Lactobacillus paracasei strain and application thereof
By using a combination of Lactobacillus paracasei strain HBUR51326 and jujube powder, the problem of high-moisture alfalfa silage was solved, the quality and nutritional value of silage were improved, production costs were reduced, and the production scale was expanded.
Patent Information
- Application Number
- CN202511452007.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2025-12-16
AI Technical Summary
Alfalfa is difficult to quickly establish an acidic environment during silage under high moisture conditions, making it difficult for lactic acid bacteria to become the dominant flora, resulting in poor silage quality. Furthermore, existing semi-dry silage technology is greatly affected by weather, has high costs, and is prone to mold growth.
Lactobacillus paracasei strain HBUR51326 was used as a fermentation agent for silage. It was applied to high-moisture alfalfa silage, and jujube powder was added. High-quality silage was prepared by vacuum packaging and room temperature fermentation.
It increases the lactic acid and acetic acid content of high-moisture alfalfa silage, increases the number of lactic acid bacteria, inhibits mold growth, improves palatability, reduces cellulose content, reduces nutrient loss, expands production scale, and reduces costs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus paracasei and its applications. 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, the high protein content and buffering capacity of alfalfa plants mean they are highly resistant to pH changes, hindering the rapid establishment of an acidic environment in the early stages of silage fermentation; secondly, the number of naturally attached lactic acid bacteria on alfalfa is relatively small, making it difficult for these bacteria 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 silage additive that can be effectively applied to alfalfa with high moisture content 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 strain of Lactobacillus paracasei, HBUR51326, and its application, so as to enrich the types of fermentation agents for alfalfa silage and solve the problem of high-moisture alfalfa silage.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application provides a lactobacillus paracasei strain HBUR51326, which is lactobacillus paracasei (LBUR51326) and is preserved in the Guangdong Microbial Culture Collection Center, 5th floor, No. 59, Guangzhou, China, on October 14, 2024, with a preservation number of GDMCC No: 65259. Lacticaseibacillus paracasei
[0007] The lactobacillus paracasei strain HBUR51326 has the following biological characteristics: the optimal growth temperature is 37℃±1℃, the culture is anaerobic or facultative anaerobic, the colony on the MS culture medium is milky white and opaque, the colony is round, the edge is thin, the middle is thick, the growth time is 48-72 hours, and the colony size is 3-4mm; the bacterial body is rod-shaped, the size is 3-4um*4-5um, it is a gram-positive bacterium, and the gram staining is purple.
[0008] The application also provides a silage fermentation agent prepared from the lactobacillus paracasei strain HBUR51326.
[0009] The application also provides a silage prepared by inoculating the raw material with the silage fermentation agent.
[0010] Further, the raw material is alfalfa or alfalfa-jujube powder.
[0011] Further, the water content of the alfalfa is 75%-80%.
[0012] Further, the water content of the alfalfa in the alfalfa-jujube powder raw material is 75%-80%.
[0013] Further, the amount of jujube powder added in the alfalfa-jujube powder raw material is 4WT%.
[0014] The preparation method of the silage includes the following steps: The lactobacillus paracasei strain HBUR51326 is prepared into a bacterial suspension, which is sprayed and inoculated into the raw material of the silage, vacuum packaged, stored at room temperature for fermentation, and the silage is obtained.
[0015] Further, the bacterial suspension is prepared by the following method: the activated lactobacillus paracasei strain HBUR51326 is inoculated into MRS liquid medium, and is cultured in a shaking bed at 37±1℃ and 200±50r / min for 45-48h, and the bacterial suspension is obtained through collection and concentration modulation.
[0016] Further, the effective viable bacterial count of the bacterial suspension is ≥5×10 8 CFU / mL.
[0017] Further, the inoculation viable cell number of the Lactobacillus paracasei strain HBUR51326 is 5*10 7 cfu / g of the silage raw material.
[0018] The application also provides application of the Lactobacillus paracasei strain HBUR51326 in preparation of a silage fermentation agent or silage.
