Kocuria carnovorans NY8 and application thereof in saline-alkali soil improvement

Abstract

The invention discloses a Kocuria carnovorans NY8 strain and application thereof in saline-alkali soil improvement, and belongs to the technical field of microbial strains and application thereof. The invention discloses a salt and alkali tolerant Kocuria carniphila NY8 (Kocuria carniphila NY8) and an application of the salt and alkali tolerant Kocuria carniphila NY8. Through sampling, separation, screening and physiological and biochemical identification from rhizosphere soil of 20-year-old elaeagnus angustifolia in Yuopu Lake County (N39 degrees 16 '14.79 E77 degrees 15' 18.02) in Kara city in the Uygur autonomous region in Xinjiang, it is determined that the saline-alkaline tolerant cocuria sarcophila provided by the invention has relatively strong saline-alkaline tolerance and a good soil improvement effect. Based on the strain, the fermentation process is further optimized, and the strain has the potential of being widely applied to the field of saline-alkali soil improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial strains and their applications, in particular to a strain of Coxsackie's bacterium NY8 and its application in improving saline-alkali soil. Background Art

[0002] Saline-alkali lands are areas where soil salt accumulates and becomes alkaline due to factors such as arid climate, high groundwater mineralization, and inappropriate irrigation. These areas are characterized by high salinity, high alkalinity, and low nutrient content, severely limiting the soil's value and ecological function. The loose soil structure, poor permeability, and low organic matter content in saline-alkali lands hinder plant growth and reduce microbial activity, severely impacting the balance of regional ecosystems and agricultural production efficiency.

[0003] my country has 550 million mu (approximately 1.5 million hectares) of saline-alkali land that can be developed and utilized. Comprehensively utilizing saline-alkali land is of strategic importance for ensuring national food security and ensuring China's rice bowl remains a safe haven. Traditional land improvement methods, such as chemical and mechanical soil amendments, can alleviate the salinization problem to a certain extent, but they are difficult to implement long-term due to their high costs, long processing times, and potential for secondary pollution. Using salt-tolerant microorganisms for soil improvement and ecological restoration has become an efficient and green approach to saline-alkali land management. Summary of the Invention

[0004] The present invention aims to provide a strain of Coxiella carnivora NY8 and its application in saline-alkali soil improvement to address the problems of the prior art described above. The present invention provides a strain of Coxiella carnivora NY8 with starch hydrolase activity and can be widely used in the field of starch hydrolysis. The present invention optimizes the fermentation formula and conditions of strain NY8, making this salt- and alkali-tolerant strain effective in improving saline-alkali soils. Therefore, Coxiella carnivora NY8 has broad application prospects in the enzyme preparation industry and saline-alkali soil improvement.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a strain of Kocuria carniphila NY8, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 3, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33699.

[0007] The second technical solution of the present invention is a microbial agent, comprising the aforementioned Coxiella carnivora NY8.

[0008] The third technical solution of the present invention is the use of the carnivorous Coxsackie bacteria NY8 or the microbial agent in improving saline-alkali soil.

[0009] A fourth technical solution of the present invention is the use of the carnivorous Coxsackie NY8 or the microbial agent in the preparation of products for improving saline-alkali soil.

[0010] The fifth technical solution of the present invention is a product for improving saline-alkali soil, comprising the aforementioned Coxiella carnivora NY8 or the aforementioned microbial agent.

[0011] The sixth technical solution of the present invention is the use of the carnivorous Coxsackie NY8 or the microbial agent in hydrolyzing starch.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The present invention provides a strain of Coxiella carnivora and its uses. This strain, through metabolic expression, is negative for proteases, cellulases, glucanases, and chitinases, and positive for starch hydrolases. Furthermore, the present invention optimizes the fermentation process of this strain, and the resulting fermentation broth demonstrates excellent results in improving the physical and chemical properties of saline-alkali soils. The salt-alkali-tolerant bacterium NY8 obtained by screening in this invention is significantly different from other previously disclosed strains of Coxiella carnivora and can be widely used in the production of starch hydrolases and saline-alkali soil improvement, possessing significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 These are the pH tolerance test results of Coxsackie's NY8.

[0016] Figure 2 These are the results of NaCl tolerance test of Coxsackie's NY8.

[0017] Figure 3 These are the results of Na2SO4 tolerance test of Coxsackie's NY8.

[0018] Figure 4 These are the results of Na2CO3 tolerance test of Coxsackie's NY8.

[0019] Figure 5 These are the results of NaHCO3 tolerance test of Coxsackie's NY8.

