A type of Bacillus subtilis for kimchi and its application

Abstract

This invention relates to the field of microbial technology, and more particularly to a strain of *Bacillus subtilis* for kimchi and its applications. The preservation number of *Bacillus subtilis* for kimchi is CGMCC No. 30790. This invention screened a strain of *Bacillus subtilis* for kimchi from saline-alkali soil. This strain possesses the ability to promote plant growth under saline-alkali stress. Applying this strain under saline-alkali stress can effectively increase root length, stem length, fresh weight, and crude protein content of plants, while significantly reducing soil salinity and pH, and increasing available phosphorus content. The *Bacillus subtilis* for kimchi provided by this invention offers a good solution for planting in saline-alkali soil, which has significant application value in the fields of ecological restoration and soil improvement.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Bacillus subtilis for kimchi and its applications. Background Technology

[0002] Soil salinization is a serious ecological and environmental problem that restricts agricultural development. In livestock farming, alfalfa is an excellent forage for herbivorous livestock such as cattle and sheep. Alfalfa, belonging to the legume family, is a perennial herbaceous plant rich in nutrients such as protein and cellulose, and is known as the "king of forage." Planting forage in saline-alkali land is an important measure to ensure the sustainable development of animal husbandry. However, moderately to severely saline-alkali land has low soil fertility, low water content, and low porosity, resulting in slow alfalfa growth and low yields, making it impossible to provide sufficient high-quality forage for livestock.

[0003] Nitrogen, phosphorus, and potassium are essential elements for the growth of all plants, including alfalfa. However, due to soil salinization, these basic elements are difficult for alfalfa to directly utilize. Saline-alkali soils are extremely poor in available phosphorus, preventing alfalfa from obtaining sufficient phosphate fertilizer; available nitrogen is even more crucial for alfalfa growth. Furthermore, salt stress interferes with alfalfa nitrogen metabolism, reducing germination and tillering, thus affecting alfalfa growth. Therefore, it is urgent to increase the content of available phosphorus and available nitrogen in saline-alkali soils.

[0004] Currently, measures to improve alfalfa cultivation in saline-alkali soils primarily involve physical, chemical, and biological approaches. Physical measures mainly regulate soil water and salt movement, reducing salt accumulation during alfalfa cultivation. Chemical measures use chemical amendments to lower soil conductivity and pH. While physical and chemical measures have some effect, they suffer from short-lived durations, limited effectiveness, and fail to fundamentally address the nutrient deficiency problem in saline-alkali lands. Biological remediation of saline-alkali lands is gaining increasing attention. Biological measures fall into two categories: one focuses on enhancing alfalfa's resistance to salt and alkali stress to enable its survival in saline-alkali soils; the other utilizes microbial management. Due to the high salinity and low water content of saline-alkali soils, the content of beneficial microorganisms in native plants is low. Therefore, applying microbial fertilizers can increase the proportion of beneficial microorganisms in the soil, allowing them to function and improve soil fertility. However, existing microbial fertilizers (such as yeast, lactic acid bacteria, actinomycetes, rhizobia, and Bacillus) do not have strong salt and alkali tolerance, resulting in poor application effects in saline-alkali lands. This is because conventional microbial fertilizers cannot survive and establish themselves in high-pH, high-salt-alkali soils, and are difficult to play a role in the growth of saline-alkali plants such as alfalfa.

[0005] In summary, the insufficient fertility of saline-alkali soils and the lack of available phosphorus and nitrogen severely restrict alfalfa growth. However, there is currently a lack of ideal microorganisms that can grow in saline-alkali environments and also possess phosphorus-solubilizing and nitrogen-fixing capabilities. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a *Bacillus subtilis* strain for kimchi and its applications.

[0007] This invention discloses the isolation of a salt-tolerant and alfalfa-promoting Bacillus subtilis from the rhizosphere soil of alfalfa in saline-alkali land, and its biological preservation. The preservation information is as follows:

[0008] Accession number: CGMCC No.30790; Classification name: Oceanobacillus skimchii; Depository institution: China General Microbiological Culture Collection Center; Depository address: Institute of Microbiology, China, No.3, No.1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Deposit date: May 28, 2024.

