated 3 times. The antibacterial effect was observed and recorded. The lactic acid bacteria supernatant was also dropped onto holly leaves to observe its inhibitory effect on powdery mildew.

[0045] The antibacterial test results of Lactobacillus short-lived HBUR51234 are as follows: Figure 5 As shown, the supernatant of the metabolites of the short-growing Lactobacillus strain HBUR51234 has a good inhibitory effect on Serratia marcescens, with an average inhibition zone diameter of (15±0.2) mm; it has no inhibitory effect on the fungi Fusarium graminearum and Botrytis cinerea; its bacterial suspension also has a certain inhibitory effect on powdery mildew on holly leaves.

[0046] Example 3: Identification of the short-growing Lactobacillus strain HBUR51234 The 16S rRNA gene sequence of several candidate strains obtained from the secondary screening was determined. The sequences of the candidate strains were compared with the standard sequences of lactic acid bacteria in the GENE BANK database. Strains that did not belong to the lactic acid bacteria group were removed, and strains that belonged to the lactic acid bacteria group were retained to obtain the target strain.

[0047] The specific steps for 16S RNA gene sequencing and phylogenetic analysis are as follows: (1) DNA extraction: After activation by YMA slant, the strain was inoculated onto TY slant. When the slant was covered with bacterial growth, the bacterial cells were washed off the slant with sterile physiological saline and transferred to a 1.5 mL Eppendorf centrifuge tube. The bacterial cells were collected by centrifugation at 10,000 rpm for 3 min. The bacterial cells were then washed three times by centrifugation with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The bacterial cells were then repeatedly frozen and thawed to lyse the cells. 600 μL of 4 M GUTC buffer was added, and the mixture was vortexed to mix thoroughly. After standing at room temperature for 15 min, 60 μL of diatomaceous earth suspension was added, and the mixture was vortexed to mix thoroughly. After standing at room temperature for 15 min, the mixture was centrifuged at 10,000 rpm for 3 min and the supernatant was discarded. 500 μL of 4 M GUTC buffer was added, and the mixture was vortexed to mix thoroughly. After standing at room temperature for 15 min, the mixture was centrifuged at 10,000 rpm for 3 min and the supernatant was discarded. 500 μL of diatomaceous earth suspension was then added. Wash twice with washing buffer by centrifugation, add 600 μL of 75% ethanol, centrifuge once and discard the supernatant, dry the precipitate in a clean bench until the diatomaceous earth turns white; finally add 50 μL of sterile ultrapure water, incubate at 55-65℃ for 10 min, centrifuge to collect the supernatant, detect by electrophoresis and store at -20℃ for later use. The primers for 16S rRNA gene sequence analysis are as follows: Pl: 5'-AGAGTTTGArCCTGGCTCAGAACGAACGCT-3'; P6: 5'-ACGGCTACCTTGTTACGACTTCACCC-3'.

[0048] (2) PCR amplification: The amplification system consisted of 5.0 μL 10×Taq Buffer, 1.0 μL 10mM dNTPs, 1.0 μL 20μM fD1, 1.0 μL 20μM rD1, 50~100 ng template DNA, 0.3 μL Taq DNA polymerase (5U / μL), and sterile ultrapure water to a final volume of 50 μL. The PCR amplification conditions were as follows: .

[0049] (3) Detection of PCR amplification products: 3 μL of 16S rRNA gene amplification product was mixed with 0.6 μL of 6× Loading Buffer and loaded onto a 1% agarose gel plate containing EB. Electrophoresis was performed at 5V / cm. After electrophoresis, the gel was scanned and photographed in a gel imaging system to check the length and concentration of the amplified fragment. After passing the test, the sample was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rRNA gene sequence of the target strain was obtained as shown in SEQ ID No.1.

[0050] The sequence results of the target strain were compared with those of Ezbiocloud (http: / / www.ezbiocloud.net / eztaxon). A model strain with high similarity was selected as a reference strain to construct a phylogenetic tree. The results showed that the target strain was similar to *Lactobacillus brevis*. Levilactobacillus brevis The 16S rRNA gene sequence has high similarity, and its model bacterium is Levilactobacillus brevis JCM1059T showed 100% similarity to the other two sequences. Based on the alignment results on Ezbiocloud, a phylogenetic tree of the 16S rRNA gene was constructed using Mega 7.0 software and the maximum likelihood method. The bootstrap value was set to 1000, and the substitution frequency at each site was 1%. The phylogenetic tree is shown below. Figure 6 .