[0020] Figure 6 This is a picture of the carnivorous Coxsackie NY8 bacteria.

[0021] Figure 7Phylogenetic tree of Coxiella carnivora NY8.

[0022] Figure 8 To detect the starch hydrolase activity of Coxsackie's NY8.

[0023] Figure 9 To detect the activity of NY8 protease of Coxiella carnivora.

[0024] Figure 10 To detect the cellulase activity of Coxsackie's NY8 bacteria.

[0025] Figure 11 To detect the glucanase activity of Coxiella carnivora NY8.

[0026] Figure 12 To detect the chitinase activity of Coxsackie's bacterium NY8.

[0027] Figure 13 Soil nutrient test results.

[0028] Figure 14 Soil enzyme activity test results. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0035] An embodiment of the present invention provides a strain of Kocuria carniphila NY8, which was deposited on March 3, 2025 at the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33699.

[0036] An embodiment of the present invention further provides a microbial agent, comprising the aforementioned Coxiella carnivora NY8.

[0037] The embodiment of the present invention also provides the use of the carnivorous Coxsackie NY8 or the microbial agent in improving saline-alkali soil.

[0038] The embodiments of the present invention also provide use of the Coxiella carnivora NY8 or the microbial agent in preparing a product for improving saline-alkali soil.

[0039] An embodiment of the present invention further provides a product for improving saline-alkali soil, comprising the Coxiella carnivora NY8 or the microbial agent.

[0040] The embodiment of the present invention also provides the use of the Coxiella carnivora NY8 or the microbial agent in hydrolyzing starch.

[0041] The present invention discloses a salt- and alkali-tolerant carniphilic bacterium, Kocuria carniphila NY8, and its applications. By sampling rhizosphere soil from 20-year-old Elaeagnus angustifolia in Yuepuhu County, Kashgar City, Xinjiang Uygur Autonomous Region (N39°16'14.79E77°15'18.02), followed by isolation, screening, and physiological and biochemical characterization, it was determined that the salt- and alkali-tolerant carniphilic Kocuria carniphila provided by the present invention has strong salt- and alkali-tolerant capabilities and excellent soil-improving effects. Based on this strain, the present invention further optimizes the fermentation process, demonstrating its potential for widespread application in saline-alkali soil improvement.

[0042] Kocuria carniphila is a Gram-positive coccus belonging to the phylum Actinobacteria, and has demonstrated strong adaptability in saline-alkali land and extreme environments. Unlike traditional actinomycetes, Kocuria carniphila does not have a branched hyphal structure, but exists in the form of a coccus, with high metabolic activity and strong stress resistance. Studies have shown that the bacterium can grow stably under high salt concentrations (15%) and strong alkalinity (pH 12.0-14.0). The active substances secreted during its metabolism can reduce soil salt concentration, neutralize alkaline substances, and improve soil structure. These characteristics give it unique advantages in the ecological restoration of saline-alkali land.

[0043] At present, research on salt-alkali tolerant functional strains mainly focuses on ecological restoration of specific environments, and Kocuria carniphila has become a potential efficient microbial remediation tool due to its excellent salt-alkali tolerance. This strain can significantly reduce soil salt stress in saline-alkali land and enhance the soil's nutrient supply capacity, thereby effectively improving the physical and chemical properties of saline-alkali soil and providing a practical solution for the ecological restoration of saline-alkali land. In addition, Kocuria carniphila can also secrete enzymes to promote the decomposition and transformation of organic matter in saline-alkali soil, thereby further improving the utilization efficiency of soil nutrients and ecological functions. Therefore, the development of salt-alkali tolerant Kocuria carniphila strains and their application technology in saline-alkali land management not only provides an efficient and safe solution for the ecological restoration of saline-alkali land, but also provides important technical support for the sustainable utilization of saline-alkali soil resources.

[0044] The main raw and auxiliary materials, reagents and instruments involved in this invention are:

[0045] Culture medium: 10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1000 ml sterile water, pH 7.0.

[0046] Key instruments and reagents included: MSSPX-250 biochemical incubator, MLS-3020 high-pressure steam autoclave, SW-CJ-1FB single-person double-sided clean bench, E360K centrifuge, HWY-100 thermostatic shaker. Eppendorf 5345 polymerase chain reaction (PCR), Bio-Rad Model 200 / 2.0 electrophoresis system, United-Bio GK-330Cplus gel imager, and PCR premix (TaKaRa Biotechnology). All other reagents were of analytical grade.