[0009] In a first aspect, the present invention provides a microbial agent comprising the aforementioned Bacillus oryzae HM-1.

[0010] Furthermore, the microbial agent is a solid microbial agent, a liquid microbial agent, or a gel microbial agent; the viable count of *Bacillus thuringiensis* HM-1 in the microbial agent is 1 × 10⁻⁶. 7-10 cfu / g.

[0011] Furthermore, the bacterial agent is one or more of solid bacterial agents, liquid bacterial agents, or gel bacterial agents.

[0012] Furthermore, the bacterial agent is prepared by the following method:

[0013] Bacillus oryzae HM-1 was cultured in liquid at 37-40℃ and 200-240 rpm in a shaker for 3-5 days.

[0014] Centrifuge the cultured Bacillus oryzae HM-1 and collect the bacterial cells, then wash them 3-5 times with sterile water.

[0015] Mix the collected bacterial cells with sterile water and shake well. The amount of sterile water should be 4-6 times the amount of liquid culture medium before centrifugation.

[0016] Secondly, the present invention provides the application of the aforementioned Bacillus oryzae HM-1 for kimchi, or the aforementioned bacterial agent, in promoting plant growth performance under salt and alkali stress.

[0017] Furthermore, the growth performance includes one or more of the following: root length, stem length, fresh weight, or crude protein content.

[0018] The present invention further provides the application of the Bacillus subtilis HM-1, or the bacterial agent, in improving saline-alkali soil environments.

[0019] Furthermore, the improvement of the saline-alkali land environment includes one or more of the following: reducing soil salinity, reducing soil pH, or increasing available phosphorus content.

[0020] Furthermore, the bacterial mud, bacterial fertilizer, or bacterial agent made from the aforementioned Bacillus oryzae HM-1 can be applied to the soil.

[0021] Furthermore, the plant is a monocotyledonous or dicotyledonous plant, preferably alfalfa.

[0022] The *Bacillus oryzae* strain HM-1 provided by this invention exhibits strong survival ability in saline-alkali environments, effectively establishing itself in saline-alkali soils and fulfilling its functions after application. Addressing the problem of extremely low available phosphorus content in saline-alkali soils, the phosphorus-solubilizing ability of strain HM-1 converts phosphorus that is difficult for alfalfa to utilize into a usable form, i.e., available phosphorus. The increase in available phosphorus allows alfalfa to survive better in saline-alkali soils and promotes its growth. Regarding the issue of low available nitrogen content in saline-alkali soils, the nitrogen-fixing ability of strain HM-1 provides alfalfa with the available nitrogen required for growth. Furthermore, salt stress interferes with alfalfa nitrogen metabolism, reducing the available nitrogen content in alfalfa tissues. The nitrogen-fixing function of strain HM-1 increases the nitrogen content within the plant while inhibiting the effects of salt stress on alfalfa nitrogen metabolism, thus increasing crude protein content. The siderophore-producing ability of strain HM-1 enhances alfalfa's salt tolerance, helping it grow normally in saline-alkali soils. Therefore, this strain has a significant effect on promoting alfalfa growth in saline-alkali soils. The strain HM-1 and inoculant provided in this invention offer a good solution for planting alfalfa in saline-alkali land and improving alfalfa growth.

[0023] The present invention has the following beneficial effects:

[0024] This invention has screened a salt-tolerant *Bacillus oryzae* strain, HM-1, from the rhizosphere soil of alfalfa in saline-alkali soil. This strain possesses the ability to solubilize phosphorus, fix nitrogen, and produce iron carriers, which has a positive effect on alfalfa growth in saline-alkali soil. The *Bacillus oryzae* strain HM-1 provided by this invention can be applied to improve saline-alkali soil environments and promote the cultivation of plants in saline-alkali soils, which is of great significance in the fields of ecological restoration and soil improvement. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1These are colony morphology and phosphorus solubilization effect diagrams of strain HM-1 provided in Example 1 of the present invention; where a is the colony morphology and b is the phosphorus solubilization effect diagram.

[0027] Figure 2 This is a graph showing the nitrogen fixation capacity and siderophore production capacity of strain HM-1 provided in Example 1 of the present invention; where a is the nitrogen fixation capacity and b is the siderophore production capacity.