[0019] The application has the following beneficial effects: The application provides a Lactobacillus paracasei strain HBUR51326, which is particularly suitable for fermentation silage of alfalfa with high water content (75%-80% water content), and the effect is better when a small amount of jujube powder is added to the silage raw material. The application effectively solves the problem of silage of alfalfa with high water content, helps to expand the production scale of alfalfa in the field, and provides a new way for the reuse of low-quality and inferior jujube waste resources.
[0020] The Lactobacillus paracasei strain HBUR51326 also increases the types of alfalfa silage and solves the problem of seasonal forage shortage of herbivorous animals in winter and spring. Specifically, the Lactobacillus paracasei strain HBUR51326 improves the quality and nutrient content of alfalfa silage, such as increasing the content of lactic acid and acetic acid in alfalfa silage, obtaining a good lactic acid / acetic acid ratio, which is beneficial to the preservation of silage. At the same time, the cellulose content in alfalfa silage and the loss of alfalfa dry matter are reduced, more nutrients are retained, the number of lactic acid bacteria is increased, the growth of mold, yeast and some other pathogenic bacteria is inhibited, and finally the quality of alfalfa silage is improved. Due to the presence of more nutrients and flavor substances such as lactic acid, the palatability of herbivorous animals is also improved, and their feeding amount is increased. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 Part of the lactic acid bacteria strains separated and purified in Example 1 grow on MRS solid medium.
[0022] Figure 2 The growth curve of the top 10 lactic acid bacterial strains in MRS liquid medium.
[0023] Figure 3 The acid production rate curve of the top 10 lactic acid bacterial strains with high acid production rate.
[0024] Figure 4 The salt tolerance gradient of the top 3 lactic acid bacterial strains with good growth.
[0025] Figure 5The images show the antibacterial effects of Lactobacillus paracasei strain HBUR51326 against Serratia marcescens and powdery mildew of holly. Figure A shows the inhibitory effect of Lactobacillus paracasei strain HBUR51326 (the treatments are marked in the figure) on Serratia marcescens, where the treatments are bacterial suspensions. Figure B shows the powdery mildew of holly before application of Lactobacillus paracasei strain HBUR51326. Figure C shows the powdery mildew of holly after application of Lactobacillus paracasei strain HBUR51326.
[0026] Figure 6 Phylogenetic tree of 16S rRNA genes of Lactobacillus paracasei strain HBUR51326 and its reference type strain.
[0027] Figure 7 Figure A shows the bacterial cells (Figure A) and colony morphology (Figure B) of Lactobacillus paracasei strain HBUR51326. Detailed Implementation
[0028] The following embodiments further illustrate the present invention in detail, but do not limit the invention in any way.
[0029] 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.
[0030] This invention relates to Lactobacillus paracasei ( Lacticaseibacillus paracasei The screening of HBUR51326 includes primary screening, secondary screening, and strain identification of lactic acid bacteria strains.
[0031] Example 1: Initial screening of lactic acid bacteria strains 1. Preparation of silage raw materials Alfalfa planted in the vicinity of Xuguan Town, Huanghua District, Cangzhou City, Hebei Province was harvested and treated with or without jujube powder. The amount of dried jujube powder added was 4%. The silage was vacuum-packed and fermented. Samples were taken at 0, 1, 3, 5, 15, 30, 45 and 60 days of fermentation to obtain silage samples.
[0032] 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 separation: 0.2 mL of the bacterial solution was taken from each dilution tube and spread evenly on MRS medium plates using a sterile spreader, with 3 repeats for each group, and labeled. The culture dishes were placed on a horizontal surface for about 15 min to allow the bacterial solution to be fully absorbed, and then the medium was cultured in each incubator upside down. After 48 h, the growth of the colonies was observed.
[0033] The MRS solid medium formula is as follows: 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.1 mL of Tween 80, 0.58 g of MgSO4·7H2O, 0.25 g of MnSO4·4H2O, 18 g of agar powder, and 1000 mL of distilled water (pH 6.2-6.6, sterilized at 115°C for 20 min).