[0051] The above results confirm that the target strain is *Lactobacillus brevis*, which was sent to the Guangdong Provincial Microbial Culture Collection Center on October 14, 2024, with accession number GDMCC No. 65258, and classified as *Lactobacillus brevis*. Levilactobacillus brevis HBUR51234, the address of which is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0052] Crystal violet was used to screen the target strain of Lactobacillus short-lived (Lactobacillus brevis). Levilactobacillus brevis After simple staining, HBUR51234 was observed under an optical microscope, and its biological characteristics are as follows: The optimal growth temperature is 37℃±1℃. It is an anaerobic or facultative anaerobic culture. On MS medium, it forms 3-5 mm, opaque, round, milky-white colonies within 48-72 hours, with thin edges and a thicker center. The bacteria are rod-shaped, measuring 3-5 μm × 4-5 μm, and are Gram-positive, staining purple with Gram stain. Figure 7 ).

[0053] Example 4: Application of the short-growing Lactobacillus strain HBUR51234 in alfalfa silage. 1. Fermentation of the short-growing Lactobacillus strain HBUR51234 The tested short-growing Lactobacillus strain HBUR51234 was transferred and preserved on MRS solid medium slant. After culturing at 37℃±1℃ for 2-5 days, a loopful of bacteria was picked and inoculated into a fermentation tube containing 10 mL of MRS liquid medium. The tube was then incubated at 37℃±1℃ and 200 r / min on a shaker for 24 h. Then, 5 mL of bacterial suspension was taken from the fermentation tube and inoculated into 95 mL of MRS liquid medium. The tube was then incubated at 37℃±1℃ and 200 r / min on a shaker until the logarithmic growth phase. Each strain was replicated in triplicate.

[0054] 2. Determination of viable lactic acid bacteria count Add 1 mL of bacterial suspension to a test tube containing 9 mL of sterile water, aspirate and shake to mix thoroughly, and prepare 10... -1 Diluent, then use a pipette to draw 10 ml of the solution. -1 Transfer 1 mL of the diluent into a test tube containing 9 mL of sterile water, and prepare the solution by pipetting and shaking. 10 -2 The diluent is diluted continuously in this manner until a final solution of 10 is prepared. -3 10 -4 10 -5 10 -6 10 -7 A series of diluted bacterial suspensions were prepared, and 0.1 mL of each suspension was added to MRS medium and spread for incubation. The culture was then incubated at 37℃±1℃ for 48 h before counting. The formula for calculating the viable cell count is as follows: ; Where C represents the average colony count at a certain dilution concentration, V represents the volume (mL) of diluent used when plating, and M represents the dilution factor.

[0055] After determining the viability count, prepare 5×10⁵ cells / day of the solution according to the specified quantity. 8 A bacterial suspension of cfu / mL.

[0056] 3. Inoculation with lactic acid bacteria and packaging of silage. Alfalfa at the initial flowering stage was harvested and divided into two groups: sun-dried and non-sun-dried. The moisture content of each group was measured. The non-sun-dried group had a moisture content of 80% (hereinafter referred to as high moisture), while the sun-dried group had a moisture content of 65% (hereinafter referred to as semi-dry). The alfalfa was then chopped into 2-3 cm long pieces using a chaff cutter. A treatment was also included in the non-sun-dried group with the addition of 4% feed meal. A specific dose of a short-growing Lactobacillus strain HBUR51234 suspension was inoculated into each silage. The inoculation dose of HBUR51234 was one times the number of viable bacteria per gram of silage. 7CFU. The bacterial suspension was thoroughly mixed with alfalfa segments, or with a mixture of alfalfa segments and jujube powder, and then packed into silage bags measuring 210 mm × 297 mm. After vacuum sealing, fermentation was carried out at room temperature. After 60 days, the bags were opened and samples were taken. An equal amount of sterile water was added to the control group. At the same time, commercial lactic acid bacteria agents such as "Yiran", "Raman" and "Yiqing No. 2" were used for inoculation according to their dosage to carry out comparative experiments.

