A Lactobacillus and its application

Abstract

The present invention is applicable to the field of microbial technology and provides a Lactobacillus and its application. The Lactobacillus is named Lactobacillus parafarraginis ZN-R1, which was deposited at the China Center for Type Culture Collection on November 23, 2023, with the deposit number CCTCC NO: M 20232305. Its morphological characteristics are as follows: Gram-positive bacilli, the cell morphology is short rod-shaped, with blunt ends at both ends, arranged in short chains, without spores, showing milky white protrusions on the MRS plate medium, with a diameter of 1-3 mm, round and regular, smooth and moist on the surface, without pigment production, and the back of the colony is white to off-white. The Lactobacillus provided by the present invention can continuously produce acid when added to saline-alkali soil, adjust the soil pH, improve the physical and chemical properties of the soil, has the ability to absorb salts from the soil, and can colonize in saline-alkali soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Lactobacillus and its application. Background Art

[0002] The domestic saline-alkali soil improvement methods and techniques are roughly as follows: (1) Physical improvement: land leveling, deep plowing, soil loosening, terrain elevation, micro-region soil improvement, etc. (2) Hydraulic improvement: flushing salt with water, underground salt drainage, etc. (3) Chemical improvement: gypsum, phosphogypsum, superphosphate, humic acid, peat, vinegar residue, etc. Some chemical improvers themselves contain substances toxic to plants, and at the same time, the improvement of the soil is only a short-term apparent effect, unable to achieve a radical or long-term effect; in addition, there are heavy metal problems in the use of fly ash and phosphogypsum, and there are parasite eggs and pathogenic microorganisms in the use of sludge, causing potential harm to the surrounding environment;

[0003] In recent years, compared with traditional soil improvement techniques, using microorganisms with important functions for saline-alkali land improvement has the advantages of high efficiency, cleanliness, and safety, and has been widely promoted and applied. The present application provides a Lactobacillus with the function of repairing saline-alkali soil. Summary of the Invention

[0004] The present invention provides a Lactobacillus to solve the above problems.

[0005] The present invention is realized as follows: a Lactobacillus named Lactobacillus parafarraginis ZN-R1 was deposited at the China Center for Type Culture Collection on November 23, 2023, with the deposit number CCTCC NO: M 20232305.

[0006] Preferably, the 16S rDNA gene sequence of the Lactobacillus is as shown in SEQ ID No.1.

[0007] Preferably, the morphological characteristics of the Lactobacillus are as follows: Gram-positive bacilli, the cell morphology is short rod-shaped, both ends are blunt round, arranged in short chains, without spores, showing milky white protrusions on the MRS plate medium, with a diameter of 1-3 mm, round and regular, the surface is smooth and moist, without pigment production, and the back of the colony is white to off-white.

[0008] The present invention also provides a microbial inoculant, including the above-mentioned Lactobacillus.

[0009] Preferably, the microbial inoculant further includes modified zeolite and modified polyethylene glycol.

[0010] Preferably, the mass ratio of the Lactobacillus, modified zeolite, and modified polyethylene glycol is 1:100-150:20-30.

[0011] Preferably, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, and perform preliminary crushing after drying; then place it in a muffle furnace and raise the temperature programatically to 1000 - 1200 °C, keep it at a constant temperature for roasting for 1 - 2 h, and grind it to a particle size of 200 - 300 mesh after cooling; then add 30 - 40% hydrochloric acid solution and soak for 2 - 3 h; filter to obtain product A, add product A to 10 - 15 times the weight of deionized water, add 20 - 30 wt% vinyltrimethoxysilane solution, and ultrasonically disperse until uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:6 - 10; then slowly add it to the mixture, raise the temperature to 60 - 80 °C, and stir and react for 3 - 5 h, filter to obtain product B, dry product B at 80 - 100 °C for 30 - 50 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 20 - 30 min under the conditions of a power frequency of 10 - 15 kHz, a working voltage of 15 - 25 kV, and a discharge power of 75 - 85 W to obtain the modified zeolite. First, roast the zeolite to remove water in different states in the zeolite crystal structure, make the internal structure loose and porous, increase the specific surface area, then perform surface corrosion modification on the zeolite through hydrochloric acid to form a stable porous structure on the surface of the zeolite particles, and then modify it with polyhydroxy glycerol to obtain zeolite with a large number of hydroxyl groups adsorbed inside, on the surface, and in the pore diameter. Finally, after treatment with a low-temperature plasma treatment device, the surface of the zeolite particles is rougher, the specific surface area is further increased, a three-dimensional network space is formed, which can adsorb and retain nutrients in the soil, making them not easy to lose, enabling the soil to better provide nutrients for plants, promoting the growth and development of plants, and can also adsorb and fix more harmful substances, avoiding their harm to plants, and helping to maintain the health of the soil ecological environment.

