Recombinant pediococcus pentosaceus with function of producing acidic xylanase and application of recombinant pediococcus pentosaceus

Abstract

The invention relates to the technical field of microorganisms, in particular to recombinant pediococcus pentosaceus with an acidic xylanase producing function and application of the recombinant pediococcus pentosaceus, and a specific construction method of the recombinant pediococcus pentosaceus comprises the following steps: inserting a xylanase Xylan gene into an expression vector of the pediococcus pentosaceus through homologous recombination to construct a recombinant expression vector; a recombinant expression vector is introduced into pediococcus pentosaceus through an electrotransformation method, recombinant pediococcus pentosaceus is obtained, and the nucleotide sequence of the xylanase Xylan gene is shown as SEQ ID NO. 1. According to the invention, a xylanase gene is introduced into pediococcus pentosaceus through a genetic engineering means, so that the pediococcus pentosaceus can efficiently express xylanase under an acidic condition. Verification is carried out in alfalfa silage, and the result shows that the recombinant pediococcus pentosaceus can improve the fermentation quality and nutritional value of the alfalfa silage.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a recombinant Pediococcus pentosaceus with acid-producing xylanase function and its applications. Background Technology

[0002] Xylan is one of the main polysaccharides in plant cell walls and is widely found in plant-based feeds such as grains and straw. The degradation of xylan is crucial for improving the nutritional value of feed and the digestibility and absorption rate of animals. However, xylan itself is not easily broken down by animal digestive enzymes, therefore microbial fermentation is necessary to improve its availability. Xylanase is an enzyme that can break down xylan, converting it into monosaccharides and oligosaccharides that are available to animals, thereby improving the nutritional value and digestibility of feed.

[0003] With the development of animal husbandry, the requirements for feed nutritional value and digestibility are becoming increasingly stringent. Traditional silage processing methods often fail to fully release the nutritional value of xylan, resulting in low feed utilization. While some progress has been made in silage fermentation using additives such as lactic acid bacteria, several technical challenges remain. Adding a single lactic acid bacteria cannot provide multiple functions; it must be used in combination with other enzymes and microorganisms. Secondly, although cellulases such as xylanase can improve feed digestibility, their stability and activity during silage may be limited by pH changes and the influence of microbial metabolites. Furthermore, the expression levels and activity of enzymes in existing technologies may be insufficient to significantly improve silage quality. Therefore, developing a microbial strain capable of efficiently degrading xylan is of great significance for improving feed quality and animal health. Summary of the Invention

[0004] To address the above problems, this invention provides a recombinant Pediococcus pentosaceus with acid-producing xylanase function and its applications.

[0005] This invention is achieved through the following technical solution: A recombinant Pediococcus pentosaccharide strain with acid-producing xylanase function, the specific construction method of which is as follows: The xylanase gene was inserted into the expression vector of lactic acid bacteria via homologous recombination to construct a recombinant expression vector.

[0006] Recombinant Pediococcus pentosaccharide was obtained by introducing the recombinant expression vector into Pediococcus pentosaccharide using an electroporation method.

[0007] The nucleotide sequence of the xylanase gene is shown in SEQ ID NO. 1.

[0008] Preferably, the expression vector for the lactic acid bacteria is pNZ8148.

[0009] Preferably, SGM17 medium is used for culturing.

[0010] Preferably, the culture temperature is 16℃~30℃.

[0011] Preferably, the culture temperature is 16°C.

[0012] Preferably, the culture is induced using nisin; the concentration of nisin is 1 ng / mL to 2 ng / mL.

[0013] Preferably, the concentration of the lactic acid nisin is 2 ng / mL. The application of the recombinant Pediococcus pentosaccharide in improving the fermentation quality of silage.