[0047] Example 1

[0048] Isolation and Culture Methods of NY8 (Kocuria carniphila)

[0049] 1 Separation

[0050] The carnivorous Coxiella NY8 strain was isolated from a 20-year-old rhizosphere soil sample from the Angustifolia elata plant in Yuepuhu County, Kashgar City, Xinjiang Uygur Autonomous Region (N41°18'25.12E 72°57'46.15). The rhizosphere soil was collected using a five-point sampling method, mixed, and placed in a sterile container. The container was then transported to the laboratory using a vehicle refrigerator. A single strain of carnivorous Coxiella NY8 was isolated using a plate dilution and spread method.

[0051] Separation steps: According to the gradient dilution method, 10 g of soil sample was weighed and added to 90 mL of sterile water. After activation at 30 °C for 30 min, gradient dilution was performed. 10 -3 , 10 -4 , 10 -5 The dilutions were spread onto plates containing 10% NaCl-containing NA medium, with three replicates per treatment, and incubated at 37°C. After colonies emerged, selected colonies of varying shape, size, and color were streaked onto fresh 10% NaCl-containing NA medium until no contaminants remained. A portion of the purified strains were stored in lyophilized ampoules, glycerol tubes, and liquid nitrogen, while a portion was stored at 4°C and used directly in subsequent studies.

[0052] 2 Culture conditions

[0053] Culture medium: NA medium: peptone 10.0 g, beef extract 3.0 g, NaCl 5.0 g, agar 15.0 g, constant volume to 1 L, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0054] The growth tolerance range of NaCl was 0.5-15%, and the optimal growth NaCl was 0-5%. The optimal growth pH was 7.0, and the optimal growth temperature was 30°C. Based on the characteristics of the carnivorous Coxiella NY8 strain provided by the present invention, its specific growth factor was determined, and the strain NY8 was inoculated and cultured. The results are shown in Table 1.

[0055] Table 1 Effects of temperature, pH and salt on the growth of strain NY8

[0056]

[0057]

[0058] Example 2

[0059] Physical and chemical properties of NY8 (Kocuria carniphila)

[0060] Physical and chemical properties testing methods:

[0061] (1) pH tolerance test

[0062] Prepare NA culture medium with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0. Pick out the purified single strains and inoculate them onto NA plates with different pH values. Incubate at 30°C for 48 hours, observe the growth and record the results. Figure 1 As shown, strain NY8 could not grow on NA plates with pH 3 to pH 6, but grew well on NA plates with pH 7 to pH 14, indicating that the optimal growth environment for this strain is neutral or alkaline conditions.

[0063] (2) Temperature tolerance test

[0064] The test bacterial liquid was inoculated into the sterilized NB liquid culture medium at a 1% inoculum size and cultured in a shaker at nine temperatures of 25, 28, 30, 33, 35, 37, 40, 45, and 50°C and a speed of 180 r / min for 48 h. The bacterial liquid cultured in a shaker at 30°C and 180 r / min for 48 h was used as a control. The growth was observed and recorded.

[0065] (3) Sodium chloride tolerance test

[0066] Prepare NA culture media with NaCl concentrations of 0.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, and 15%. Inoculate purified strains onto NA plates containing different NaCl concentrations and incubate at 30°C for 48 h. Observe and record growth. Figure 2 The results showed that the strain NY8 had strong salt tolerance and could grow normally at a NaCl concentration of 0.5% to 15%.

[0067] (4) Sodium sulfate tolerance test

[0068] Prepare NA culture medium with Na2SO4 concentrations of 0.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, and 15%. Pick out the purified single strains and inoculate them onto NA plates with different Na2SO4 concentrations. Incubate at 30℃ for 48h, observe the growth and record the results. Figure 3 As shown, strain NY8 can adapt to an environment with a Na2SO4 concentration of 0.5% to 9%, but cannot grow under conditions of a concentration of 10% or above.

[0069] (5) Sodium carbonate tolerance test

[0070] Prepare NA culture medium with Na2CO3 concentrations of 0.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, and 15%. Pick out the purified single strains and inoculate them onto NA plates with different Na2CO3 concentrations. Incubate at 30℃ for 48h, observe the growth and record the results. Figure 4 As shown, strain NY8 can grow normally on NA plates with a concentration of 0.5% to 10% Na2CO3, but its growth is significantly inhibited as the concentration of Na2CO3 increases, and stops growing under conditions of a concentration of 11% to 15% Na2CO3.