[0057] Example 5: Determination of Physicochemical Indicators of Silage The alfalfa silage samples obtained in Example 4 were subjected to silage quality testing, including determination of dry matter content, carbohydrates, pH, organic acids (lactic acid, acetic acid, butyric acid, propionic acid), and ammonia nitrogen. The specific testing methods are as follows: Take 10 g of fresh alfalfa silage sample, add 90 mL of sterile water and stir well. After homogenizing with a tissue homogenizer for 1 min, filter through four layers of gauze and then through qualitative filter paper. Collect the leachate for relevant index determinations: pH value was measured using a pH meter; ammonia nitrogen (NH3-N) and total nitrogen (TN) were determined using a Kjeldahl nitrogen analyzer; lactic acid (LA), acetic acid (AA), and butyric acid (BA) contents were determined by HPLC using a KC-811 ion chromatography column at 50℃, with a mobile phase of 3 mmol / L HClO4 solution and an injection volume of 5 μL; the remaining sample was dried at 105℃ to determine dry matter (DM) content using the constant mass method; water-soluble carbohydrates (WSC) content was determined using the anthrone-sulfuric acid colorimetric method; crude protein content was calculated as total nitrogen × 6.2; acid detergent fiber content was determined by... The content of fiber (ADF) and neutral detergent fiber (NDF) was determined using a cellulose analyzer.

[0058] (1) Dry matter (DM%) data (Table 1) obtained by constant weight determination after drying showed that the dry matter content of alfalfa silage under different treatment conditions after inoculation with the short-growing Lactobacillus strain HBUR51234 was higher than that of the control, and there was no significant difference in dry matter content between the two inoculated with commercial strains. The dry matter content of silage made from alfalfa with 80% moisture content after inoculation with the short-growing Lactobacillus strain HBUR51234 was higher than that of the commercial strains "Yiran" and "Yiqing". Under the same high moisture conditions, the dry matter content of alfalfa silage after the addition of jujube powder was higher than that of the corresponding treatment without jujube powder.

[0059] Table 1. Statistical table of mean dry matter content (60 days) of alfalfa silage (2) The results of the determination of soluble carbohydrates (Table 2) show that the soluble carbohydrate content of alfalfa silage under different treatment conditions after inoculation with Lactobacillus brevis strain HBUR51234 is higher than that of the control. The soluble carbohydrate content of silage made from alfalfa with 80% moisture content after inoculation with Lactobacillus brevis strain HBUR51234 is higher than that of silage inoculated with the commercial strain "Yiran"; the soluble carbohydrate content of silage made from alfalfa / jujube powder with 80% moisture content after inoculation with Lactobacillus brevis strain HBUR51234 is higher than that of silage inoculated with the commercial strains "Yiran" and "Raman". Under the same high moisture conditions, the dry matter content of alfalfa silage after the addition of jujube powder is higher than that of the corresponding treatment without jujube powder, and can even reach the level comparable to that of semi-dry alfalfa silage.

[0060] Table 2. Statistical Table of Mean Soluble Carbohydrate Values ​​in Alfalfa Silage (3) The pH measurement results (Table 3) show that the pH value of silage under the three treatment conditions was lower than that of the control and the commercial inoculum after inoculation with the short-growing Lactobacillus strain HBUR51234. The effect remained unchanged after the addition of jujube powder raw material.

[0061] Table 3. Statistical table of average pH values ​​for alfalfa silage (4) Determination of crude fiber As shown in Table 4, without the addition of any microbial agent, the neutral detergent fiber (NDF) content in silage decreased with increasing alfalfa raw material moisture content, indicating that high moisture content in alfalfa silage is not conducive to NDF production. However, under the same high moisture conditions, the NDF content of alfalfa silage alone after adding the short-growing Lactobacillus strain HBUR51234 of this invention was higher than that of the control or some commercial microbial agent treatments. The addition of jujube powder reduced the NDF content, which may be related to the nutritional composition of jujube powder.

[0062] Table 4. Statistical Table of Mean Neutral Detergent Fiber (NDF, %DM) in Alfalfa Silage As shown in Table 5, without the addition of any microbial agents, the acid detergent fiber content in silage increases with the increase of moisture content in alfalfa raw materials. However, after inoculation with the *Lactobacillus short-lived* HBUR51234 microbial agent of this invention, the acid detergent fiber content decreases. The addition of jujube powder slightly increases the acid detergent fiber content, possibly due to the sugar content in jujube powder. Furthermore, the silage produced from high-moisture alfalfa raw materials inoculated with the *Lactobacillus short-lived* HBUR51234 microbial agent of this invention is superior to that produced by commercial microbial agents "Yiran" and "Yiqing".

[0063] Table 5. Statistical table of mean values ​​of acid detergent fiber (ADF, %DM) in alfalfa silage (5) Determination of crude protein As shown in Table 6, without any added microbial agents, the crude protein content in silage decreased with decreasing moisture content of alfalfa raw materials, indicating that moisture is beneficial for the preservation of crude protein. When high-moisture alfalfa raw materials were inoculated with lactic acid bacteria, the crude protein content of the silage decreased, indicating that microbial growth consumes some crude protein. However, in silage treated with semi-dry conditions and those with added jujube powder, the crude protein content after inoculation with the *Lactobacillus brevis* HBUR51234 of this invention was higher than that after inoculation with other commercial microbial agents or without any added microbial agents, indicating that the *Lactobacillus brevis* HBUR51234 of this invention is more effective than commercial strains.