[0014] Preferably, the specific application method involves inoculating the recombinant Pediococcus pentosaceus into silage raw materials for fermentation; the inoculation amount is at least 1×10⁻⁶ per gram of silage raw material. 6 CFU strains.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a recombinant *Pediococcus pentosaceus* strain with acidic xylanase production function. The specific construction method of the recombinant *Pediococcus pentosaceus* strain is as follows: the xylanase gene is inserted into the expression vector of *Pediococcus pentosaceus* via homologous recombination to construct a recombinant expression vector; the recombinant expression vector is introduced into *Pediococcus pentosaceus* via electroporation to obtain the recombinant *Pediococcus pentosaceus* strain; the nucleotide sequence of the xylanase gene is shown in SEQ ID NO. 1. *Pediococcus pentosaceus* is a Gram-positive bacterium widely distributed in nature, including the animal intestines. This strain possesses multiple enzymatic activities and can utilize xylanase to decompose various complex carbohydrates. This invention obtains an excellent *Pediococcus pentosaceus* strain through preliminary screening and verification; further, the designed acidic xylanase gene is introduced into this strain, and culture conditions are screened to achieve high expression of xylanase. The results are verified in an alfalfa silage application experiment, showing that the recombinant *Pediococcus pentosaceus* strain can improve the fermentation quality and nutritional value of alfalfa silage. By adding recombinant Pediococcus pentosaceus with xylanase function, the degradation efficiency of xylan in silage can be significantly improved, thereby increasing the nutritional value of the feed and the digestibility and absorption rate of animals. Furthermore, fermented feed also has advantages such as improving animal intestinal health and reducing environmental pollution. Producing fermented silage with high nutritional value and high digestibility provides technical support for the development of animal husbandry. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram illustrating the Escherichia coli synthesis strategy of this invention; Figure 1 In the diagram, A is the plasmid synthesis diagram; B is the plasmid synthesis unfolding diagram.

[0018] Figure 2 This is a diagram illustrating the lactic acid bacteria synthesis strategy of the present invention; Figure 2 In the diagram, A is the plasmid synthesis diagram; B is the plasmid synthesis unfolding diagram.

[0019] Figure 3 For the PCR verification of recombinant Escherichia coli in this invention, lanes 3 and 4 are positive clones of the expected size, and lanes 1, 2, 5, 6, 7, and 8 are blank lanes.

[0020] Figure 4 This invention provides an SDS-PAGE electrophoresis analysis of xylanase in recombinant Escherichia coli. Lanes 1 and 2 are the supernatants of two correctly sequenced colonies for SDS-PAGE analysis.

[0021] Figure 5 To determine the activity of recombinant Escherichia coli xylanase.

[0022] Figure 6 This is for PCR validation of recombinant lactic acid bacteria; the left white-framed lane contains positive clones of recombinant lactococcus lactis that match the expected size; the right white-framed lane contains positive clones of recombinant Pediococcus pentosaceus that match the expected size.

[0023] Figure 7 SDS-PAGE electrophoresis analysis of recombinant lactic acid bacteria; lane 1 is for SDS-PAGE analysis of supernatant of recombinant lactococcus with correct sequencing, and lane 2 is for SDS-PAGE analysis of supernatant of recombinant Pediococcus pentosaccharide with correct sequencing.

[0024] Figure 8 The results of screening culture conditions for recombinant lactic acid bacteria xylanase activity are shown. NE represents recombinant Lactococcus lactis, and PE represents recombinant Pediococcus pentosus. Figure 8In the figures, (a) shows the results of yeast xylanase activity assay of recombinant *Lactococcus lactis*, where CK is NZ9000 and represents unrecombined pure bacteria; (b) shows the results of yeast xylanase activity assay of recombinant *Pediococcus pentosaceus*, where CK is PP and represents unrecombined pure bacteria; (c) shows the results of yeast xylanase activity assay of recombinant *Lactococcus lactis* at different nisin concentrations; (d) shows the results of yeast xylanase activity assay of recombinant *Pediococcus pentosaceus* at different nisin concentrations; (e) shows the results of yeast xylanase activity assay of recombinant *Lactococcus lactis* at different temperatures; (f) shows the results of yeast xylanase activity assay of recombinant *Pediococcus pentosaceus* at different temperatures; (g) shows the results of yeast xylanase activity assay of recombinant *Lactococcus lactis* in different culture media; and (h) shows the results of yeast xylanase activity assay of recombinant *Pediococcus pentosaceus* in different culture media. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0028] Example 1 1. Materials and Methods 1.1 The main reagents are shown in Table 1. Table 1 Main reagents used in the experiment 1.2 The main equipment is shown in Table 2. Table 2 Main Instruments and Equipment 1.3 Strains and Plasmids Escherichia coli DH5α and BL21 (DE3) were purchased from Shanghai Angyu Biotechnology.

[0029] The Pentosaccharidococcus used in this invention ( Pediococcus pentosaceusThe information is disclosed in the literature “[1] Xueyan B, Haoran F, Gang G, et al. Effects of laccase and lactic acid bacteria on thefermentation quality, nutrient composition, enzymatic hydrolysis, and bacterial community of alfalfa silage [J]. Frontiers in Microbiology, 2022, 131035942-1035942.”. In this invention, it is represented by PP. The applicant undertakes to release the biological material to the public within twenty years from the date of the application and provides the means of obtaining the biological material (such as the address for obtaining it).