[0071] (6) Sodium bicarbonate tolerance test

[0072] Prepare NA culture medium with NaHCO3 concentrations of 0.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, and 15%. Pick out the purified single strains and inoculate them on NA plates with different NaHCO3 concentrations. Incubate at 30℃ for 48h, observe the growth and record the results. Figure 5 As shown, strain NY8 can grow normally on NA plates with a concentration of 0.5% to 10% NaHCO3. However, as the concentration of Na2HCO3 increases, its growth is significantly restricted and it completely stops growing under the conditions of a Na2HCO3 concentration of 11% to 15%.

[0073] (7) Citrate utilization test

[0074] Inoculate the test strain onto citrate medium (0.5 g NaCl, 0.02 g MgSO₄, 0.1 g NH₄H₂PO₄, 0.1 g K₂HPO₄, 0.5 g sodium citrate, 4 mL bromothymol blue (0.2%), 2 g agar powder, 100 mL distilled water). Use uninoculated citrate medium as a control and incubate at 30°C for 48 hours. A positive result is observed if bacterial growth is observed and the medium changes from light green to blue; a negative result is observed if no growth occurs.

[0075] (8) MR test

[0076] Inoculate the test bacteria into a glucose-peptone water culture medium (0.5 g glucose, 0.5 g peptone, 0.2 g K₂HPO₄, 100 mL distilled water). Use an uninoculated culture medium as a control and incubate at 30°C for 48 hours. Add methyl red reagent dropwise to each culture medium, mix thoroughly, and observe. If the color turns red, it is positive; if it turns yellow, it is negative.

[0077] (9) H2S test

[0078] The test bacteria were inoculated into Chessner medium (1 g of peptone, 0.05 g of ferric citrate, 1.8 g of agar, and 100 mL of distilled water), cultured at 30°C for 48 h, and then observed to see if melanin was produced. If melanin was produced, hydrogen sulfide was produced; otherwise, no hydrogen sulfide was produced.

[0079] (10) VP test

[0080] Inoculate the test bacteria into a glucose-peptone culture medium (0.5 g glucose, 0.5 g peptone, 0.2 g K₂HPO₄, 100 mL distilled water). Use an uninoculated culture medium as a control and incubate at 30°C for 48 hours. Mix the culture medium with 40% NaOH and add a small amount of creatine. A positive test is considered if the culture medium turns red.

[0081] Physical and chemical properties of strain NY8 were tested, revealing strong salt and alkali tolerance, and the strain was able to grow normally in 15.0% NaCl and pH 14. Other physical and chemical properties are shown in Table 2.

[0082] Table 2 Physiological and biochemical characteristics of strain NY8

[0083]

[0084]

[0085] Note: “+”: positive; “-”: negative.

[0086] Example 3

[0087] Identification of carnivorous Coxsackie strains

[0088] 1 Morphological identification

[0089] The carniphilic Kocuria NY8 strain screened by the present invention is a Gram-positive bacterium with medium-sized colonies that are yellow to orange-yellow and flat. It grows well on ordinary agar medium, has a dense texture, and lacks aerial hyphae. Microscopic observation reveals that the cells are spherical to oval, lack hyphal structures, and do not form spores. See the attached diagram for the colony morphology of Kocuria carniphila NY8 CGMCC No. 33699. Figure 6 .

[0090] 2 Molecular biological identification

[0091] The single colony obtained by screening was inoculated into NB culture medium and placed at 37°C and 180 r·min -1The culture was shaken on a shaker for 12-24 hours, and genomic DNA was extracted using a bacterial genomic DNA extraction kit. Using DNA as a template, the PCR amplification primers were 27F (SEQ ID NO. 1: 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (SEQ ID NO. 2: 5′-CGGTTACCTTGTTACGACTTC-3′). The PCR reaction system (50 μL) is shown in Table 3. PCR reaction conditions: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 40 seconds, and 72°C for 1.5 minutes, 35 cycles; 72°C for 5 minutes. The resulting PCR amplification products were verified by 1% agarose gel electrophoresis and subsequently sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. A phylogenetic tree was constructed using the neighbor-joining method of Saitou and Nei using MEGA 7.0 software. The results are shown in the attached figure. Figure 7 ), this strain is on the same branch as Kocuria carniphila, indicating their closest relationship. Sequence alignment further revealed that the NY8 strain shared the highest homology with Kocuria carniphila. Based on microbial classification methods, the NY8 strain was preliminarily identified as Kocuria carniphila, belonging to the phylum Actinobacteria.

[0092] Table 3 PCR reaction system

[0093]

[0094] The present invention isolated a carniphila strain Kocuria carniphila NY8, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 3, 2025, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33699.