[0064] Table 6. Statistical Table of Mean Crude Protein (%DM) in Alfalfa Silage (6) Determination of crude fat The lower the crude fat content in silage, the better it is for reducing fat accumulation in fed animals. As shown in Table 7, without the addition of any microbial agents, the crude fat content in silage increases with the increase of moisture content in alfalfa raw materials, indicating that the moisture content in alfalfa raw materials is beneficial for preserving the crude fat content of silage. Although inoculation with lactic acid bacteria agents increases the corresponding crude fat content of silage, under high moisture conditions in alfalfa raw materials, the crude fat content of silage inoculated with the *Lactobacillus brevis* HBUR51234 strain of this invention is significantly lower than that of commercial strains, indicating that the *Lactobacillus brevis* HBUR51234 strain of this invention is more effective than commercial strains.

[0065] Table 7. Statistical Table of Mean Crude Fat (%DM) in Alfalfa Silage (7) Determination of organic acids Lactic acid is an important indicator of alfalfa silage quality, and its content reflects the overall quality of the silage. Under three treatment conditions, silage inoculated with the *Lactobacillus short-growing* strain HBUR51234 showed significantly higher lactic acid content than the control and commercial inoculum treatments. Particularly under high moisture conditions in alfalfa feed, the lactic acid content of silage inoculated with HBUR51234 was significantly higher than that inoculated with commercial inoculum. Furthermore, under high moisture conditions in alfalfa feed, the lactic acid content of silage with the addition of jujube meal was significantly higher than that without jujube meal. These results indicate that, in terms of lactic acid production, the *Lactobacillus short-growing* strain of this invention is superior to commercial strains.

[0066] Table 8. Statistical Table of Mean Lactic Acid (%DM) in Alfalfa Silage Acetic acid is crucial in the later stages of silage production and plays a vital role in the long-term preservation of silage microorganisms. An appropriate acetic acid content is beneficial for silage preservation and may be related to heterotrophic fermentation by lactic acid bacteria. As shown in Table 9, without any added microbial agents, the acetic acid content in silage increases significantly with increasing moisture content of alfalfa raw materials. Under high moisture conditions, inoculation with lactic acid bacteria reduces the acetic acid content in silage. However, after inoculating semi-dry alfalfa raw materials with the *Lactobacillus short-lived* HBUR51234 microbial agent of this invention, the acetic acid content of the silage is higher than that inoculated with other commercial microbial agents, but the overall quantity is significantly lower than that under high moisture conditions.

[0067] The above results indicate that inoculation with *Lactobacillus shortii* and the addition of jujube powder plays an important role in the preservation of silage and can be promoted as a model. The inoculation results are shown in Table 9.

[0068] Table 9. Statistical Table of Mean Values ​​of Acetic Acid (%DM) in Alfalfa Silage As shown in Table 10, no propionic acid was produced in the silage prepared from alfalfa raw materials under semi-dry conditions. However, the propionic acid content increased with the increase of the moisture content of alfalfa raw materials. After inoculation with lactic acid bacteria agents, the content was significantly lower than that of the control, and was significantly lower than that of commercial agents "Yiran", "Yiqing" and "Raman".

[0069] Table 10. Statistical Table of Mean Propionic Acid (%DM) in Alfalfa Silage Butyric acid is the main volatile acid that contributes to the flavor of silage. Appropriate butyric acid levels promote animal feed intake and increase palatability, but excessive levels can negatively impact the stability and nutritional value of silage. Table 11 shows that no butyric acid is produced in silage prepared from semi-dry alfalfa raw materials. However, the butyric acid content increases with the moisture content of the alfalfa raw materials, especially after inoculation with lactic acid bacteria. However, compared to silage inoculated with commercial inoculants "Yiran," "Yiqing," and "Raman," the butyric acid content in silage inoculated with the *Lactobacillus short-growing* strain HBUR51234 of this invention is significantly lower (except for the treatment with added jujube powder and inoculation with lactic acid bacteria, where the butyric acid content was slightly higher than with commercial inoculants). In summary, this demonstrates that adding the *Lactobacillus short-growing* strain HBUR51234 of this invention has a good control effect on the production of propionic and butyric acids.