[0030] The Lactococcus lactis used in this invention is the standard Lactococcus lactis NZ9000, purchased from Wuhan Miaoling Biotechnology. In this invention, NZ9000 is used as the designation.

[0031] The plasmid pUC57-pNZ8148-Usp45 was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and the target gene Xylan was designed and then synthesized by BGI Genomics.

[0032] 1.4 Preparation of Culture Media and Buffer Solutions (1) LB liquid medium: 1g tryptone, 0.5g yeast extract, 1g sodium chloride, and distilled water to a final volume of 100mL. LB solid medium is prepared by adding 2g agar powder to the above formula.

[0033] (2) 50×TAE electrophoresis buffer: 20mL TAE, ddH2O to a final volume of 1L. Store at 4℃.

[0034] (3) 5% stacking gel (5mL): 0.83mL of 30% gel preparation solution, 2.5mL of 1M Tris-HCl (pH 8.8), 50µL of 10% SDS, 75µL of 10% APS, 3.42mL of ddH2O, and finally 7.5µL of LTEMED coagulant.

[0035] (4) 10% separating gel (10 mL): 3.3 mL of 30% gel preparation solution, 0.625 mL of 1M Tris-HCl (pH 6.8), 100 µL of 10% SDS, 100 µL of 10% APS, 4 mL of ddH2O, and finally 10 µL of LTEMED coagulant.

[0036] (5) 5×SDS PAGE gel electrophoresis buffer: Tris 15.1g, glycine 94g, 5g SDS, distilled water to a final volume of 1L (working solution diluted to 1×). Store at room temperature.

[0037] (6) Coomassie Brilliant Blue R-250 staining solution: 0.25 g Coomassie Brilliant Blue, 45 mL methanol, 10 mL glacial acetic acid, distilled water to a final volume of 100 mL, store at room temperature.

[0038] (7) Coomassie brilliant blue decolorizing solution: 423 mL anhydrous ethanol, 50 mL glacial acetic acid, and 522 mL distilled water, mix well and store at room temperature.

[0039] (8) PBS buffer: 0.27 g potassium dihydrogen phosphate, 1.42 g disodium hydrogen phosphate, 8 g sodium chloride, 0.2 g potassium chloride, and distilled water to a final volume of 1 L. Autoclave at 121 °C for 20 min and store at 4 °C.

[0040] (9) 10× Transfer Buffer: Tris 30g, glycine 144g, sterile water to a final volume of 1L (working solution diluted to 1×). Store at room temperature.

[0041] (10) Erythromycin (250mg / mL): 1.25g of erythromycin is dissolved in 5mL of anhydrous ethanol, filtered and sterilized in a laminar flow hood, and stored at -20℃.

[0042] (11) GM17 medium: 4.225g M17 broth, 0.5% glucose (v / v), and distilled water to a final volume of 100mL. Autoclave at 115℃ for 15min. Store at room temperature. If preparing a solid medium, add 2% agar powder (w / v).

[0043] (12) GSGM17 medium: 2% glycine, 0.5M sucrose, 4.225g M17 broth, 0.5% glucose, dissolved in 100mL distilled water (glycine was sterilized separately at 121℃), autoclaved at 115℃ for 15min, and stored at room temperature.

[0044] (13) SGM17MC resuscitation medium: 0.5M sucrose, M17 broth 4.225g, 0.5% glucose (v / v), 20mM magnesium chloride, 2mM calcium chloride, dissolved in 100mL distilled water (calcium chloride was sterilized separately at 121℃), autoclaved at 115℃ for 15min, and stored at 4℃.

[0045] (14) Lactococcus lactis electroporation buffer I: 0.5M sucrose, 10% glycerol (v / v), and distilled water to a final volume of 500mL. Autoclave at 121℃ for 20min and store at 4℃.

[0046] (15) Lactococcus lactis electroporation buffer II: 0.5M sucrose, 10% glycerol (v / v), 0.05M EDTA, and distilled water to a final volume of 500mL. Autoclave at 121℃ for 20min and store at 4℃.

[0047] (16) 1 mg / mL glucose standard solution: Weigh an appropriate amount of glucose and dry it at 105℃ to constant weight. Weigh 1 g of anhydrous glucose dried to constant weight, add a small amount of distilled water to dissolve it, and then make up to 1 L. Store for later use.