[0095] Example 4

[0096] Screening of enzyme activity of carnivorous Coxsackie strains

[0097] The plate clearing zone method was used to screen enzyme-producing strains for five enzyme activities: amylolytic enzyme, protease, cellulase, glucanase, and chitinase. Test strains were spotted onto screening plates and incubated at 37°C for 3 days before observation. The enzyme activity assay medium and its components are shown in Table 4.

[0098] Table 4 Enzyme activity detection medium and its components

[0099]

[0100] The enzyme activity detection methods are as follows:

[0101] Screening for starch hydrolyzing enzymes: Sterilize the culture medium to 1L, set the pH to 7.0-7.2, and sterilize at 121°C for 20 minutes. Pour the culture medium onto the plate, inoculate the bacteria, and incubate at 30°C for 72 hours. Stain with iodine solution and observe the clearing zone. Positive colonies will have clearing zones around them; negative colonies will have no clearing zones. To prepare the iodine solution: Dissolve 2g KI in 10ml of water, add 1g I2, mix thoroughly, and add water to 300ml. Protect from light. Dilute the solution 2-fold before use.

[0102] Protease screening: Sterilize the culture medium to 1 L, pH 7.0-7.2, and autoclave at 121°C for 20 minutes. Pour the culture medium onto the plate, inoculate the bacteria, and incubate at 30°C for 72 hours. Observe the clearing zone. A positive colony will be surrounded by a clearing zone; a negative colony will be negative.

[0103] Cellulase Screening: Sterilize the culture medium to 1 L, pH 7.0-7.2, and autoclave at 121°C for 20 minutes. After sterilization, cool the culture to 60°C, pour the plate, inoculate, and incubate at 30°C for 72 hours. Observe for clearing zones. Positive colonies will have clearing zones around them; negative colonies will lack clearing zones. Congo Red Staining: Stain with 0.5% Congo Red for 1 hour, decolorize with 1M NaCl for 3 hours, rinse, and observe for clearing zones.

[0104] Glucanase Screening: Sterilize the culture medium to 1 L, pH 7.0-7.2, and sterilize at 121°C for 20 minutes. Inoculate the culture medium and incubate at 30°C for 72 hours. Observe the hydrolysis zone. The presence of a hydrolysis zone is considered positive, while the absence of a hydrolysis zone is considered negative.

[0105] Chitinase screening: Sterilize the culture medium to 1 L, pH 7.0-7.2, and sterilize at 121°C for 20 minutes. Pour the culture medium onto the plate, inoculate the bacteria, and incubate at 30°C for 72 hours. Observe the clearing zone directly; the presence of a clearing zone indicates a positive result, while the absence of a clearing zone indicates a negative result.

[0106] The enzyme activity screening was carried out according to the above method, and the results are shown in Table 5 and Figure 8-12 shown.

[0107] Table 5 Enzyme activity screening results

[0108]

[0109] Note: “+”: positive; “-”: negative.

[0110] Example 5

[0111] Development of fermentation process for carnivorous Coxsackie strain

[0112] (1) Preparation of strain fermentation broth

[0113] A single colony was picked and inoculated into 100 mL of NB medium, placed in a shaking incubator at 37°C and 180 rpm for 18 h.

[0114] (2) Single factor screening of carbon sources

[0115] Add 10 g / L of different carbon sources (glucose, sucrose, molasses, and white sugar), sterilize, and then add 1% (v:v) NY8 fermentation broth. Measure the OD of the fermentation broth by UV spectrophotometry. 600 The initial fermentation conditions were natural pH, 100 mL / 250 mL of liquid volume, and shaking culture at 28°C and 180 rpm for 2 days.

[0116] Through single factor screening of carbon sources, it was found that none of the four carbon sources would produce precipitation to block the spray bottle nozzle. By analyzing the OD 600 The results are shown in Table 6. The fermentation broth concentration of the molasses group (2.46) was significantly higher than that of the other carbon source groups. The fermentation broth concentrations of the other carbon source groups were white sugar > sucrose > glucose > CK. Molasses was selected as the carbon source in subsequent experiments.

[0117] Table 6 Single factor screening of carbon sources

[0118]

[0119] (3) Single factor screening of nitrogen sources

[0120] 10 g / L of different nitrogen sources (yeast extract, beef extract powder, peptone, soy peptone) were added, and after sterilization, 1% (v:v) NY8 fermentation broth was added. The fermentation broth concentration detection method and initial fermentation conditions were the same as above.