[0034] 3. Purification and preservation of the bacterial strain Single colonies with different morphological characteristics were picked and subjected to plate streaking or dilution purification culture to obtain single colonies, which were inoculated into slant tube medium, and then glycerol tubes were prepared using 20% glycerol liquid medium. The slant tubes were placed in a refrigerator at 4°C, and the glycerol tubes were placed in a refrigerator at -80°C for preservation.
[0035] Through the above separation and purification, a total of 50 strains were obtained.
[0036] The colonies of the lactic acid bacteria were milky white, off-white, smooth in surface, regular in edge, and varied in size. As shown in the following table, the growth of some lactic acid bacteria strains on MRS solid medium is shown. Figure 1
[0037] Example 2: Rescreening of lactic acid bacteria The strains obtained by the preliminary screening were subjected to growth curve determination, acid production rate determination, salt tolerance determination, acid and alkali tolerance determination, and antibacterial activity determination, respectively. According to these indicators, the top 3-10 strains were screened.
[0038] 1. Growth curve determination of the lactic acid bacterial strain (1) Activation of the strain: the lactic acid bacterial strain obtained by separation and purification was transferred from the glycerol tube to a plate and cultured in an inverted manner in a 37±1°C incubator; after 48 h, the colonies grown on the plate were picked and streaked onto a slant tube, which was cultured in a 37±1°C incubator, and after 48 h, the growth of the slant was observed for standby use; (2) Preparation of the seed fermentation broth: one ring of bacteria was picked from the test tube slant and inoculated into 100 mL of MRS liquid medium, which was cultured for 24 h; (3) Determination of growth curve of lactic acid bacteria: Take the seed fermentation liquor of lactic acid bacteria, each according to 5% inoculation amount, inoculate into MRS liquid medium. Place under the condition of 37℃, cultivate, take 5mL fermentation liquor of different strains every 2h, under the condition of 600nm, use ultraviolet spectrophotometer to determine the absorbance value of fermentation liquor at different time, take MRS liquid medium as blank. Draw the growth curve, in which the horizontal coordinate is different time point, and the vertical coordinate is the corresponding absorbance value.
[0039] The growth curve of the top 10 lactic acid bacteria in MRS liquid medium is shown in Figure 2 Most of the strains grow rapidly after the start of fermentation, enter the logarithmic phase from 2h, enter the stationary phase after 12h of fermentation, after that, the growth rate slows down, the OD600 value changes little, even after 22h of fermentation, the OD600 value decreases. Some strains grow slowly after the start of fermentation, the lag phase is longer, but after a period of cultivation, they will enter the logarithmic growth phase from different time periods, and then the growth rate slows down, entering the stationary phase.
[0040] Determination of acid production rate of lactic acid bacteria strains Take the seed fermentation liquor of lactic acid bacteria, each according to 5% inoculation amount, inoculate into MRS liquid medium. Place under the condition of 37℃, cultivate for 24h, take 5mL fermentation liquor of different strains every 2h, determine the pH value of fermentation liquor, draw the acid production rate curve, in which the horizontal coordinate is different time point, and the vertical coordinate is the corresponding pH value.
[0041] As shown in Figure 3 , the initial pH value of MRS liquid medium is 6.35, after 24h of cultivation of different lactic acid strains, the pH value of lactic acid bacteria decreases consistently overall, different lactic acid bacteria strains grow rapidly in 0-6h, the pH value decreases rapidly, the pH value continues to decrease in 6-14h, and finally tends to be stable, among which 10 strains of lactic acid bacteria have pH value below 4.00, with good acid production rate.
[0042] Determination of stress resistance of lactic acid bacteria strains (1) Salt tolerance test Prepare MRS liquid medium with different NaCl mass fraction (1%, 3%, 5%, 7%, 9%), take the seed fermentation liquor of lactic acid bacteria, each according to 5% inoculation amount, inoculate into MRS liquid medium. Place under the condition of 37℃, cultivate for 24h, under the condition of 600nm, use ultraviolet spectrophotometer to determine the absorbance value.