[0012] Preferably, the preparation method of the modified polyethylene glycol is as follows: Under stirring conditions, drop 10 - 16 parts by weight of polyethylene glycol into 40 - 50 parts of deionized water, raise the temperature to 50 - 60 °C, then add 4 - 8 parts of 3 - amino - 1,2 - propanediol and 3 - 9 parts of diethylamine, raise the temperature to 80 - 90 °C and stir for 30 - 50 min, add 2 - 4 parts of sodium dodecylbenzenesulfonate and 3 - 5 parts of sodium alginate, and continue to stir for 20 - 40 min to obtain the modified polyethylene glycol. By grafting an amino group at the end of the polyethylene glycol, the reaction activity of the polyethylene glycol can be effectively enhanced. When compounded with sodium dodecylbenzenesulfonate and sodium alginate, a macromolecular network structure is formed through mutual reaction. On the one hand, it can coat and protect microorganisms, endowing the microbial inoculant with a slow-release property. On the other hand, it can combine with the soil to form soil aggregates, improving the soil structure and air permeability, and being beneficial to the root growth of plants and the reproduction of microorganisms.

[0013] The present invention also provides a preparation method of the above-mentioned microbial inoculant, including the following steps:

[0014] Weigh each raw material according to the ratio;

[0015] Aseptically open the Lactobacillus, pick the bacterial strain and streak it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0016] Pick the activated strain and inoculate it into an enrichment medium for enrichment culture;

[0017] Inoculate the enriched strain into a liquid medium to obtain a liquid seed culture;

[0018] Perform solid-state fermentation on the liquid seed culture to obtain a solid-state fermentation product;

[0019] Mix the solid-state fermentation product with modified zeolite and modified polyethylene glycol to obtain a microbial inoculant.

[0020] The present invention also provides the application of the above-mentioned Lactobacillus or microbial inoculant in repairing saline-alkali soil.

[0021] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0022] The Lactobacillus provided by the present invention can continuously produce acid when added to saline-alkali soil, adjust the soil pH, improve the physical and chemical properties of the soil, has the ability to absorb salt from the soil, and can colonize in saline-alkali soil. Description of the Drawings

[0023] Figure 1 It is a morphological diagram of the Lactobacillus ZN-R1 strain cultured on MRS medium provided by the present invention;

[0024] Figure 2 It is a microscopic morphological diagram of the Lactobacillus ZN-R1 strain stained by Gram provided by the present invention;

[0025] Figure 3 It is an electrophoresis diagram of the Lactobacillus ZN-R1 provided by the present invention;

[0026] Figure 4 It is a phylogenetic tree of the Lactobacillus ZN-R1 provided by the present invention. Detailed Embodiments

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application pertains; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "comprising" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0028] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears at various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0029] Embodiment 1

[0030] An embodiment of the present invention provides a Lactobacillus named Lactobacillus parafarraginis ZN-R1, which was deposited at the China Center for Type Culture Collection on November 23, 2023, with the deposit number CCTCC NO: M 20232305.

[0031] I: Morphological identification

[0032] The colony morphology of the strain individuals is as Figure 1 shown. The strain ZN-R1 presents a milky white convex shape on the MRS plate medium, with a diameter of 1-3 mm, round and regular, smooth and moist surface, no pigment production, and the back of the colony is white to off-white.

[0033] Gram staining

[0034] Gram-positive bacilli, with the cell morphology being short rod-shaped, blunt at both ends, arranged in short chains, and without spores, as Figure 2 shown.