[0121] Through single factor screening of nitrogen sources, it was found that none of the four nitrogen sources would produce precipitation to block the spray bottle nozzle. By analyzing the OD 600 The results are shown in Table 7. The fermentation broth concentration of the yeast extract group (2.37) was significantly higher than that of the other nitrogen source groups. The fermentation broth concentrations of the other nitrogen source groups were in the order of soy peptone > peptone > beef extract powder > CK from high to low. Yeast extract was selected as the nitrogen source in subsequent experiments.

[0122] Table 7 Single factor screening of nitrogen sources

[0123]

[0124] (4) Carbon-nitrogen ratio screening

[0125] The carbon and nitrogen sources screened above were added to the initial fermentation medium according to the ratio of CN1 to CN16, and then sterilized and added with 1% (v:v) NY8 fermentation broth. The fermentation broth concentration detection method and initial fermentation conditions were the same as above.

[0126] The results of carbon-nitrogen ratio screening are shown in Table 8. The fermentation liquid OD under CN10 treatment 600 It was significantly higher than other carbon-nitrogen ratio treatments, and 1.5% molasses and 1% yeast extract were subsequently selected for experiments.

[0127] Table 8 Carbon-nitrogen ratio settings

[0128]

[0129]

[0130] (5) Single factor screening of inorganic salts

[0131] The carbon and nitrogen sources obtained by the above screening were added into the initial fermentation medium according to the ratio obtained by screening (4), and 0.5 g / L of different inorganic salts (MnCl2, MnSO4·7H2O, (NH4)2SO4, NaCl, K2HPO4, MgSO4, KCl, FeCl2, NaH2PO4, FeSO4, Na2HPO4, KH2PO4) were added. After sterilization, 1% (v:v) NY8 fermentation broth was added. The fermentation broth concentration detection method and initial fermentation conditions were the same as above.

[0132] The results of single factor screening of inorganic salts are shown in Table 9. The OD of fermentation liquid under NaH2PO4 treatment 600 The fermentation formula was significantly higher than that of the other inorganic salt treatments, followed by (NH4)2SO4 > K2HPO4 > MnCl2 > FeSO4 > FeCl2 > KCl > Na2HPO4 > KH2PO4 > Nacl2 > MgSO4 > MnSO4·7H2O. The first four were selected for subsequent experiments. The final fermentation formula was: peptone 5g / L (from the initial culture medium), NaCl 1g / L (from the initial culture medium), molasses 15g / L, yeast extract 15g / L (containing 5g of yeast extract from the initial culture medium), NaH2PO4 0.5g / L, (NH4)2SO4 0.5g / L, K2HPO4 0.5g / L, and MnCl2 0.5g / L.

[0133] Table 9 Single factor screening of inorganic salts

[0134]

[0135] (6) pH single factor screening

[0136] The selected formulations were prepared into five pH gradient fermentation shake flasks (3, 5, 7, 9, and 11). The pH of the culture medium was retested after sterilization and before inoculation to confirm that the pH was not affected by high-temperature sterilization. 1% (v:v) NY8 fermentation broth was then inoculated. Otherwise, the pre-sterilization pH was adjusted based on the pH change before and after sterilization until the post-sterilization pH met the five experimental requirements. The fermentation broth concentration testing method and initial fermentation conditions were the same as above.

[0137] (7) Temperature single factor screening

[0138] The fermentation shake flasks were prepared with the above-screened formula, adjusted to the screened pH conditions, sterilized, and added with 1% (v:v) NY8 fermentation broth. The flasks were placed in a shaker at different temperatures (28, 30, 33, 35, 37, 40, 45, and 50°C). The fermentation broth concentration was detected using the same method and rotation speed as above.

[0139] (8) Speed ​​single factor screening

[0140] The above-screened formula was prepared into fermentation shake flasks, sterilized, and then added with 1% (v:v) NY8 fermentation broth. The flasks were placed on a shaker with different speeds (100, 120, 140, 160, 180, and 200 r / min). The shaker temperature was set to the screened temperature, and the fermentation broth concentration was tested in the same manner as above.

[0141] The results of single factor screening of fermentation conditions are shown in Table 10. The OD value of fermentation liquid under pH 7.0 treatment was 600 Significantly higher than other pH treatments, 4 groups of 5 pH gradients can grow normally, and the subsequent experiment was conducted at pH 7.0; the fermentation liquid OD under 33℃ treatment 600 Significantly higher than other temperature treatments, under 9 temperature treatments, 7 groups of strains can grow normally, and the fermentation liquid OD under 30-37℃ 600 Significantly higher than the fermentation broth OD at 25-28℃ and 40-50℃ 600 The fermentation liquid OD under the treatment of 160r / min speed was 600 Higher than other speed treatments, among which the fermentation liquid OD under 180r / min treatment was 600 Slightly lower than the fermentation liquid OD under 160r / min treatment 600 The results were negative, but no significant difference was found between the two. Therefore, 160 rpm was selected for subsequent experiments. The final fermentation conditions were: pH 7.0, 33°C, and 160 rpm. The final fermentation recipe is shown in Table 11.