[0043] The lactic acid bacteria strains preliminarily screened are subjected to salt tolerance test. By determining the OD600 value of lactic acid bacteria under the addition of different NaCl concentration, the salt tolerance is detected. From Figure 4It 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, and when the NaCl concentration reaches 9%, they cannot grow at all. In comparison, strains HBU51326, HBU51235, and HBU51281 have higher salt tolerance than other strains and can grow even at a NaCl concentration of 7%.
[0044] (2) Acid and alkali resistance test MRS liquid culture media with different pH values (3, 4, 6, 8, 9, 10) were prepared. Seed fermentation broth of lactic acid bacteria was inoculated into each medium at a rate of 5%. The media were then incubated at 37°C for 24 hours, and the absorbance was measured using a UV spectrophotometer at a wavelength of 600 nm.
[0045] Acid and alkali resistance tests revealed that most strains were acid-resistant, reaching a pH of 3.0, but had poor tolerance to alkalis, failing to grow at pH 8.0.
[0046] 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 ).
[0047] (2) Preparation of lactic acid bacteria metabolites The obtained strains 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. Then, one loopful of bacteria was picked and inoculated into MRS liquid medium and incubated at 37℃ and 200 rpm for 24 h. 1 mL of the liquid 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 prepare a sterile filtrate, which was stored at 4℃ for later use.
[0048] (3) Preparation of indicator bacteria and antibacterial test Using a sterile scalpel, cut a cube approximately 0.5 cm in diameter from a Fusarium graminearum or Botrytis cinerea colony. Place the side with mycelial growth on a PDA solid plate and incubate at 25°C for 3-5 days. Use a sterile bamboo stick to pick up a small amount of mycelium into a sterile test tube, add a small amount of sterile water, stir with the bamboo stick to release the spores, and filter through sterile absorbent cotton into another sterile test tube to prepare a spore suspension. Adjust the OD60 to 0.6-0.8. Add the suspension to the unconsolidated PDA solid culture medium at a ratio of 1:50, mix well, and pour the plate over.
[0049] PDA medium: potato 200 g, sucrose (or glucose) 20 g, agar 15-20 g, water 1000 ml, pH 6.2-6.6, sterilized at 115°C for 20 min.
[0050] (4) Preparation of bacterial suspension The Serratia marcescens slant that has been transferred and grown well is inoculated on NA solid medium by streaking method, and cultured at 37±1°C for 24-48h, and then transferred to 10 mL of NA liquid medium, and cultured at 37°C and 200 rpm for 24h. The bacterial suspension is taken, and the OD600 is adjusted to 0.6-0.8 with 10 mL of NA liquid medium. The un-solidified NA solid medium is added in a ratio of 1:50, mixed, and poured into plates as a control.
[0051] (5) Bacteriostatic experiment of lactic acid bacterial metabolites A sterile puncher is used to punch holes in the PDA medium added with spore suspension and the NA medium added with bacterial suspension. MRS liquid medium is used as a blank control. 100 μL of lactic acid bacterial fermentation supernatant is added into the holes, and placed at 25°C and 37°C for culture. Each strain is repeated in three groups. The bacteriostatic conditions are observed and recorded. The lactic acid bacterial supernatant is dropped on the leaves of wintergreen, and the inhibitory effect on powdery mildew is observed. The results are shown in Figure 5 .
[0052] The lactic acid bacterial strain HBUR51326 has a good inhibitory effect on Serratia marcescens, and the diameter of the bacteriostatic circle is (5±0.5) mm. However, it has no inhibitory effect on the fungi Fusarium graminearum and Botrytis cinerea. The bacterial suspension of lactic acid bacteria also has an inhibitory effect on powdery mildew of the diseased wintergreen leaves.
[0053] Example 3 Identification of lactic acid bacterial strain HBUR51326 The several candidate strains obtained by re-screening are subjected to 16S rRNA gene sequence determination. The sequences of the candidate strains are compared with the standard sequences of lactic acid bacterial groups in the GENEBANK database. The strains not belonging to the lactic acid bacterial groups are excluded, and the strains belonging to the lactic acid bacterial groups are retained to obtain the target strain.
