Brown sugar culture medium suitable for salt-tolerant composite microbial inoculant and application thereof

Abstract

The application discloses a brown sugar culture medium suitable for a salt-tolerant composite microbial inoculant and application thereof, and remarkably improves the propagation efficiency of the salt-tolerant composite microbial inoculant and the growth promoting effect on wheat by optimizing the brown sugar culture medium. The culture medium provided by the application can effectively support the growth of strains, and in the range of 0.2-2 g / L, the biomass of the strains increases with the increase of the concentration, and is better than that of an LB control group. After the wheat is treated by the salt-tolerant composite microbial inoculant cultivated by the culture medium provided by the application, the salt-tolerant composite microbial inoculant shows excellent growth promoting capacity, the winter wheat seedling length and root length are longer than those of the control group, and the spring wheat seedling length and root length are also better than those of other treatment groups, thereby significantly promoting the root development and the growth of the above-ground part of crops under a salt stress environment, and showing a broad application prospect of the technology in the improvement of saline-alkali land or the development of biological fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the technical field of adapting microbial nutritional needs. Background Technology

[0002] For a long time, in industrial fermentation and agricultural microbial preparation production, the cost of culture media, especially carbon source costs, has accounted for a large proportion of the total cost. Traditional pure sugars (such as glucose and sucrose), although their composition is well-defined, are expensive, severely limiting their economic feasibility in large-scale production. Therefore, finding inexpensive and efficient alternative carbon sources has become a key focus in this field. Brown sugar and its pre-refining byproduct molasses, as widely available and inexpensive natural raw materials, have been proven by numerous studies and practices to be an ideal alternative carbon source due to their unique chemical composition and significant economic benefits, and have shown application potential exceeding that of pure sugar in the cultivation of various microorganisms.

[0003] The nutritional value of brown sugar lies in its comprehensive composition. It not only provides readily available carbon and energy, primarily sucrose, but is also rich in various minerals (such as potassium, calcium, magnesium, and iron), vitamins (especially B vitamins), and trace growth factors such as amino acids. These substances are key components of microbial enzymes and coenzymes, effectively promoting cell growth, metabolic activity, and spore formation. Notably, the organic acids and minerals in brown sugar also buffer the pH of the fermentation system. Its naturally occurring ionic components are essential for salt-tolerant microorganisms to maintain cell osmotic pressure, potentially reducing reliance on exogenous inorganic salts during fermentation. In terms of cost, as a minimally processed product, brown sugar is significantly cheaper than refined sugar, typically reducing carbon source costs by 30% to 50% or more. This economic advantage makes it highly attractive in cost-conscious applications.

[0004] Compared to other inexpensive carbon sources, brown sugar also exhibits unique advantages. Compared to starchy raw materials (such as corn flour), brown sugar is a directly usable sugar, requiring no complex saccharification pretreatment, thus shortening the fermentation cycle and reducing energy consumption. Compared to other industrial and agricultural wastes (such as cassava residue and straw hydrolysate), brown sugar has a relatively stable composition with minimal batch-to-batch variation, and generally does not contain toxic substances such as lignin degradation products that may inhibit microbial growth. This makes the fermentation process more stable and reliable, with a higher success rate.

[0005] Current research largely focuses on the fermentation of single bacterial strains on optimized culture media (such as those containing sucrose or yeast extract), or the growth of known probiotics (such as lactic acid bacteria) in brown sugar. However, for compound microbial agents specifically designed for saline-alkali land remediation, the synergistic growth relationships and competitive inhibition among different strains (such as phosphate-solubilizing bacteria, nitrogen-fixing bacteria, and biocontrol bacteria) in pure brown sugar culture media are largely unexplored. Although brown sugar contains various trace elements, its content of key nutrients such as nitrogen and phosphorus is usually low and in fixed proportions, which may lead to prolonged fermentation cycles and lower microbial activity, requiring increased dosage to achieve the desired effect. Therefore, rigorous evaluation data is still lacking regarding the overall economic efficiency of using brown sugar culture media to produce salt-tolerant compound microbial agents, and its comparison with the comprehensive benefits of using traditional culture media. Summary of the Invention