[0028] Figure 3 This is a comparison chart of alfalfa growth with and without the application of microbial fertilizer, provided in Embodiment 2 of the present invention.

[0029] Figure 4 This is a comparison chart of alfalfa root length, stem length, fresh weight, and crude protein content with and without the application of microbial fertilizer, provided in Embodiment 2 of the present invention; where a is the comparison of root length, b is the comparison of stem length, c is the comparison of fresh weight, and d is the comparison of crude protein content.

[0030] Figure 5 This is a comparison chart of soil salinity, pH value and available phosphorus content with and without the application of microbial fertilizer provided in Embodiment 2 of the present invention; where a is the comparison of soil salinity, b is the comparison of pH value, and c is the comparison of available phosphorus content.

[0031] Figure 6 This is a comparison chart of alfalfa root length, stem length, fresh weight, and crude protein content with and without the application of microbial fertilizer, provided in Example 3 of the present invention; where a is the comparison of root length, b is the comparison of stem length, c is the comparison of fresh weight, and d is the comparison of crude protein content.

[0032] Figure 7 This is a comparison chart of soil salinity, pH value, and available phosphorus content with and without the application of microbial fertilizer provided in Example 3 of the present invention; wherein, a is a comparison of soil salinity, b is a comparison of pH value, and c is a comparison of available phosphorus content. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0035] Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available. For example, in Example 1...

[0036] 1. Screening and isolation of salt-tolerant strain HM-1

[0037] 1.1 This invention collects rhizosphere soil samples of alfalfa from saline-alkali land in Huanghua, Cangzhou, Hebei Province, and aliquots them into sterile sealed bags, storing them in a refrigerator at 4℃. A screening medium is prepared, containing the following components: 10g tryptone, 5g yeast extract, 100g NaCl, pH 8.5, and 1L distilled water, sterilized at 121℃ for 20 min. 10g of alfalfa rhizosphere soil sample is weighed and added to a conical flask containing 100mL of sterile water, shaken thoroughly, and then incubated in a 90℃ water bath for 10 min. 100μL of the supernatant is added to 100mL of the screening medium and incubated at 37℃ and 200rpm on a shaker for 3 days. After 3 days, the screening medium becomes turbid. 1mL of bacterial culture is taken from the conical flask containing the bacterial strain and diluted with 9mL of sterile water in a test tube to a concentration of 10. -1 From a concentration of 10 -1 Take 1 mL of bacterial culture in a test tube, add 9 mL of sterile water and continue diluting. The concentration is now 10. -2 The dilution method follows the same logic, diluting to 10... -5 Proceed to the next step. Spread 200 μL of each dilution onto a plate and incubate at 37°C for 48 hours. Perform three replicates for each dilution. Select single colonies of different colors, shapes, and morphologies and streak them onto plates for purification. Repeat streak purification three times and preserve the strain. The HM-1 colony morphology is shown below. Figure 1 As shown in 'a'.

[0038] 1.2 Salt tolerance assessment of HM-1

[0039] LB solid culture media with different NaCl concentrations were prepared: 10 g / L, 50 g / L, 100 g / L, 150 g / L, 200 g / L, and 250 g / L. The pH of the culture medium was 8.5 (adjusted to 8.5 with 1 mol / L NaOH). Strains were spotted onto the media with different NaCl concentrations using the spotting method and incubated at 37℃ for 48 h. Their growth was then observed. The salt tolerance results of HM-1 are shown in Table 1.

[0040] Table 1 Salt tolerance test of HM-1 strain

[0041] NaCl concentration 10g / L 50g / L 100g / L 150g / L 200g / L 250g / L HM-1 - + + + - -

[0042] Note: "+" indicates that the strain is growing; "-" indicates that the strain is not growing.

[0043] 1.3 Identification of the phosphorus solubility of HM-1

[0044] The Mongkina organophosphate bacteria culture medium was used. The strain was inoculated into test plates and incubated at 37°C for 72 hours. The appearance of a turbidity zone after 72 hours indicated phosphorus solubilization ability. The phosphorus solubilization ability of HM-1 was as follows: Figure 1 As shown in b in the figure.