[0054] The specific steps of 16S RNA gene sequence determination and phylogenetic analysis are as follows: (1) DNA extraction: the strain was activated on YMA slant, inoculated on TY slant, and then the bacterial body was washed from the slant with sterile normal saline after the slant was covered with bacterial lawn, and was collected in a 1.5 mL Eppendorf centrifuge tube by centrifugation at 10,000 rpm for 3 min; washed with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 3 times, then repeatedly freeze-thawed several times to break the cells, and then 600 μL of 4M GUTC buffer was added, vortexed to mix thoroughly, and then placed at room temperature for 15 min, 70 μL of diatomite suspension was added, vortexed to mix thoroughly, and then placed at room temperature for 15 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was discarded; 500 μL of 4M GUTC buffer was added, vortexed to mix thoroughly, and then placed at room temperature for 15 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was discarded; washed with 500 μL of washing buffer for 2 times, then washed with 600 μL of 75% ethanol for 1 time, and the supernatant was discarded; dried the precipitate in the clean bench until the diatomite turned white, and finally 50 μL of sterile ultrapure water was added, and the mixture was incubated at 55-65°C for 10 min, and then the supernatant was collected by centrifugation, and then stored at -20°C after electrophoresis detection.
[0055] 16S rRNA gene sequence analysis primers are as follows: Pl: 5'-AGAGTTTGArCCTGGCTCAGAACGAACGCT-3'; P6: 5'-ACGGCTACCTTGTTACGACTTCACCC-3'.
[0056] (2) PCR amplification, the amplification system is as follows: 5.0 μL of 10×Taq Buffer, 1.0 μL of 10 mM dNTPs, 1.0 μL of 20 μM fD1, 1.0 μL of 20 μM rD1, 50-100 ng of template DNA, 0.3 μL of Taq DNA polymerase (5 U / μL), and sterile ultrapure water to 50 μL; the PCR amplification conditions are as follows: .
[0057] (3) Detection of PCR amplification product: 3 μL of 16S rRNA gene amplification product was mixed with 0.6 μL of 6×Loading Buffer, and then was loaded on a 1% agarose gel (containing EB) plate for electrophoresis detection at a voltage of 5 V / cm; after electrophoresis, the gel was scanned in a gel imaging instrument, the length and concentration of the amplified fragment were checked, and then the qualified product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the obtained 16S rRNA gene sequence of the target strain is shown as SEQ ID No. 1.
[0058] The sequence results of the target strain were compared in Ezbiocloud (http: / www. ezbiocloud.net / eztaxon), and the similar model bacteria were selected as reference strains to construct the phylogenetic tree. The results showed that the 16S rRNA gene sequence of strain HBUR51326 had the highest similarity with that of Lactobacillus paracasei Lacticaseibacillus paracasei , with a similarity of 100%. Using the alignment results of the sequence in Ezbiocloud, the 16S rRNA gene phylogenetic tree was constructed by the maximum likelihood method (Maximun likelihood method) of Mega7.0 software, with a bootstrap value of 1000 and a substitution frequency of 1% for each base. The phylogenetic tree is shown in Figure 6 .
[0059] From the above results, it can be determined that the target strain is Lactobacillus paracasei. The strain was sent to the Guangdong Microbial Culture Collection Center on October 14, 2024, with the accession number GDMCC No: 65259, and the classification and naming of Lactobacillus paracasei Lacticaseibacillus paracasei ) HBUR51326, and the preservation address is No. 59, Building 5, 100, Martyrs' Road, Guangzhou.
[0060] After simple staining of the target strain Lactobacillus paracasei Lacticaseibacillus paracasei ) HBUR51326 selected by crystal violet, its characteristics were observed under an optical microscope, and its biological characteristics are as follows: The optimal growth temperature is 37℃±1℃, and the culture is anaerobic or facultative anaerobic. The colony on the MS medium is milky white and opaque. The colony is round, with thin edges and thick middle. The growth time is 48-72 hours, and the colony size is 3-4mm. The bacterial cell is rod-shaped, with a size of 3-4μm×4-5μm. It is a gram-positive bacterium, and the gram staining is purple. Figure 7 ).