[0006] Given the lack of existing technologies regarding the use of brown sugar as the sole carbon source in salt-tolerant compound microbial agent culture media, the purpose of this invention is to improve the propagation efficiency of microbial agents, reduce propagation costs, and optimize the practicality of the culture medium. This aims to solve problems such as insufficient nutrient compatibility, high cost, and complex operation in traditional microbial agent propagation, providing technical support for the large-scale, low-cost production and application of microbial agents. This application significantly improves the propagation efficiency of salt-tolerant compound microbial agents and their growth-promoting effect on wheat by optimizing the brown sugar culture medium. The culture medium provided by this application effectively supports the growth of the strains. After treatment, wheat exhibits excellent growth-promoting ability; the seedling and root lengths of winter wheat are longer than those of the control group, and the seedling and root lengths of spring wheat are also superior to other treatment groups, significantly promoting root development and aboveground growth of crops under salt stress.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention provides a brown sugar culture medium suitable for salt-tolerant compound bacterial agents. The culture medium uses 0.2-25 g / L brown sugar and 5 g / L yeast powder, and is sterilized by high-pressure steam at 121℃ and 0.1 MPa for 20 min.

[0009] The aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents contains 0.75 g / L urea and 1.5 g / L potassium dihydrogen phosphate.

[0010] The salt-tolerant compound bacterial agent is *Gordonella hydrophobicum* (…). Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any combination of one or more of the following.

[0011] Furthermore, this application also provides the application of the aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents in promoting wheat growth under salt stress conditions.

[0012] Preferably, the application uses hydrophobic Gordon's bacteria ( Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any one or more of the following are cultured in brown sugar medium suitable for salt-tolerant compound bacterial agents until the bacterial solution OD reaches 0.02, and then wheat seeds are soaked in the solution.

[0013] Preferably, the wheat seeds are selected from Xinchun 26 and Xindong 18 wheat seeds.

[0014] Furthermore, this application also provides the application of the aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents in the improvement of saline-alkali land or the development of bio-fertilizers.

[0015] Compared with the prior art, the present invention has the following features: (1) This invention provides a brown sugar culture medium suitable for salt-tolerant compound bacterial agents. This application significantly improves the propagation efficiency of salt-tolerant compound bacterial agents and their growth-promoting effect on wheat by optimizing the brown sugar culture medium. The culture medium provided by this application can effectively support hydrophobic Gordon's bacteria (… Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis The strains in the salt-tolerant compound bacterial agent constructed by this application showed good growth. In the brown sugar culture medium suitable for the salt-tolerant compound bacterial agent provided in this application, brown sugar, as a single carbon source, can support the efficient propagation of mixed strains at an appropriate concentration. Furthermore, the supplementation of exogenous nitrogen sources and phosphorus and potassium elements effectively compensates for the deficiency of key nutrients in brown sugar, optimizes the C / N / P ratio of the culture medium, and thus significantly promotes the overall growth performance of the mixed strains.

[0016] (2) The brown sugar culture medium provided by the present invention is suitable for salt-tolerant compound bacterial agent. After soaking wheat seeds under salt stress, it showed excellent growth promotion ability compared with the control group (CK, sterile water soaking treatment). The seedling length and root length of winter wheat were longer than those of the control group, and the seedling length and root length of spring wheat were also better than those of other treatment groups. It significantly promoted the root development and aboveground growth of crops under salt stress, showing the broad application prospects of this technology in saline-alkali land improvement or bio-fertilizer development. Attached Figure Description

[0017] Figure 1 The figure shows the growth curves of mixed bacterial samples in different gradients of basic brown sugar culture medium.

[0018] Figure 2 The figure shows the growth curves of mixed bacterial samples in different gradient enhanced brown sugar culture media.

[0019] Figure 3 The figure shows the growth curves of mixed bacterial samples in basic brown sugar medium and enhanced brown sugar medium.

[0020] Figure 4 The image shows the effect of different culture media on wheat germination.

[0021] Figure A shows the growth of spring wheat seedlings after soaking in bacterial solutions cultured in different culture media; Figure B shows the growth of spring wheat roots after soaking in bacterial solutions cultured in different culture media; Figure C shows the growth of winter wheat seedlings after soaking in bacterial solutions cultured in different culture media; Figure D shows the growth of winter wheat roots after soaking in bacterial solutions cultured in different culture media.