[0048] (17) DNS solution: Weigh 6.3 g DNS, add 500 mL distilled water, and add 250 mL of 2 M / L sodium hydroxide in a 45 °C water bath while stirring until the solution becomes transparent. Add 182 g potassium sodium tartrate tetrahydrate, 5 g phenol and anhydrous sulfite to a 45 °C water bath, and continue to add water at 45 °C while stirring continuously until all the material is dissolved. Cool to room temperature, make up to 1 L with distilled water, and store in a brown bottle in a dark place.

[0049] (18) 1 mol / L citrate buffer (pH 4.5): Weigh 10.5 g of citric acid, dissolve it in 40 mL of distilled water, adjust the pH of the solution to 4.5, and bring the volume to 50 mL. Store at 4 °C.

[0050] 1.5 Expression of xylanase gene in Escherichia coli 1.5.1 Design and Synthesis of Xylanase Gene The target gene, xylan, a xylanase, has a full-length gene sequence of 660 bp (NCBI accession number: KY849859.1). Sequence design was based on the codon preference of Lactococcus lactis, and the designed sequence is shown in SEQ ID NO. 1. The designed Xylan xylanase sequence was used to construct the pet28a(+)-Xylan (E. coli) plasmid, which was synthesized at the company and transformed into E. coli DH5α. The E. coli expression plasmid is shown below. Figure 1 As shown. The synthesized and preserved bacterial strain was streaked with a line. A single strain was placed in LB broth, shaken at 37°C, and the fresh bacterial cells were removed. Plasmid DNA was extracted. E. coli Xylan is expressed in BL21.

[0051] SEQ ID NO. 1:

[0052] 1.5.2 Transformation of xylanase gene in Escherichia coli (1) BL21 (DE3) competent cells were thawed on ice.

[0053] (2) Add 10 μL of the recombinant product to 100 μL of the recipient cells, gently shake to mix (do not shake to mix), and place on ice for 30 min.

[0054] (3) Heat in a water bath at 42°C for 45 seconds, then immediately place on ice to cool for 3 minutes.

[0055] (4) Add 900 μL (antibiotic-free) LB medium and shake at 37°C (200 rpm) for 1 hour.

[0056] (5) Spread on kanamycin-resistant LB plates and incubate overnight at 37°C.

[0057] Single colonies were placed in LB (100 μg / mL kanapenem) and shaken for 8 hours. PCR verification was then performed, and the resulting samples were sent for sequencing. The obtained sequence information was then subjected to further genetic transformation through BLAST alignment.

[0058] The PCR reaction system is shown in Table 3 below: Table 3 PCR reaction system The PCR reaction procedure is shown in Table 4 below: Table 4 PCR reaction procedure Note: " / " indicates that this item is not present.

[0059] 1.5.3 Fermentation of Recombinant Escherichia coli 1.5.3.1 Induced expression of recombinant Escherichia coli Select recombinant Escherichia coli with normal sequences, activate them, and inoculate them into LB solution with a kanamycin concentration of 100 µg / mL. Incubate at 200 r / min and 37 °C until the OD value reaches 0.8. Then add isopropyl β-D-1-thinogalactoside (IPTG) solution to a final concentration of 500 mg / mL and induce culture at 20 °C for 8 h.

[0060] 1.5.3.2 SDS-PAGE detection of recombinant Escherichia coli (1) Sample processing After centrifugation at 8000 rpm for 10 min at 4 °C, the enzyme solution was collected and resuspended in PBS buffer. The crude enzyme solution was then sonicated on an ice plate for 20 min, followed by centrifugation at 8000 rpm for 5 min. The supernatant was collected and analyzed by SDS-PAGE electrophoresis. The separated protein solution was placed in an ultrafiltration tube and centrifuged at 4000 rpm for 15 min at 4 °C, repeated 6 times. The protein was then detected by SDS-PAGE gel electrophoresis.

[0061] (2) SDS-PAGE electrophoresis Add the prepared 10% (w / w) separating gel to the gel casting plate, immediately add ddH2O and press the separating gel flat. After 30 min, pour out the ddH2O (at this point, the top edge of the separating gel should be a horizontal straight line). Blot off any remaining moisture with absorbent paper, add 5% (w / w) stacking gel, and immediately insert the comb. Let stand for 30 min, then add 10 µL of sample processing solution to each well. Perform electrophoresis at 80 V for 30 min, then at 120 V for 90 min. After electrophoresis, remove the gel and stain with Coomassie Brilliant Blue solution for 30 min. Destain the stained gel with destaining solution (changing the destaining solution every 30 min) until the gel is free of blue. After destaining, analyze the electrophoresis results using a gel imaging system.