[0061] Example 4 Application of Lactobacillus paracasei strain HBUR51326 in alfalfa silage feed 1. Fermentation of Lactobacillus paracasei Lacticaseibacillus paracasei strain HBUR51326 The test bacteria Lactobacillus paracasei Lacticaseibacillus paracasei) The strain HBUR51326 was transferred and preserved on MRS solid medium slant, and after 2-5 days of incubation at 37±1°C, a ring of bacteria was picked and inoculated into a fermentation tube containing 10 mL of MRS liquid medium, which was incubated at 37±1°C in a 200 r / min shaker for 24 h. 5 mL of bacterial suspension of different strains was taken from the fermentation tube using a pipette and inoculated into 95 mL of MRS liquid medium in three replicates. The culture was incubated at 37°C in a 200 r / min shaker until the logarithmic phase.
[0062] 2. Determination of viable count of lactic acid bacteria 1 mL of bacterial solution was taken and added to a test tube containing 9 mL of sterile water, and the bacterial solution was mixed thoroughly by blowing and sucking several times, to prepare a 10 -1 dilution solution. Then 10 -1 mL of the dilution solution was taken and transferred to a test tube containing 9 mL of sterile water, and the bacterial solution was mixed thoroughly by blowing and sucking, to prepare a 10 -2 dilution solution. In this way, a series of bacterial suspensions with different dilution concentrations were prepared, such as 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , etc. Then 0.1 mL of each gradient of bacterial suspension was taken and spread on MRS plates, which were incubated at 37±1°C for 48 h before counting. The formula for calculating the viable count is as follows: ; wherein C is the average number of colonies at a certain dilution concentration, V represents the volume of the dilution solution used when spreading the plates (mL), and M represents the dilution factor.
[0063] After the determination of the viable count, the bacteria were prepared into a bacterial suspension with a concentration of 5×10 8 cfu / mL according to the number.
[0064] 3. Inoculation of lactic acid bacteria inoculum and packaging of silage material The initial flowering alfalfa was selected for mowing and divided into two groups, air-drying and non-air-drying, and the water content was determined, wherein the non-air-drying group had a water content of 75% (hereinafter referred to as high moisture), and the air-drying group had a water content of 65% (hereinafter referred to as semi-dry). Then the grass was cut into 2-3 cm long straw using a grass cutter. Another non-air-drying group was added with feed jujube powder (addition amount of 4%). The three groups were inoculated with a certain dose of bacterial suspension of Lactobacillus paracasei strain HBUR51326. The Lactobacillus paracasei strain HBUR51326 was inoculated with a viable count of 1 dose, which contained 5×10 7cfu. The bacterial suspension was evenly sprayed on alfalfa straw or a mixture of alfalfa straw and feed date powder, and was filled into a silage bag (specification: 210 mm x 297 mm), vacuum sealed, and fermented at room temperature. After 60 days, the sample was taken out of the bag. The control group was added with the same amount of sterile water; at the same time, the commercial bacterial agents "Yiran", "Laman" and "Yiqing No. 2" were used for inoculation to carry out comparative tests, and the dosage was according to the usage instruction.
[0065] Example 5: Determination of physical and chemical indexes of silage The dry matter content, pH, organic acids (lactic acid, acetic acid, butyric acid, propionic acid), ammonia nitrogen and other indexes of the alfalfa silage samples obtained in Example 4 were determined to characterize the quality, and the specific determination methods are as follows: 10 g of the alfalfa silage sample was taken, 90 mL of sterile water was added and stirred uniformly, and was stirred with a tissue crusher for 1 min. First, it was filtered with gauze and filter paper, and the leaching liquid was reserved for relevant index determination. The pH value was detected by a pH meter; the ammonia nitrogen (NH3-N) and total nitrogen (TN) were detected by a Kjeldahl nitrogen determination instrument; the lactic acid (LA), acetic acid (AA) and butyric acid (BA) contents were detected by HPLC, using a KC-811 ion chromatographic column, a column temperature of 50°C, a mobile phase of 3 mmol / L HCLO4 solution and a sample injection amount of 5 μL; the remaining sample was subjected to dry matter (DM) content determination by the constant mass method of oven drying at 105°C; the water-soluble carbohydrates (WSC) content was determined by the anthrone-sulfuric acid colorimetric method; the crude protein content = total nitrogen x 6.2; the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents were determined by a cellulose determination instrument.