[0022] CK was the control group treated with sterile water soaking. LB, J2, and Z2 were treated with mixed bacterial cultures cultured in LB medium, basal brown sugar medium, and enhanced brown sugar medium, respectively. One-way ANOVA was used, with different superscript letters indicating statistically significant differences between groups. p <0.05) Figure 5 The image shows the germination of spring wheat seeds after soaking in a mixed bacterial solution cultured on different culture media for 7 days.

[0023] Figure 6 The image shows the germination of winter wheat seeds after soaking in a mixed bacterial solution cultured in different culture media for 7 days. Detailed Implementation

[0024] The following examples are provided to further illustrate the content of this invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are within the scope of this invention.

[0025] The instruments and equipment used in this application are: electronic balance, autoclave, pipette, sterile pipette tip, 96-well cell culture plate (sterile disposable), ELISA reader (for continuous monitoring of growth curve), solid culture medium dishes (90 mm in diameter), and liquid culture medium conical flasks (250 mL and 500 mL).

[0026] The reagents used in this application are: tryptone, yeast extract, sodium chloride, yeast powder, urea, and potassium dihydrogen phosphate, all of which are commercially available products and can be purchased by the general public from biotechnology companies.

[0027] The hydrophobic Gordon's bacterium used in this invention (Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis All five strains were harmless and safe, including *Gordonella hydrophobicum*. Gordonia hydrophobica ), Epidermal short bacillus ( Brevibacterium epidermidis ), Streptomyces montana ( Streptomyces monticola Seabed biogenic microorganisms ( ) can be purchased through other public channels such as the China General Microbiological Culture Collection Center (CGMCC); Planomicrobium okeanokoites It can be purchased through other public channels such as the Weihai Marine Microbial Resource Center of Shandong University; *Glaucoma glabra* ( Luteimonas huabeiensis It can be purchased through other public channels such as Beijing Biowell Biotechnology Co., Ltd.

[0028] In this application, the following embodiments use the term "hydrophobic Gordon's bacteria ( Gordonia hydrophobica ")" is abbreviated as "Hydrophobic Gordon's bacterium"; "Streptomyces montanatum ... Streptomyces monticola "Montage Mountain Streptomyces" is abbreviated as "Montage Mountain Streptomyces"; "Seabed Motile Microbes ... Planomicrobium okeanokoites ")" is abbreviated as "seabed motile microbes"; "epidermal short bacilli" ( Brevibacterium epidermidis The abbreviation for "epidermal short bacillus" is recorded as "epidermal short bacillus".

[0029] The Xinchun 26 and Xindong 18 wheat seeds used in this application were donated by the Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences. The general public can obtain them through the Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences, or seed companies.

[0030] The LB medium used in this application was obtained by: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 1.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] Example 1: A brown sugar culture medium suitable for salt-tolerant compound bacterial agents and its application This invention provides a brown sugar culture medium suitable for salt-tolerant compound bacterial agents. The culture medium uses 0.2-25 g / L brown sugar and 5 g / L yeast powder, and is sterilized by high-pressure steam at 121℃ and 0.1 MPa for 20 min.

[0033] The aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents contains 0.75 g / L urea and 1.5 g / L potassium dihydrogen phosphate.

[0034] The salt-tolerant compound bacterial agent is *Gordonella hydrophobicum* (…). Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any combination of one or more of the following.

[0035] Furthermore, this application also provides the application of the aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents in promoting wheat growth under salt stress conditions.

[0036] Preferably, the application uses hydrophobic Gordon's bacteria ( Gordonia hydrophobica ), Streptomyces montana ( Streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any one or more of the following are cultured in brown sugar medium suitable for salt-tolerant compound bacterial agents until the bacterial solution OD reaches 0.02, and then wheat seeds are soaked in the solution.

[0037] Preferably, the wheat seeds are selected from Xinchun 26 and Xindong 18 wheat seeds.

[0038] Furthermore, this application also provides the application of the aforementioned brown sugar culture medium suitable for salt-tolerant compound bacterial agents in the improvement of saline-alkali land or the development of bio-fertilizers.