[0062] 1.5.3.3 Xylanase Activity Assay Dilute the bacterial culture to an appropriate ratio, add 1 mL of the test solution to two test tubes, add 1 mL of 1% xylan substrate to one test tube, and add 1 mL of phosphate buffer to the other test tube. Place the tubes in a 39°C water bath and start timing. React for 30 min (shake well every 10 min). After removing the tubes, add 3 mL of DNS solution to the control tube, shake for 3 s to mix, incubate in a boiling water bath for 7 min, and then cool rapidly. Determine the xylanase activity using the DNS method.

[0063] 1.6 Expression of xylanase in Lactococcus lactis and Pediococcus pentosaceus Using plasmid DNA extracted from *E. coli* as a template, primers were designed and homologous arms were added to amplify the full-length sequence of the xylanase gene. The primers are shown in Table 5. The xylanase gene Xylan was expressed in *Lactococcus lactis* and *Pediococcus pentosaceus*, and the plasmid pUC57-pNZ8148-SP310--Xylan (lactic acid bacteria) was constructed. The lactic acid bacteria expression plasmid is shown in Table 5. Figure 2 As shown.

[0064] 1.6.1 Cloning of the xylanase gene Table 5 Primers used Note: The underlined part is the homologous arm primer.

[0065] The PCR reaction system is shown in Table 6 below: Table 6 PCR Reaction System The PCR reaction procedure is shown in Table 7 below: Table 7 PCR reaction procedure Note: " / " indicates that this item is not present. Take 5 µL of PCR product and run it on a 1% agarose gel electrophoresis to check if it is a single band. Then purify it using a DNA purification kit.

[0066] 1.6.2 Expression Vector Linearization The Xylan gene itself does not have a signal peptide, so the signal peptide SP310 of the vector pUC57-pNZ8148 was selected for ligation. The primer sequences are shown in Table 8.

[0067] Table 8 Primer sequences The PCR reaction system is the same as in Table 6.

[0068] The PCR reaction procedure is shown in Table 9 below: Table 9 PCR reaction procedure Note: " / " indicates that this item is not present.

[0069] The PCR products were collected and purified by electrophoresis and gel electrophoresis.

[0070] 1.6.3 Construction of Recombinant Plasmids The primers designed for the Xylan target gene contain a partial sequence that is inversely complementary to the vector, which can be used as homologous arms to perform homologous recombination ligation with the vector. The enzyme ligation system is shown in Table 10.

[0071] Table 10 Enzyme ligation system React at 37°C for 30 minutes in a PCR instrument, then cool to 4°C or immediately place on ice to cool.

[0072] 1.6.4 Transformation of Lactococcus lactis expression host Homemade Lactococcus lactis competent cells: (1) Activate Lactococcus lactis, pick a single colony and inoculate it into 5 mL of GM17 liquid medium, and incubate at 30°C overnight.

[0073] (2) Repeat step 1 to reactivate and culture the above culture medium.

[0074] (3) The bacterial culture was inoculated into 100 mL of GSGM17 liquid medium at a volume fraction of 5%, and incubated at 30°C until OD600=0.6.

[0075] (4) Place the bacterial solution in an ice bath for 10 minutes, transfer it to a pre-cooled sterile centrifuge tube, centrifuge at 4000 r / min for 10 minutes at 4℃, and discard the supernatant.

[0076] (5) Add 50 mL of pre-cooled electroporation buffer I to resuspend the bacterial cells, centrifuge at 4000 r / min for 10 min at 4℃, and discard the supernatant.

[0077] (6) Add 25 mL of pre-cooled electroporation buffer II to resuspend the bacterial cells, pre-ice for 15 min, centrifuge at 4000 r / min for 10 min at 4℃, and discard the supernatant.

[0078] (7) Repeat step 6.

[0079] (8) Suspend the bacteria in 1 mL of solution I to prepare competent cells. Quickly aliquot the competent cells into 100 µL portions into sterile pre-cooled 1.5 mL centrifuge tubes, freeze them in liquid nitrogen, and quickly transfer them to a -80 °C freezer for storage.