[0035] II: Physiological and biochemical identification

[0036] 1. Catalase test: Drop freshly prepared 3% H2O2 on a glass slide with a dropper, pick a single colony with an inoculation loop and place it in the H2O2 droplet, and observe whether there are bubbles generated within 30 seconds. The strain ZN-R1 does not produce bubbles, indicating a negative result.

[0037] 2. Gelatin liquefaction test: Use an inoculation loop to pick colonies and stab-inoculate them into gelatin medium. Incubate at 37°C for 48 h. After taking it out, place it in a 4°C refrigerator for 30 min. If the content does not solidify and is in a liquid state, it is positive; if the content solidifies and does not flow, it is negative. The content of strain ZN-R1 solidified and did not flow, so it was negative.

[0038] 3. Hydrogen sulfide test: Use an inoculation loop to pick colonies and stab-inoculate them into hydrogen sulfide medium. Incubate at 37°C for 48 h. As a result, the color of the medium of strain ZN-R1 did not change, so it was negative.

[0039] 4. Methyl red test: Take an appropriate amount of agar culture and inoculate it into a buffered glucose peptone water (MR-VP) biochemical tube. Incubate at 37°C for 24 h. Add 5 drops of methyl red reagent and immediately observe the result. As a result, the color of the medium of strain ZN-R1 turned yellow, so it was negative.

[0040] 5. V-P test: Use an inoculation loop to pick single colonies and inoculate them into MRS broth medium. Incubate at 37°C for 24 h. Add 0.6 ml (6 drops) of solution A of the V-P test kit and mix well with the culture, then add 0.2 ml (2 drops) of solution B and mix well. Shake the test tube and let it stand for 0.5 - 2 h. Observe the result. There was no color change in strain ZN-R1, so it was negative.

[0041] 6. Hippurate hydrolysis test: Use an inoculation loop to pick a large amount of bacterial growth and inoculate it into a biochemical tube. Mix well and incubate at 37°C for 4 h. After the incubation is over, slowly add 0.2 mL of ninhydrin solution along the wall of the test tube without shaking. Place it in a 37°C incubator for 10 min and then read the result. As a result, strain ZN-R1 showed a deep purple reaction, so it was positive;

[0042] 7. Sugar fermentation test: Use an inoculation loop to pick colonies from the plate and inoculate them into biochemical tubes. Incubate at 37°C for 24 h and observe the color change. As a result, strain ZN-R1 can ferment raffinose, maltose, mannitol, sucrose, lactose, inulin, and salicin, but cannot ferment esculin, sorbitol, and cellobiose.

[0043] Summary: Strain ZN-R1 is a Gram-positive bacterium. The results of hydrogen peroxide, gelatin liquefaction, hydrogen sulfide, methyl red, and V-P tests were negative, and the hippurate hydrolysis test was positive. It can ferment raffinose, maltose, mannitol, sucrose, lactose, inulin, and salicin, but cannot ferment esculin, sorbitol, and cellobiose.

[0044] Item Result Item Result Esculin － Salicin ＋ Sucrose ＋ Cellobiose － Lactose ＋ Inulin ＋ Mannitol ＋ Maltose ＋ Sorbitol － Raffinose ＋ Catalase Test － Gelatin Liquefaction Test － Hydrogen Sulfide Test － V-P Test － Methyl Red Test － Hippurate Hydrolysis ＋

[0045] III: 16S rDNA identification

[0046] Electrophoresis diagram of 16S rDNA of strain ZN-R1

[0047] Using the genomic DNA of strain ZN-R1 as a template, its 16S rDNA was amplified by PCR, electrophoresed on 1% agarose gel at a voltage of 5 V / cm with 1×TAE as the electrophoresis buffer, and observed after 25 min. The results are as Figure 3 shown. Compared with the MK band, a clear band appeared at 1500 bp for strain ZN-R1, indicating that the sequence size of strain ZN-R1 was about 1500 bp.