[0070] Table 11. Statistical Table of Mean Values ​​of Alfalfa Silage Butyric Acid (%DM) (8) Ammonia nitrogen is an indicator of silage spoilage; the lower the ammonia nitrogen content, the better the silage quality. As shown in Table 12, without the addition of any microbial agents, the ammonia nitrogen content in silage increases significantly with the increase of alfalfa raw material moisture content. When alfalfa raw material is in a semi-dry state, inoculation with commercial lactic acid bacteria agents will significantly reduce the ammonia nitrogen content in silage. Under high moisture conditions of alfalfa raw material, even with inoculation with commercial lactic acid bacteria agents, the ammonia nitrogen content is still higher than under semi-dry conditions. Under the same high moisture conditions of alfalfa raw material, after inoculation with the *Lactobacillus short-growing* strain HBUR51234 of this invention, the ammonia nitrogen content of silage is reduced and lower than the control. After adding jujube powder to alfalfa silage raw material and inoculating with the *Lactobacillus short-growing* strain HBUR51234 of this invention, the ammonia nitrogen content in silage is also significantly reduced and lower than that inoculated with commercial microbial agents "Yiran", "Yiqing", and "Raman".

[0071] The above results indicate that inoculation with the short-growing Lactobacillus agent of the present invention and the addition of jujube powder improve the quality of silage. This provides a method for improving the quality of silage from high-moisture alfalfa raw materials and can be promoted as a model.

[0072] Table 12. Statistical table of mean ammonia nitrogen (DM%) in alfalfa silage The test results of crude ash content are shown in Table 13. It can be seen that the crude ash content of silage after inoculation with the Lactobacillus short-life HBUR51234 of the present invention is not much different from that after inoculation with commercial inoculum. In alfalfa high-moisture silage, the ash content is reduced after inoculation with the Lactobacillus short-life HBUR51234 of the present invention, and its effect is better than that of inoculation with commercial inoculum.

[0073] Table 13. Statistical Table of Average Coarse Ash Content in Alfalfa Silage In summary, through the determination of nutritional and physicochemical properties of alfalfa and silage with added jujube powder, it was confirmed that the addition of the short-growing-promoting Lactobacillus strain HBUR51234 of this invention significantly improved important indicators such as lactic acid content, pH value, and ammonia nitrogen content in the silage compared to the control and three commercial inoculants. Other indicators, such as crude protein, crude fat, dry matter, and crude fiber content, were also comparable to those of commercial inoculants. This indicates that the short-growing-promoting Lactobacillus strain HBUR51234 of this invention has reached or even surpassed the effects of commercial inoculants. Especially under high moisture conditions (80%) in alfalfa raw materials, the various indicators of the silage after adding jujube powder were comparable to those of silage under semi-dry conditions (65% moisture), and some indicators were even better than those of silage under semi-dry conditions. This further demonstrates that the short-growing-promoting Lactobacillus strain HBUR51234 of this invention is an excellent lactic acid bacteria strain and can be used as a lactic acid bacteria additive for silage fermented with alfalfa / jujube powder.

Claims

1. A short-growing Lactobacillus strain, HBUR51234, characterized in that, It is a short-lived lactobacillus ( Levilactobacillus brevis HBUR51234, deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 14, 2024, with accession number GDMCCNo. 65258.

2. The application of the short-growing Lactobacillus strain HBUR51234 as described in claim 1 in the preparation of alfalfa silage.

3. The application according to claim 2, characterized in that, The raw materials for the alfalfa silage also contain jujube powder.

4. The application according to claim 3, characterized in that, The content of the jujube powder is 4%.

5. The application according to any one of claims 2-4, characterized in that, The process includes the following steps: preparing the short-growing Lactobacillus strain HBUR51234 into a bacterial suspension, spraying it onto the silage raw material, vacuum packaging it, and storing it at room temperature for silage fermentation.

6. The application according to claim 5, characterized in that, The bacterial suspension was prepared by the following method: the activated short-growing Lactobacillus strain HBUR51234 was inoculated into MRS liquid medium and cultured in a shaker at 37±1℃ and 200±50r / min for 45~48h. The bacterial suspension was obtained by collection and concentration adjustment.

7. The application according to claim 5, characterized in that, The effective viable bacteria count of the bacterial suspension is ≥5×10⁻⁶. 8 CFU / mL.

8. The application according to claim 5, characterized in that, The inoculation count of the short-growing Lactobacillus strain HBUR51234 was 5 × 10⁻¹. 7 cfu / g silage raw materials.

9. A lactic acid bacteria additive, characterized in that, The active ingredient includes the short-growing Lactobacillus strain HBUR51234 as described in claim 1.

Images