[0080] (9) The method for preparing Pediococcus pentosaceus competent cells is the same as described above. Thaw NZ9000 and PP competent cells on ice, add the plasmid to be transformed, and quickly transfer to a pre-cooled 0.2cm electroporation cuvette for 5 min. Verify the correctness of the transformation by using recombinant plasmids NZ9000 and PP under the following conditions: pulse voltage 2200V, pulse resistance 100Ω, pulse capacitance 25μF, pulse duration 3s. Incubate at 30℃ for 2 h, spread on ampicillin-resistant plates, and incubate overnight at 30℃. Pick a single colony, inoculate it on M17 (100µg / mL ampicillin), shake culture for about 8 h, extract the plasmid, verify by PCR, and send the verified product for sequencing. Analyze the sequencing results using BLAST.

[0081] 1.7 Fermentation of recombinant Lactococcus lactis and recombinant Pediococcus pentosaceus 1.7.1 Induced expression of recombinant Lactococcus lactis and recombinant Pediococcus pentosaceus Recombinant *Lactococcus lactis* (NE) and recombinant *Pediococcus pentosus* (PE) with correct sequencing were selected, reactivated, and single colonies were picked and cultured in liquid M17 with an ampicillin concentration of 100 μg / mL at 200 rpm and 30°C until the OD value reached 0.8. Then, 2 ng / mL of nisin was added, and the culture was induced at 20°C for another 8 h. The SDS-PAGE detection method was consistent with that used for recombinant *Escherichia coli*.

[0082] 1.7.2 Xylanase activity assay: same as 2.2.2.3 1.7.3 Screening of culture conditions for recombinant Lactococcus lactis and recombinant Pediococcus pentosaceus (1) Optimal induction concentration Recombinant Lactococcus lactis and recombinant Pediococcus pentosus with the highest enzyme activity were selected. The recombinant Lactococcus lactis and recombinant Pediococcus pentosus were cultured at 30℃ until the OD value reached 0.8. Five induction concentrations of nisin were set at 1ng / mL, 2ng / mL, 4ng / mL, 8ng / mL and 10ng / mL. The cultures were incubated overnight at 30℃. The bacterial cultures were collected and the xylanase activity was measured.

[0083] (2) Optimal induction temperature Cultivate the bacteria according to the above method until the OD value reaches 0.8, add the optimal concentration of nisin, and then incubate overnight at 16℃, 25℃ and 30℃ respectively. Collect the bacterial culture and measure the xylanase activity.

[0084] (3) Different culture media Three culture media were set up: M17 medium, GSGM17 medium and SGM17 resuscitation medium. The optimal induction concentration and induction temperature were used. After overnight culture, the bacterial solution was collected and the xylanase activity was measured.

[0085] 2 Results 2.1 Expression of xylanase gene in Escherichia coli 2.1.1 Identification of plasmids in recombinant Escherichia coli Plasmids were extracted and transformed into Escherichia coli BL21(DE3). Positive clones were screened, and the results were obtained by PCR in the bacterial culture followed by agarose gel electrophoresis. Figure 3 As shown, the size of the single amplified band was approximately 1000 bp. The electrophoresis result matched the size of the target band (1003 bp). The remaining PCR products of the same size were sent to Sangon Biotech for sequencing, and the sequencing results were compared using BLAST.

[0086] 2.1.2 SDS-PAGE electrophoresis analysis of xylanase in recombinant Escherichia coli After culturing the correctly sequenced colonies, the supernatant of the recombinant Lactococcus lactis fermentation broth and the supernatant of the cell disruption broth were analyzed by SDS-PAGE using a His tag designed on the Xylan xylanase sequence. The results are as follows: Figure 4 As shown in the figure, the target band appeared in both the supernatant of the fermentation broth and the supernatant of the cell wall disruption broth of the recombinant strain, indicating that Xylan xylanase was successfully expressed through secretion.

[0087] 2.1.3 Determination of xylanase activity in recombinant Escherichia coli Recombinant Escherichia coli was cultured statically at 16°C for 24 hours, and then xylanase activity was measured. Figure 5 As shown, the enzyme activity assay results indicate that recombinant Escherichia coli successfully achieved the secretory expression of Xylan xylanase.

[0088] 2.2 Expression of xylanase gene in recombinant Lactococcus lactis and recombinant Pediococcus pentosus 2.2.1 Identification of plasmids in recombinant Lactococcus lactis and recombinant Pediococcus pentosaceus Recombinant lactic acid bacteria colonies were selected after electroporation, and positive clones were screened. After PCR of the bacterial culture, the results were analyzed by agarose gel electrophoresis. Figure 6 As shown, the size of the single amplified band was approximately 2000 bp. The electrophoresis result matched the size of the target band (1997 bp). The remaining PCR products of the same size were sent to Sangon Biotech for sequencing, and the sequencing results were compared using BLAST.