[0048] Sequence determination of 16S rDNA of strain ZN-R1

[0049] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit. The forward primer was 27f (5′-AGAGTTTGATCCTG-GCTCAG-3′), and the reverse primer was 1492r (5′-TACGGY-TACCTTTGTTACGACTT-3′). Then PCR amplification was carried out. The amplification reaction system was 1.0 μl of DNA template, 12.5 μl of Taq PCR Master mix, 9.5 μl of ddH2O, 1.0 μl of upstream primer, and 1.0 μl of downstream primer, with a total volume of 25 μL. The PCR amplification reaction program was denaturation at 95°C for 5 s, annealing at 55°C for 10 s, extension at 72°C for 25 s, for 35 cycles, and incubation at 4°C. And it was sequenced, and the sequencing results are as follows. The sequence length is 1446 bp:

[0050]

[0051] Construction of the phylogenetic tree of the 16S rDNA of strain ZN-R1

[0052] By performing sequence alignment in the NCBI database, a phylogenetic tree of the 16S rRNA gene sequences of ZN-R1 and its related type strains was constructed. The results are as follows Figure 4 shown. Strain ZN-R1 and ON076809.1 Lentilactobacillus parafarraginis converge to one branch and have the closest genetic relationship. At the same time, combined with morphological identification and physiological and biochemical experiments, this strain was determined to be Lentilactobacillus parabranchiatus.

[0053] Example 2

[0054] The embodiment of the present invention provides a microbial inoculant, which includes Lactobacillus ZNR1 of Example 1, and also includes modified zeolite and modified polyethylene glycol. The mass ratio of the Lactobacillus, modified zeolite and modified polyethylene glycol is 1:100:20. The preparation method of the microbial inoculant includes the following steps:

[0055] Weigh each raw material according to the ratio;

[0056] Sterilely open the Lactobacillus, pick the strain and streak it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0057] Pick the activated strain and inoculate it into an enrichment medium for enrichment culture;

[0058] Inoculate the enriched strain into a liquid medium to obtain a liquid seed solution;

[0059] Perform solid-state fermentation on the liquid seed solution to obtain a solid-state fermentation product;

[0060] Mix the solid-state fermentation product with modified zeolite and modified polyethylene glycol to obtain the microbial inoculant.

[0061] Among them, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, and after drying, conduct preliminary crushing; then place it in a muffle furnace and program the temperature to 1000 °C, keep it at a constant temperature for roasting for 1 h, and after cooling, grind it to a particle size of 200 mesh; then add 30% hydrochloric acid solution and soak for 2 h; filter to obtain product A, add product A to 10 times the weight of deionized water, add 20 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:6; then slowly add it to the mixture, heat up to 60 °C, stir and react for 3 h, filter to obtain product B, dry product B at 80 °C for 30 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 20 min under the conditions of a power frequency of 10 kHz, a working voltage of 15 kV, and a discharge power of 75 W to obtain the modified zeolite.

[0062] In this embodiment, the preparation method of the modified polyethylene glycol is as follows: Under a stirring state, drop 10 parts of polyethylene glycol by weight into 40 parts of deionized water, heat up to 50 °C, then add 4 parts of 3-amino-1,2-propanediol and 3 parts of diethylamine, heat up to 80 °C and stir for 30 min, add 2 parts of sodium dodecylbenzenesulfonate and 3 parts of sodium alginate, and continue to stir for 20 min to obtain the modified polyethylene glycol.

[0063] Example 3

[0064] The embodiment of the present invention provides a microbial inoculum, which includes Lactobacillus ZN-R1 of Example 1, and also includes modified zeolite and modified polyethylene glycol. The mass ratio of Lactobacillus, modified zeolite and modified polyethylene glycol is 1:110:23. The preparation method of the microbial inoculum includes the following steps:

[0065] Weigh each raw material according to the ratio;

[0066] Aseptically open Lactobacillus, pick the bacterial strain and streak-inoculate it onto a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0067] Pick the activated strain and inoculate it into an enrichment medium for enrichment culture;

[0068] Inoculate the enriched strain into a liquid medium to culture and obtain a liquid seed solution;

[0069] Perform solid-state fermentation on the liquid seed solution to obtain a solid-state fermentation product;

[0070] Mix the solid-state fermentation product with the modified zeolite and the modified polyethylene glycol to obtain the microbial inoculum.