[0045] Nitrogen fixation capacity identification of 1.4HM-1

[0046] Ashby medium was used. Strain HM-1 was inoculated into test plates and incubated at 37°C for 72 hours. The appearance of a clear zone after 72 hours indicated nitrogen fixation ability. The nitrogen fixation ability of HM-1 was as follows: Figure 2 As shown in 'a'.

[0047] 1.5HM-1's ability to generate iron carriers

[0048] CAS detection medium was used. The strain was inoculated into test plates and incubated at 37°C for 72 hours. The appearance of a yellow halo after 72 hours indicated the ability to produce siderophores. The siderophore-producing capacity of HM-1 is as follows: Figure 2 As shown in b in the figure.

[0049] Example 2

[0050] This invention verifies the ability of strain HM-1 to promote alfalfa growth under salt stress conditions, specifically including the following process:

[0051] 1. The saline-alkali soil used for potted plants was taken from Huanghua, Cangzhou City, Hebei Province. After being air-dried at room temperature, it was sieved through a 100-mesh sieve for later use. Select uniformly sized and plump alfalfa seeds and sterilize them with 1% NaClO (sodium hypochlorite). Rinse with sterile water and soak for a period of time. Place the seeds in a petri dish lined with double layers of sterile filter paper and germinate in the dark.

[0052] 2. Incubate HM-1 in liquid for 3 days. Centrifuge the cultured HM-1 and collect the bacterial cells, washing them three times with sterile water. Mix the collected bacterial cells with sterile water (4 times the volume of the liquid culture medium before centrifugation) and set aside. Divide Huanghua saline-alkali soil into two equal portions. Add 240 mL of sterile water to one portion and mix well, then dispense into 5×7×7.8 cm plastic flowerpots. Add 240 mL of the prepared microbial fertilizer to the other portion, mix thoroughly, and dispense into 5×7×7.8 cm plastic flowerpots.

[0053] 3. Transplant the germinated alfalfa seeds into plastic flowerpots, planting 10 alfalfa seeds in each pot. Harvest the alfalfa after 30 days. During the entire cultivation period, no additives are used except for sterile deionized water. Regular watering is necessary to maintain soil moisture.

[0054] 4. After harvest, statistical analysis of various growth performance characteristics of alfalfa was conducted to obtain the following results: Figures 3-5 The results shown are as follows:

[0055] Figure 3 The comparison shows that alfalfa growth was better with and without the application of microbial fertilizer.

[0056] Figure 4 The comparison shows the root length, stem length, fresh weight, and crude protein content of alfalfa with and without the application of microbial fertilizer. Figure 4 As shown in Figure a, the root length of alfalfa after applying microbial fertilizer was significantly higher than that of the group without microbial fertilizer. Figure 4 Figure b shows a comparison of alfalfa stem length, which also demonstrates a significant increase in alfalfa stem length after application of the microbial fertilizer prepared with HM-1. Alfalfa fresh weight comparison... Figure 4 As shown in c, compared to the unfertilized group, the fresh weight of alfalfa increased significantly after fertilization. The crude protein content of alfalfa was compared to... Figure 4 As shown in d, compared with the unfertilized group, the application of microbial fertilizer significantly increased the crude protein content of alfalfa.

[0057] Figure 5 The study compared soil salinity, pH value, and available phosphorus content with and without the application of microbial fertilizer. Figure 5 In the figure, 'a' represents the comparison of soil salinity. After applying microbial fertilizer, the soil salinity decreased by 6.57%. Figure 5 In the figure, 'b' represents the comparison of soil pH; the application of microbial fertilizer significantly reduced soil pH. Results regarding soil available phosphorus content are as follows: Figure 5 As shown in c, the application of microbial fertilizer increased the available phosphorus content in the soil to 37.16 mg / kg, which was 1.42 mg / kg higher than the unfertilized group, showing a significant effect.

[0058] In conclusion, Bacillus oryzae HM-1, after application, effectively improved soil fertility and promoted alfalfa growth.