[0089] 2.2.2 SDS-PAGE electrophoresis analysis of xylanase in recombinant Lactococcus lactis and recombinant Pediococcus pentosus After culturing the correctly sequenced bacterial colonies, the supernatant of the recombinant lactococcus fermentation broth was analyzed by SDS-PAGE. The results are as follows: Figure 7 As shown in the figure, the target band appeared in both the supernatant of the fermentation broth and the supernatant of the cell wall disruption broth of the recombinant strain, indicating that Xylan xylanase was successfully expressed through secretion.

[0090] 2.2.3 Screening results of culture conditions for xylanase activity in recombinant Lactococcus lactis and recombinant Pediococcus pentosus NE and PE bacteria showed significant xylanase activity, such as Figure 8 As shown in (a) and (b) above, after induction with different concentrations of nisin, 1 ng / mL to 2 ng / mL of nisin showed better induction effects, with 2 ng / mL of nisin showing the best induction effect. Figure 8 As shown in (c) and (d) in the figure. It exhibits xylanase activity at temperatures ranging from 16℃ to 30℃, with the optimal induction effect observed at the lower temperature of 16℃. Figure 8 As shown in (e) and (f) in the diagram. Enzyme activity is optimal in SGM17 medium, as... Figure 8 As shown in (g) and (h).

[0091] Example 2: Verification of the practical application effect of recombinant Pediococcus pentosaceus in silage. I. Experimental Methods The alfalfa used in this invention, 'SR 4030' (originating in Canada), was planted in the experimental field of the College of Animal Science, Shanxi Agricultural University (37°25'08"N", 112°35'25"E", altitude 783 m). The alfalfa was harvested at the budding stage, with a stubble height controlled at 5 cm. After 5 hours of natural drying, the dry matter content reached approximately 35%. Subsequently, the alfalfa raw material was chopped to approximately 2 cm using a chaff cutter, thoroughly mixed, and four random samples (200g each) were taken for chemical composition determination (Table 11). The remaining portion was used for silage preparation. Before the experiment, each bacterial strain was inoculated into liquid culture medium for activation culture and cultured under anaerobic conditions in a constant temperature shaking incubator for 36 hours to achieve a bacterial concentration of 1×10⁻⁶. 6 CFU / mL.

[0092] Table 11 Nutritional components of alfalfa Note: *DM, Dry Matter; FW, Fresh Weight; CP, Crude Protein; NDF, Neutral Detergent Fiber; ADF, Acid Detergent Fiber; WSC, Water-Soluble Carbohydrates; 1. Experimental Design Configure the processing group as follows: Control group (C).

[0093] Lactococcus lactis group (N).

[0094] Recombinant Lactococcus lactis group (NE).

[0095] Lactobacillus pentosus (LPE) group.

[0096] Group P. pentosaceus (PP).

[0097] Recombinant Lactococcus lactis + Lactobacillus pentosus group (NE+LPE).

[0098] Recombinant Lactococcus lactis + Pediococcus pentosus group (NE+PP).

[0099] Recombinant Pediococcus pentosaceus group (PE).

[0100] 2. Silage production 150g of chopped alfalfa was thoroughly mixed with the lactic acid bacteria suspension to ensure uniform distribution of the inoculum. The mixture was then placed into a 15.5cm × 23cm sterile polyethylene bag with a one-way valve, vacuum-sealed, and used for dynamic monitoring of silage fermentation (7d, 15d, 30d). Another 500g of the mixed alfalfa was placed in a 25cm × 35cm vacuum bag of the same type for monitoring the fermentation quality of the silage product at 60d. Four biological replicates (128 samples in total) were set up for each treatment group at each time point, with the ambient temperature maintained at 25℃, at a ratio of 1×10⁻⁶. 6 The standard addition was CFU / g fresh weight. The control group was treated with an equal volume of sterile distilled water.

[0101] 3. Measurement Indicators and Methods Chemical composition analysis Sample processing procedure: (1) Take 150g of field-dried alfalfa (4 portions) and silage samples from different fermentation stages (150g / repeat for each treatment) and put them into constant weight envelopes. (2) Place in a 65℃ forced-air drying oven and continue drying for 72 hours; (3) After taking it out, equilibrate at room temperature for 24 hours, record the weight and calculate the initial moisture content.

[0102] The dried sample was ground by a pulverizer, then sealed and stored in a desiccator for the determination of the following indicators: The determination methods for bound water content, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and water-soluble carbohydrates (WSC) in alfalfa silage are shown in Table 12.