[0071] Among them, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, dry it and then conduct preliminary crushing; then place it in a muffle furnace, program the temperature to rise to 1000 °C, keep it at a constant temperature for roasting for 1 h, cool it and grind it to a particle size of 200 mesh; then add 30% hydrochloric acid solution and soak for 2 h; filter to obtain product A, add product A to 10 times the weight of deionized water, add 20 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:6; then slowly add it to the mixture, raise the temperature to 60 °C, stir and react for 3 h, filter to obtain product B, dry product B at 80 °C for 30 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 20 min under the conditions of a power frequency of 10 kHz, a working voltage of 15 kV, and a discharge power of 75 W to obtain the modified zeolite.

[0072] In this embodiment, the preparation method of the modified polyethylene glycol is as follows: Under a stirring state, drop 10 parts of polyethylene glycol by weight into 40 parts of deionized water, raise the temperature to 50 °C, then add 4 parts of 3-amino-1,2-propanediol and 3 parts of diethylamine, raise the temperature to 80 °C and stir for 30 min, add 2 parts of sodium dodecylbenzenesulfonate and 3 parts of sodium alginate, and continue to stir for 20 min to obtain the modified polyethylene glycol.

[0073] Example 4

[0074] The embodiment of the present invention provides a microbial inoculant, which includes Lactobacillus ZN-R1 of Example 1, and also includes modified zeolite and modified polyethylene glycol. The mass ratio of Lactobacillus, modified zeolite and modified polyethylene glycol is 1:125:25. The preparation method of the microbial inoculant includes the following steps:

[0075] Weigh each raw material according to the ratio;

[0076] Sterilely open Lactobacillus, pick the bacterial strain and streak inoculate it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0077] Pick the activated strain and inoculate it in an enrichment medium for enrichment culture;

[0078] Inoculate the enriched strain in a liquid medium to culture to obtain a liquid seed solution;

[0079] Perform solid-state fermentation on the liquid seed solution to obtain a solid-state fermentation product;

[0080] Mix the solid-state fermentation product with the modified zeolite and the modified polyethylene glycol to obtain the microbial inoculant.

[0081] Among them, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, and perform preliminary crushing after drying; then place it in a muffle furnace and program the temperature to 1100 °C, keep it at a constant temperature for roasting for 1.5 h, and grind it to a particle size of 200 mesh after cooling; then add 35% hydrochloric acid solution and soak for 2.5 h; filter to obtain product A, add product A to 12.5 times the weight of deionized water, add 25 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:8; then slowly add it to the mixture, raise the temperature to 70 °C, stir and react for 4 h, filter to obtain product B, dry product B at 90 °C for 40 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 25 min under the conditions of a power frequency of 12 kHz, a working voltage of 20 kV, and a discharge power of 80 W to obtain the modified zeolite.

[0082] In this embodiment, the preparation method of the modified polyethylene glycol is as follows: Under a stirring state, 13 parts by weight of polyethylene glycol are added dropwise to 45 parts of deionized water, the temperature is raised to 55 °C, then 6 parts of 3-amino-1,2-propanediol and 6 parts of diethylamine are added, the temperature is raised to 85 °C and stirred for 40 min, 3 parts of sodium dodecylbenzenesulfonate and 4 parts of sodium alginate are added, and stirring is continued for 30 min to obtain the modified polyethylene glycol.

[0083] Example 5

[0084] The embodiment of the present invention provides a microbial inoculant, which includes the Lactobacillus ZN-R1 of Example 1, and also includes modified zeolite and modified polyethylene glycol. The mass ratio of the Lactobacillus, modified zeolite and modified polyethylene glycol is 1:140:27. The preparation method of the microbial inoculant includes the following steps:

[0085] Weigh each raw material according to the ratio;

[0086] Aseptically open the Lactobacillus, pick the strain and streak it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0087] Pick the activated strain and inoculate it into an enrichment medium for enrichment culture;

[0088] Inoculate the enriched strain into a liquid medium to culture and obtain a liquid seed solution;

[0089] Perform solid-state fermentation on the liquid seed solution to obtain a solid-state fermentation product;

[0090] Mix the solid-state fermentation product with the modified zeolite and the modified polyethylene glycol to obtain the microbial inoculant.