[0066] (1) Dry matter content of alfalfa silage Through the determination of the oven drying constant weight of the alfalfa silage, it was found that after inoculation of the Lactobacillus paracasei strain HBUR51326 bacterial agent, the dry matter content of each group was improved compared with the control group, and the effect of each group was better than that of the three commercial bacterial agent groups. And the addition of date powder can increase the dry matter content. It is shown that the inoculation of Lactobacillus paracasei strain HBUR51326 is better than that of the commercial bacterial agent, and the addition of date powder makes the effect more significant. The data are shown in Table 1.
[0067] Table 1: Average statistical table of dry matter content of alfalfa silage (60d) (2) The content of crude protein and crude fat After inoculating the Lactobacillus paracasei strain HBUR51326 inoculant, the crude fat content (shown in Table 2) and the crude protein content (shown in Table 3) of each group were higher than those of the control group. For the high-moisture content group (with or without jujube powder added), the effect of the inoculant of the application was better than that of each commercial inoculant group (Yiran, Yiqing and Laman).
[0068] Table 2 Average statistical table of crude fat of alfalfa silage (% DM) Table 3 Average statistical table of crude protein of alfalfa silage (% DM) (3) The content of soluble carbohydrates From the test results, it can be seen that the higher the water content of the silage raw material, the lower the carbohydrate content. Under the same water content, the addition of jujube powder can increase the carbohydrate content of the obtained feed. Inoculation of the Lactobacillus paracasei strain HBUR51326 inoculant can increase the content of soluble carbohydrates in each group (75% water content, 65% water content and 80% water content with jujube powder added) compared with the control group, and the effect is better than that of the commercial inoculant group (Yiran, Yiqing and Laman). The data are shown in Table 4 below.
[0069] Table 4 Average statistical table of soluble carbohydrates of alfalfa silage (4) The content of crude fiber The test showed that after inoculation of the Lactobacillus paracasei strain HBUR51326, the neutral detergent fiber increased with the decrease of the water content of alfalfa, and the addition of jujube powder was more conducive to the increase of the content of neutral detergent fiber, and the addition of the Lactobacillus paracasei strain HBUR51326 was better than that of the commercial inoculant. After inoculation of the Lactobacillus paracasei strain HBUR51326, the acid detergent fiber content of each treatment was lower than that of the control and the commercial bacteria, indicating that it was better than the commercial bacteria, and the decrease of the water content was conducive to the reduction of the content of acid detergent fiber. The addition of jujube powder also reduced the content of acid detergent fiber, indicating that the addition of jujube powder was conducive to the silage of high-moisture alfalfa and was beneficial to the reduction of the content of acid detergent fiber. The results are shown in Table 5 below.
[0070] Table 5 Average statistical table of crude fiber of alfalfa silage (5) Determination of pH and crude ash The results show that the moisture content directly affects the pH value, the pH value of the material with less moisture content decreases rapidly, and reaches the pH value of the silage feed, and after inoculating the Lactobacillus paracasei strain HBUR51326, the pH value is obviously reduced, and the treatment of adding jujube powder has no difference with the semi-dry alfalfa, which shows that adding the feed jujube powder has an improvement effect on the silage of high-moisture alfalfa, and can replace the drying link of alfalfa, and at the same time, the pH value of the silage material of each treatment is lower than that of the commercial bacteria, which shows that the acid production of the bacteria is better than that of the commercial bacteria, and the results are shown in Table 6.