[0039] Example 2: Treatment of the target strain Strain purification culture: Five experimental strains were inoculated onto the surface of solid culture medium (LB medium) and purified by streak plating. They were then incubated in a 30℃ constant temperature incubator for 24-48 hours until single colonies were formed.

[0040] Activation and mixing of bacterial strains: Single colonies with consistent morphology were picked from the purified solid culture medium and transferred to Erlenmeyer flasks containing 50 mL of liquid culture medium (LB medium). The colonies were activated by incubation at 30 °C and 150 r / min for 24 h on a shaker. After activation, the five bacterial strains were diluted to the same concentration and then mixed in equal volume ratios to prepare a mixed bacterial solution for later use.

[0041] Example 3: Culture medium preparation Basic brown sugar culture medium gradient settings: Set up 5 brown sugar concentration gradients (200mL water) of 2g, 5g, 10g, 15g and 20g respectively, and keep the yeast powder 5g / L unchanged; prepare 200mL of each gradient, put them into Erlenmeyer flasks, and autoclave them at 121℃ and 0.1MPa for 20min, and cool to room temperature for use.

[0042] Preparation of enhanced brown sugar culture medium: After the optimal brown sugar gradient is selected from the basic brown sugar culture medium, add 0.75 g / L urea and 1.5 g / L potassium dihydrogen phosphate to prepare 200 mL of enhanced brown sugar liquid culture medium. Sterilize at 121℃ and 0.1 MPa for 20 min and then cool for later use.

[0043] Control group setup: The control group was prepared with LB liquid culture medium and treated under the same sterilization conditions (121℃, 0.1MPa, 20min), and then cooled before use.

[0044] Example 4: Inoculation of bacterial culture and monitoring of growth curve Two ml of the prepared mixed bacterial culture was inoculated into each gradient of brown sugar medium. LB medium inoculated with an equal volume of the mixed bacterial culture was used as a control. The microbial growth curves (OD values) of each group were continuously monitored using a 96-well plate and a microplate reader to screen for the optimal brown sugar concentration.

[0045] Growth curve determination: After inoculation, the 96-well plates were placed in a microplate reader, the incubation temperature was set to 30℃, and the absorbance (OD) at 600nm wavelength was measured every 2 hours using dynamic monitoring mode. 600 (Value), continuously monitored for 48 hours.

[0046] The effect of different brown sugar concentration gradients on microbial community growth was investigated: OD values ​​of five brown sugar concentration gradients (2g, 5g, 10g, 15g, and 20g) and the LB control group were continuously monitored over 48 hours using a 96-well plate. 600 The changes in values ​​were analyzed to plot growth curves and assess bacterial biomass. See the appendix for results. Figure 1 As shown, the bacterial biomass increased with increasing brown sugar concentration from 2g to 5g, 10g, 15g, and 20g. In this experiment, the mixed bacterial strain exhibited better growth rate and final biomass in the basal brown sugar medium than the LB control group, indicating that brown sugar, as a single carbon source, can support efficient propagation of the mixed bacterial strain at appropriate concentrations.

[0047] Example 5: Validation Experiment of Enhanced Brown Sugar Culture Medium Based on the optimal brown sugar concentration screened by the basic brown sugar concentration gradient experiment, the same volume of mixed bacterial solution was inoculated into the prepared enhanced brown sugar medium (with urea 0.75 g / L and potassium dihydrogen phosphate 1.5 g / L). The growth curve was monitored using an enzyme-linked immunosorbent assay (ELISA) reader and compared with the corresponding gradient brown sugar medium and LB medium control group to evaluate the nutrient fortification effect.

[0048] The effect of different brown sugar concentration gradients on microbial community growth was investigated: the OD values ​​of five brown sugar concentration gradients (2g, 5g, 10g) and the LB control group were continuously monitored over 48 hours using a 96-well plate. 600 The changes in values ​​were analyzed to plot growth curves and assess bacterial biomass. See the appendix for results. Figure 2 As shown, the bacterial biomass increased with increasing brown sugar concentration from 2g to 5g and then to 10g. In this experiment, the mixed bacterial strain exhibited better growth rate and final biomass in the enhanced brown sugar medium than the LB control group, indicating that the enhanced brown sugar medium at suitable concentrations can also support efficient propagation of the mixed bacterial strain.