[0091] Among them, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, dry it and then conduct preliminary crushing; then place it in a muffle furnace and raise the temperature programatically to 1200 °C, keep it at a constant temperature and bake for 2 h, and grind it to a particle size of 300 mesh after cooling; then add 40% hydrochloric acid solution and soak for 3 h; filter to obtain product A, add product A to 15 times the weight of deionized water, add 30 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:10; then slowly add it to the mixture, raise the temperature to 80 °C, stir and react for 5 h, filter to obtain product B, dry product B at 100 °C for 50 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 30 min under the conditions of a power frequency of 15 kHz, a working voltage of 25 kV, and a discharge power of 85 W to obtain the modified zeolite.

[0092] In this embodiment, the preparation method of the modified polyethylene glycol is as follows: Under stirring, add 16 parts of polyethylene glycol dropwise to 50 parts of deionized water, raise the temperature to 60 °C, then add 8 parts of 3-amino-1,2-propanediol and 9 parts of diethylamine, raise the temperature to 90 °C and stir for 50 min, add 4 parts of sodium dodecylbenzenesulfonate and 5 parts of sodium alginate, and continue to stir for 40 min to obtain the modified polyethylene glycol.

[0093] Example 6

[0094] The embodiment of the present invention provides a microbial inoculum, which includes the Lactobacillus ZN-R1 of Example 1, and also includes modified zeolite and modified polyethylene glycol. The mass ratio of the Lactobacillus, modified zeolite and modified polyethylene glycol is 1:150:30. The preparation method of the microbial inoculum includes the following steps:

[0095] Weigh each raw material according to the ratio;

[0096] Sterilely open the Lactobacillus, pick the strain and streak it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies;

[0097] Pick the activated strain and inoculate it into an enrichment medium for enrichment culture;

[0098] Inoculate the enriched strain into a liquid medium to culture and obtain a liquid seed solution;

[0099] Perform solid-state fermentation on the liquid seed solution to obtain a solid-state fermentation product;

[0100] Mix the solid-state fermentation product with the modified zeolite and the modified polyethylene glycol to obtain the microbial inoculum.

[0101] Among them, the preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, dry it and then conduct preliminary crushing; then place it in a muffle furnace and raise the temperature programatically to 1200 °C, keep it at a constant temperature for roasting for 2 h, and grind it to a particle size of 300 mesh after cooling; then add 40% hydrochloric acid solution and soak for 3 h; filter to obtain product A, add product A to 15 times the weight of deionized water, add 30 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:10; then slowly add it to the mixture, raise the temperature to 80 °C, stir and react for 5 h, filter to obtain product B, dry product B at 100 °C for 50 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 30 min under the conditions of a power frequency of 15 kHz, a working voltage of 25 kV, and a discharge power of 85 W to obtain the modified zeolite.

[0102] In this embodiment, the preparation method of the modified polyethylene glycol is as follows: Under stirring conditions, add 16 parts of polyethylene glycol dropwise to 50 parts of deionized water by weight, raise the temperature to 60 °C, then add 8 parts of 3-amino-1,2-propanediol and 9 parts of diethylamine, raise the temperature to 90 °C and stir for 50 min, add 4 parts of sodium dodecylbenzenesulfonate and 5 parts of sodium alginate, and continue to stir for 40 min to obtain the modified polyethylene glycol.

[0103] Comparative Example 1: Compared with Example 4, the modified zeolite is replaced with ordinary zeolite.

[0104] Comparative Example 2: Compared with Example 4, the modified polyethylene glycol is replaced with ordinary polyethylene glycol.

[0105] Comparative Example 3: Compared with Example 4, the modified zeolite is replaced with ordinary zeolite and the modified polyethylene glycol is replaced with ordinary polyethylene glycol.

[0106] Comparative Example 4: A commercially available microbial agent for repairing saline-alkali soil.

[0107] Test

[0108] Test site: Saline-alkali soil in Gansu. The pH value of the soil at the test site is about 8.8, and the total salt content is about 5.25 g / kg;

[0109] Control group: Conventional fertilization, without adding a repairing microbial agent;

[0110] 9 experimental groups: Conventional fertilization + respectively applying the microbial agents of Examples 1-5 and Comparative Examples 1-4. The microbial agent is applied at 300 kg / mu. After 60 days, the pH values and total salt contents of the control group and the experimental groups are measured as follows in the table:

[0111]

[0112] As can be seen from the above results, after the microbial inoculant prepared by the present invention acts on saline-alkali soil, the physical and chemical properties of the saline-alkali soil are significantly improved. In particular, the addition of modified zeolite and modified polyethylene glycol has a synergistic effect and can further repair saline-alkali soil.