[0142] Table 10 Single factor screening of fermentation conditions

[0143]

[0144] Table 11 Optimal fermentation conditions and formulation results

[0145]

[0146] Example 6

[0147] Improvement and remediation effects of Coxsackie NY8 on soil

[0148] Soil Type: This experiment used two different soil types: nutrient soil and saline-alkali soil. Different soil conditions were simulated through different mixes and treatments. See Table 12 for a detailed overview of the experimental treatments.

[0149] Table 12 Overview of soil improvement experimental treatments

[0150]

[0151] Test plants: Elaeagnus angustifolia plants in the seedling stage with relatively consistent growth.

[0152] Experimental facilities: The pot size was 18.9 cm long, 18.9 cm wide, and 18.9 cm high. The experiment set up two treatment groups and two control groups, with three replicates in each group.

[0153] Soil physical and chemical properties were tested twice during the experiment: the first test was conducted on the second day after transplanting (0d), and the second test was conducted on the 30th day of the experiment.

[0154] During the experiment, soil nutrient properties were tested once on the 30th day of the experiment.

[0155] During the experiment, soil enzyme activity (S-ALP, S-DHA, S-NAG) was determined once: on the 30th day of the experiment.

[0156] The experimental groups were divided into treatment group and control group.

[0157] The soil types of the control group were: A1 nutrient soil + saline-alkali soil (1:1) (v / v); A2 pure saline-alkali soil.

[0158] Before planting, the sterile blank fermentation liquid of the present invention was applied, diluted 5 times to 500 mL, sprayed on the ground and stirred, and no additional fermentation liquid was applied. The sterile blank fermentation liquid was applied once during the entire experimental period.

[0159] The soil types of the treatment groups were: B1 nutrient soil + saline-alkali soil (1:1) (v / v); B2 pure saline-alkali soil.

[0160] Before planting, apply the fermented NY8 fermentation liquid of the present invention, dilute it 5 times to 500mL (about 10 8CFU / mL), sprayed on the ground and stirred, and no additional fermentation liquid was applied. The microbial fermentation liquid of the present invention was applied once during the entire experimental period.

[0161] Groups A1 and B1, and groups A2 and B2 with the same soil types were selected for analysis respectively. The results are shown in Table 13.

[0162] Table 13 Test results of soil physical and chemical properties

[0163]

[0164] In terms of bulk density: the bulk density of group B1 on day 0 was 1.27 g / cm 3 , the 30th day is 1.25g / cm 3 , decreased by 0.02g / cm 3 Compared with group A1, the bulk density of group A1 at day 0 and day 30 decreased by 0.09 g / cm 3 and 0.17g / cm 3 The bulk density of group B2 on day 0 was 1.35 g / cm 3 , the 30th day is 1.38 g / cm 3 , increase by 0.03g / cm 3 Compared with group A1, the bulk density of group A1 decreased by 0.09 g / cm on the 0th and 30th day, respectively. 3 and 0.17g / cm 3 .

[0165] In terms of electrical conductivity, the conductivity of Group B1 decreased from 3.02 ms / cm on day 0 to 2.98 ms / cm on day 30, a decrease of 0.04 ms / cm. Compared with Group A1, the conductivity decreased by 0.43 ms / cm and 1.42 ms / cm on day 0 and day 30, respectively. The conductivity of Group B2 decreased from 3.95 ms / cm on day 0 to 3.92 ms / cm on day 30, a decrease of 0.03 ms / cm. Compared with Group A2, the conductivity decreased by 0.21 ms / cm and 0.26 ms / cm on day 0 and day 30, respectively.

[0166] In terms of pH, the pH of group B1 on day 30 was 6.91, a decrease of 0.06 from 7.02 on day 0. Compared with group A1, the pH of group B1 on day 0 and day 30 decreased by 0.69 and 0.79, respectively. The pH of group B2 on day 30 was 8.02, a decrease of 0.09 from 8.11 on day 0. Compared with group A2, the pH of group B2 on day 0 and day 30 decreased by 0.51 and 0.57, respectively.