[0071] Table 6: pH value of alfalfa silage After inoculating the lactic acid bacteria of the application, the crude ash of the silage feed of each group is obviously lower than that of the control group. After inoculating the commercial bacteria, the crude ash of the silage feed of each group is higher than that of the control group, so the bacteria of the application is better than the commercial bacteria. The results are shown in Table 7. Table 7: Crude ash of alfalfa silage (6) Content of organic acid After 60 days of alfalfa silage, the test results of lactic acid content show that the lactic acid content of the Lactobacillus paracasei strain HBUR51326 bacteria group is higher than that of the control group and each commercial bacteria group, especially the 75% moisture content group with or without jujube powder; at the same time, the acetic acid content of the Lactobacillus paracasei strain HBUR51326 bacteria group is lower than that of the control group, especially the semi-dry group and the 75% moisture content group with jujube powder, which shows that the bacteria of the application is better than the commercial bacteria in improving the lactic acid / acetic acid ratio. The results are shown in Table 8.
[0072] Table 8: Lactic acid and acetic acid production of alfalfa silage Among the organic acids, the content of propionic acid and butyric acid is less in all silage treatments. Under high-moisture conditions, the addition of jujube powder or not, the treatment of using the bacteria of the application is lower than that of the control group, which shows that inoculating the bacteria of the application can reduce the two organic acids. See Table 9.
[0073] Table 9: Propionic acid and butyric acid production of alfalfa silage (7) Content of ammonia nitrogen Ammonia nitrogen is an important index for evaluating silage material, after inoculating the Lactobacillus paracasei strain HBUR51326 inoculant of the application, the ammonia nitrogen content of the silage material is obviously reduced, thereby being beneficial to improving the quality of the silage feed, and the effect is obviously better than that of the commercial bacteria. It can be seen that the use effect of the application is obviously better than that of the commercial bacteria. See Table 10.
[0074] Table 10: Average statistics table of ammonia nitrogen content of alfalfa silage The water content of alfalfa is crucial for alfalfa silage, the alfalfa with 65% water content produces less ammonia nitrogen and lower pH value than the alfalfa with 75% water content during silage, but the crude fat and crude protein are less, and the dry matter content is obviously increased. Under the condition of 75% water content, the lactic acid content of alfalfa silage is the highest after inoculating the Lactobacillus paracasei strain HBUR51326 inoculant and adding jujube powder.
[0075] The application of the Lactobacillus paracasei strain HBUR51326 inoculant is better than that of the commercial bacteria Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus rhamnosus in many indexes, and the lactic acid production and pH value are also better than those without adding, which indicates that the Lactobacillus paracasei strain HBUR51326 inoculant has good effect and can be used as a fermentation agent for alfalfa silage, and the addition of jujube powder is helpful for lactic acid production, which doubles the quality of alfalfa silage material.
Claims
1. A strain of Lactobacillus paracasei, HBUR51326, characterized in that, The Lactobacillus paracasei strain HBUR51326 is Lactobacillus paracasei ( Lacticaseibacillus paracasei HBUR51326, deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposited on October 14, 2024, with accession number GDMCC No: 65259.
2. A silage fermentation agent, characterized in that, The silage fermentation agent was prepared from the Lactobacillus paracasei strain HBUR51326 as described in claim 1.
3. A type of silage, characterized in that, The silage is obtained by fermenting raw materials by inoculating them with the silage fermentation agent described in claim 2.
4. The silage according to claim 3, characterized in that, The raw material is alfalfa or alfalfa-jujube powder.
5. The silage according to claim 4, characterized in that, The alfalfa has a moisture content of 75%-80%.
6. The silage according to claim 4, characterized in that, The alfalfa-jujube powder raw material has a moisture content of 75%-80%.
7. The silage according to claim 6, characterized in that, The amount of jujube powder added is 4 wt.
8. The silage according to any one of claims 3 to 7, characterized in that, The silage is prepared by a method comprising the following steps: The Lactobacillus paracasei strain HBUR51326 was prepared into a bacterial suspension, sprayed onto the raw materials of silage, vacuum-packed, and stored at room temperature for fermentation to obtain the silage.
9. The silage according to claim 8, characterized in that, The bacterial suspension was prepared by the following method: activated Lactobacillus paracasei strain HBUR51326 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.
10. The use of the Lactobacillus paracasei strain HBUR51326 as described in claim 1 in the preparation of silage fermentation inoculant or silage.