[0113] It should be noted that for the foregoing embodiments, for the sake of simple description, they are all expressed as a series of action combinations. However, those skilled in the art should know that the present invention is not limited by the described action sequence, because according to the present invention, certain steps may be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0114] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the protection scope of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still, without conflict, make combinations, additions, deletions or other adjustments to the features in the embodiments of the present invention according to the situation without creative efforts, so as to obtain different technical solutions that are essentially not divorced from the concept of the present invention, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A preparation method of a microbial inoculum, characterized in that, It includes the following steps: Weigh each raw material according to the ratio; Sterilely open Lactobacillus, pick the strain and streak inoculate it on a solid slant medium, and culture it in a constant temperature incubator at 30 °C for 36 hours until the slant is covered with colonies; Pick the activated strain and inoculate it into an enrichment medium for enrichment culture; Inoculate the enriched strain into a liquid medium to obtain a liquid seed culture; Perform solid-state fermentation on the liquid seed culture to obtain a solid-state fermentation product; Mix the solid-state fermentation product with modified zeolite and modified polyethylene glycol to obtain a microbial inoculant; The name of the Lactobacillus is Lactobacillus ( Lentilactobacillus parafarraginis ) ZN-R1, which was deposited at the China Center for Type Culture Collection on November 23, 2023, with the deposit number of CCTCC NO: M 20232305; The preparation method of the modified zeolite is as follows: Take zeolite, clean the surface, dry it and then conduct preliminary crushing; then place it in a muffle furnace and program the temperature to 1000 - 1200 °C, keep it at a constant temperature for roasting for 1 - 2 h, cool it and grind it to a particle size of 200 - 300 mesh; then add 30 - 40% hydrochloric acid solution and soak it for 2 - 3 h; filter to obtain product A, add product A to 10 - 15 times the weight of deionized water, add 20 - 30 wt% vinyltrimethoxysilane solution, and ultrasonically disperse it until it is uniform to obtain a mixture; mix glycerol and deionized water according to a mass ratio of 1:6 - 10; then slowly add it to the mixture, raise the temperature to 60 - 80 °C, stir and react for 3 - 5 h, filter to obtain product B, dry product B at 80 - 100 °C for 30 - 50 min and then evenly spread it on the ground electrode in a low-temperature plasma treatment device, and treat it for 20 - 30 min under the conditions of a power frequency of 10 - 15 kHz, a working voltage of 15 - 25 kV, and a discharge power of 75 - 85 W; The preparation method of the modified polyethylene glycol is as follows: Under stirring conditions, drop 10 - 16 parts by weight of polyethylene glycol into 40 - 50 parts of deionized water, raise the temperature to 50 - 60 °C, then add 4 - 8 parts of 3 - amino - 1,2 - propanediol and 3 - 9 parts of diethylamine, raise the temperature to 80 - 90 °C and stir for 30 - 50 min, add 2 - 4 parts of sodium dodecylbenzenesulfonate and 3 - 5 parts of sodium alginate, and continue to stir for 20 - 40 min.

2. The preparation method of the microbial inoculum according to claim 1, characterized in that, The 16S rDNA gene sequence of the Lactobacillus is as shown in SEQ ID No.

1.

3. The preparation method of the microbial inoculum according to claim 1, characterized in that, The morphological characteristics are as follows: Gram-positive bacillus, the cell morphology is short rod-shaped, both ends are blunt round, arranged in short chains, without spores, presenting milky white protrusions on the MRS plate medium, with a diameter of 1 - 3 mm, round and regular, the surface is smooth and moist, without pigment production, and the back of the colony is white to off-white.

4. The preparation method of the microbial inoculum according to claim 1, characterized in that, The mass ratio of the Lactobacillus, the modified zeolite and the modified polyethylene glycol is 1:100 - 150:20 - 30.

5. Application of the microbial inoculant prepared by the preparation method of the microbial inoculant according to any one of claims 1 - 4 in repairing saline-alkali soil.

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