[0103] Table 12 Methods for detecting nutrients in alfalfa silage 4. Silage fermentation quality determination Accurately weigh 20g of silage sample and place it in a 150mL Erlenmeyer flask. Add 60mL of deionized water at a ratio of 1:3 (w / v). Seal the flask with sealing film and extract at 4℃ for 24h. After extraction, filter the filtrate sequentially through four layers of sterile gauze and quantitative filter paper (medium speed). Divide the filtrate into two parts: immediately measure the pH of one part using a precision pH meter; aliquot the other part into 2mL centrifuge tubes and freeze at -20℃ for subsequent determination of fermentation parameters such as lactic acid (LA), volatile fatty acid (VFA), and ammoniacal nitrogen (NH3-N). The determination methods are shown in Table 13.

[0104] Table 13 Methods and Instruments for Detecting the Quality of Alfalfa Silage Fermentation 5. Observation using a scanning electron microscope The microstructure analysis of the samples was completed by Shanxi Bocui Biotechnology Co., Ltd., and the ultrastructure of the stem surface was characterized and observed using a Regulus 810 field emission scanning electron microscope (FE-SEM).

[0105] 6. Diversity analysis of silage After thawing the samples extracted with physiological saline and frozen for fermentation for 60 days, centrifugation at 8000×g for 10 min was performed at 4℃, and the precipitate was collected. The precipitate was resuspended in 5 mL of phosphate buffer (pH 7.4) and sent to Guangzhou Gediao Biotechnology Co., Ltd. for high-throughput sequencing analysis of the 16S rRNA gene.

[0106] 7. Data Processing and Statistical Analysis Statistical analysis was performed using SPSS 26.0 software. A two-way ANOVA model was established for the dynamic process of silage fermentation. The time factor included four levels: 3d, 7d, 15d, and 60d. The treatment factors included a blank control (CK) and seven additive treatments. Duncan's method was used for multiple comparisons, with a significance level of P < 0.05.

[0107] 4. Test Results Recombinant Pediococcus pentosaceus can produce high levels of xylanase under acidic conditions after fermentation. In alfalfa silage fermentation experiments, the recombinant Pediococcus pentosaceus group improved the silage fermentation quality. Experimental results after fermentation showed that the recombinant Pediococcus pentosaceus can produce xylanase under acidic conditions. After screening, the lyophilized xylanase, diluted in water, could reach 50 U / mL under slightly acidic conditions.

[0108] In the alfalfa silage fermentation experiment, the recombinant Pediococcus pentosate group showed the best treatment effect, with the highest crude protein content of 197 g / kg DM, the highest lactic acid content of 58 g / kg DM, low NH3-N content, and the lowest pH of 4.40.

[0109] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A recombinant Pediococcus pentosaceus with acid-producing xylanase function, characterized in that, The specific construction method of the recombinant Pediococcus pentosaceus is as follows: The xylanase gene was inserted into the expression vector of Pediococcus pentosus via homologous recombination to construct a recombinant expression vector; Recombinant Pediococcus pentosaccharide was obtained by introducing the recombinant expression vector into Pediococcus pentosaccharide using an electroporation method. The nucleotide sequence of the xylanase gene is shown in SEQ ID NO.

1.

2. The recombinant Pediococcus pentosaceus according to claim 1, characterized in that, The expression vector for Pediococcus pentosaceus was pNZ8148.

3. The recombinant Pediococcus pentosaceus according to claim 1, characterized in that, Cultured using SGM17 medium.

4. The recombinant Pediococcus pentosaceus according to claim 3, characterized in that, The culture temperature is 16℃~30℃.

5. The recombinant Pediococcus pentosaceus according to claim 4, characterized in that, The culture temperature was 16°C.

6. The recombinant Pediococcus pentosaceus according to claim 3, characterized in that, The culture process uses nisin for induction; the concentration of nisin is 1 ng / mL to 2 ng / mL.

7. The recombinant Pediococcus pentosaceus according to claim 6, characterized in that, The concentration of the lactic acid nisin is 2 ng / mL.

8. The application of the recombinant Pediococcus pentosaccharide according to claim 1 in improving the fermentation quality of silage.

9. The application according to claim 8, characterized in that, The specific application method involves inoculating the recombinant Pediococcus pentosaccharide into silage raw materials for fermentation; the inoculation amount is 1×10⁻⁶ bacteria per gram of silage raw material. 6 Recombinant Pediococcus pentosaccharide CFU.

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