[0059] Example 3

[0060] This invention further verifies the ability of strain HM-1 to promote alfalfa growth under salt stress conditions, specifically including the following process:

[0061] 1. The saline-alkali soil used for potted plants was taken from Huanghua, Cangzhou City, Hebei Province. After being air-dried at room temperature, it was sieved through a 100-mesh sieve for later use. Select uniformly sized and plump alfalfa seeds and sterilize them with 1% NaClO (sodium hypochlorite). Rinse with sterile water and soak for a period of time. Place the seeds in a petri dish lined with double layers of sterile filter paper and germinate in the dark.

[0062] 2. Culture HM-1 in liquid for 5 days. Centrifuge the cultured HM-1 and collect the bacterial cells, washing them three times with sterile water. Mix the collected bacterial cells with sterile water (5 times the volume of the liquid culture medium before centrifugation) and set aside. Divide Huanghua saline-alkali soil into two equal portions. Add 300 mL of sterile water to one portion and mix well, then dispense into 5×7×7.8 cm plastic flowerpots. Add 300 mL of the prepared microbial fertilizer to the other portion, mix thoroughly, and dispense into 5×7×7.8 cm plastic flowerpots.

[0063] 3. Transplant the germinated alfalfa seeds into plastic flowerpots, planting 10 alfalfa seeds in each pot. Harvest the alfalfa after 30 days. During the entire cultivation period, no additives are used except for sterile deionized water. Regular watering is necessary to maintain soil moisture.

[0064] 4. After harvest, statistical analysis of various growth performance characteristics of alfalfa was conducted to obtain the following results: Figures 6-7 The results shown are as follows:

[0065] like Figure 6 As shown, the comparison of alfalfa root length, stem length, fresh weight, and crude protein content with and without the application of microbial fertilizer can be observed. Figure 6 The results in group a showed that the root length of alfalfa after applying microbial fertilizer was significantly higher than that of the group without microbial fertilizer. Figure 6 The comparison of alfalfa stem length shown in b also demonstrates a significant increase in alfalfa stem length after application of microbial fertilizer prepared by HM-1. Figure 6 Figure c shows the comparison of alfalfa fresh weight. Compared to the unfertilized group, the fresh weight of alfalfa increased significantly after fertilization. The crude protein content of alfalfa is compared to... Figure 6 As shown in d, compared with the unfertilized group, the application of microbial fertilizer significantly increased the crude protein content of alfalfa.

[0066] Figure 7 The study compared soil salinity, pH value, and available phosphorus content with and without the application of microbial fertilizer. Figure 7 The comparison of soil salinity shown in Figure a reveals that the application of microbial fertilizer reduced soil salinity by 6.88%. Figure 7 In the figure, 'b' represents the comparison of soil pH; the application of microbial fertilizer significantly reduced soil pH. The results for soil available phosphorus content are as follows: Figure 7 As shown in c, the application of microbial fertilizer increased the available phosphorus content in the soil to 37.76 mg / kg, which was 2.14 mg / kg higher than the unfertilized group, showing a significant effect.

[0067] In conclusion, Bacillus thuringiensis HM-1 for kimchi performed its function after application, significantly improving soil fertility and promoting alfalfa growth.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Oceanobacillus kimchii HM-1, characterized in that, The preservation number of the Bacillus oryzae HM-1 in the kimchi is: CGMCC No.30790.

2. A microbial agent, characterized in that, Includes the Bacillus oryzae HM-1 of claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a solid, liquid, or gel bacterial agent; the viable count of *Bacillus thuringiensis* HM-1 in the bacterial agent is 1 × 10⁻⁶. 7-10 cfu / g.

4. The application of Bacillus oryzae HM-1 of claim 1, or the inoculant of any one of claims 2-3, in promoting the growth performance of alfalfa under salt-alkali stress.

5. The application according to claim 4, characterized in that, The growth performance includes one or more of the following: root length, stem length, fresh weight, or crude protein content.

6. The application of the Bacillus oryzae HM-1 of claim 1, or the bacterial agent of any one of claims 2-3, in improving the saline-alkali land environment.

7. The application according to claim 6, characterized in that, The improvement of the saline-alkali land environment includes one or more of the following: reducing soil salinity, reducing soil pH, or increasing available phosphorus content.

8. The application according to any one of claims 4-7, characterized in that, The microbial agent made from the aforementioned Bacillus oryzae HM-1 was applied to the soil.

Images