[0167] Specific soil nutrient test results are shown in Table 14 and Figure 13 .

[0168] Table 14 Soil nutrient test results

[0169]

[0170] Total nutrient content indicators: In terms of total nitrogen content, the content of group B1 was significantly higher than that of the other three groups, and increased by 0.13g / kg compared with group A1. The total nitrogen content of group B2 was 0.46g / kg, which was 0.03g / kg higher than that of group A2. In terms of total phosphorus content, the total phosphorus content of group B1 was significantly higher than that of the other three groups, and there was no significant difference between groups A1, A2 and B2. Compared with group A1, the total phosphorus content of group B1 increased by 9.21%. In terms of total potassium content, the total potassium content of group B2 was higher than that of the other three groups, and there was no significant difference between the three groups. Compared with group A2, the total potassium content of group B2 increased by 2.1g / kg. Among the three total nutrient content indicators, the B1 and B2 treatment groups showed an upward trend in nutrient content compared with the A1 and A2 control groups, respectively.

[0171] Available nutrient indicators: In terms of alkaline nitrogen content, group B1 increased by 21.04 mg / kg compared to group A1, and group B2 increased by 22.44 mg / kg compared to group A2. In terms of available phosphorus, group B1 increased by 12.56 mg / kg compared to group A1, and group B2 increased by 1.00 mg / kg compared to group A2. In terms of available potassium, the B1 and B2 treatment groups had significantly higher levels of available potassium than the A1 and A2 control groups, with increases of 11.16 mg / kg and 4.60 mg / kg, respectively. Across all three available nutrient indicators, both the B1 and B2 treatment groups showed an upward trend in nutrient content compared to the A1 and A2 control groups.

[0172] The A1 and B1 groups, and A2 and B2 groups with the same soil type were selected for analysis (see Appendix for details). Figure 14 ).

[0173] The activities of S-ALP, S-DHA, and S-NAG in groups B1 and B2, which received NY8 fermentation broth, were significantly higher than those in groups A1 and A2, which did not receive NY8 fermentation broth. In terms of S-ALP, the S-ALP enzyme activity in group B1 (33.41 μmol / d / mg) increased by 14.09 μmol / d / mg compared to group A1 (19.32 μmol / d / mg); and the S-ALP enzyme activity in group B2 (19.21 μmol / d / mg) increased by 11.53 μmol / d / mg compared to group A2 (7.68 μmol / d / mg). In terms of S-DHA, the enzyme activity of group B1 (20.12 μmol / d / mg) increased by 30.48% compared with group A1 (15.42 μmol / d / mg); in group B2 (13.76 μmol / d / mg), it increased by 97.70% compared with group A2 (6.96 μmol / d / mg). In terms of S-NAG, the enzyme activity of group B1 (39.15 μmol / d / mg) increased by 15.00 μmol / d / mg compared with group A1 (24.15 μmol / d / mg); and in group B2 (21.63 μmol / d / mg), it increased by 1.52 μmol / d / mg compared with group A2 (20.11 μmol / d / mg).

[0174] The above results show that strain NY8 has a significant effect on promoting enzyme activity in soil and can enhance the decomposition of organic matter. Groups B1 and B2, which were treated with NY8 strain fermentation broth, showed significant increases in enzyme activities such as S-ALP, S-DHA, and S-NAG, indicating that it has a significant improvement effect on saline-alkali soil. In contrast, the enzyme activities of groups A1 and A2 were lower, with significant differences, further verifying that the blank fermentation medium had a limited effect on soil enzyme activity. Strain NY8 has shown good results in soil improvement and can significantly increase soil enzyme activity, thereby enhancing soil sustainability and agricultural productivity.

[0175] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Kocuria carniphila NY8, characterized in that: The strain was deposited in the General Microbiology Center of China Culture Collection Administration on March 3, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33699.

2. A microbial agent, characterized in that: The invention comprises the carnivorous Coxsackie bacterium NY8 according to claim 1.

3. Use of the Coxiella carnivora NY8 according to claim 1 or the microbial agent according to claim 2 in improving saline-alkali soil.

4. Use of the Coxiella carnivora NY8 according to claim 1 or the microbial agent according to claim 2 in preparing a product for improving saline-alkali soil.

5. A product for improving saline-alkali soil, characterized in that: The method comprises the carnivorous Coxsackie species NY8 according to claim 1 or the microbial agent according to claim 2.

6. Use of the Coxiella carnivora NY8 according to claim 1 or the microbial agent according to claim 2 in hydrolyzing amylase.

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