[0049] Based on the above test results, the growth-promoting effects of enhanced and basic culture media were compared. At a cost-effective brown sugar concentration (2g), the effects of basic and enhanced brown sugar culture media on bacterial growth were compared (the control group was LB medium). Results are attached. Figure 3 As shown, the cell growth rate and maximum OD value in the enhanced medium were significantly higher than those in the basic brown sugar medium ( p <0.05). This indicates that supplementing with exogenous nitrogen and phosphorus / potassium effectively compensated for the deficiency of key nutrients in brown sugar, optimized the C / N / P ratio of the culture medium, and thus significantly promoted the overall growth performance of the mixed strain.

[0050] Example 6: Wheat Germination Experiment Using spring wheat 'Xinchun 26' and winter wheat 'Xindong 18' seeds as materials, wheat seeds were soaked in a mixed bacterial solution (bacterial solution OD 0.02) cultured on different culture media, and then placed in sterile petri dishes containing 1.2% NaCl aqueous solution for seed germination experiments. Seeds soaked in sterile water and cultured in 1.2% NaCl aqueous solution served as a control group. Root length and seedling length results were recorded.

[0051] See the appendix for the results of wheat growth promotion measurement. Figure 4 Appendix Figure 5 Appendix Figure 6 As shown. Compared with the control group (CK, aseptic water soaking treatment), all three bacterial solutions significantly promoted root length and seedling length in both spring and winter wheat. p <0.05). Among them, the enhanced brown sugar medium (Z2) treatment showed the most significant growth-promoting effect, with root length and seedling length significantly higher than those of the LB group and the basic brown sugar group (J2).p The value <0.05 indicates that the bacterial solution cultured in this medium has a better effect on promoting the salt-tolerant growth of crops.

[0052] Regarding spring wheat seedling length, the seedlings in group Z2 were significantly longer than those in other groups, while the seedlings in groups LB and J2 were similar in length and longer than those in group CK. As for spring wheat root length, group LB had the longest roots, followed by groups Z2 and J2, with group CK having the shortest. The differences between groups were relatively small.

[0053] Regarding winter wheat seedling length, the seedlings in group Z2 were significantly longer than those in other groups, followed by group J2, then group LB, with group CK being the shortest. As for winter wheat root length, group Z2 had the longest roots, followed by group J2, while groups LB and CK had relatively shorter roots.

[0054] In summary, regarding seedling length, winter wheat seedlings were generally longer than spring wheat seedlings across all treatment groups. Regarding root length, winter wheat root length increased significantly under the Z2 and J2 treatments, while the differences among spring wheat treatments were relatively small. The Z2 treatment showed the best performance in promoting seedling and root growth in both spring and winter wheat. The LB and J2 treatments also had a positive effect on the growth of both wheat types, but the effect was slightly less than that of the Z2 treatment.

[0055] In conclusion, the Z2 treatment was most effective in improving the growth indicators (including seedling height and root length) of spring and winter wheat.

[0056] This application provides a brown sugar culture medium suitable for salt-tolerant compound microbial agents. Brown sugar demonstrates its potential as a primary carbon source in the propagation of mixed microorganisms. Furthermore, by optimizing the culture medium and supplementing it with nitrogen, phosphorus, and potassium, the biomass and activity of the microbial agent are enhanced. This enhanced brown sugar culture medium, while maintaining low cost, significantly promotes strain growth and enhances the promoting effect of the bacterial solution on wheat seedling growth under salt stress conditions, showing promising prospects for agricultural applications.

[0057] The above embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A brown sugar culture medium suitable for salt-tolerant compound bacterial agents, characterized in that, The culture medium used was made of 0.2-25 g / L brown sugar and 5 g / L yeast powder, and was autoclaved at 121℃ and 0.1 MPa for 20 min.

2. The brown sugar culture medium suitable for salt-tolerant compound bacterial agents as described in claim 1, characterized in that, The culture medium also includes 0.75 g / L urea and 1.5 g / L potassium dihydrogen phosphate.

3. The brown sugar culture medium suitable for salt-tolerant compound bacterial agents as described in claim 1, characterized in that, The salt-tolerant compound bacterial agent is *Gordonella hydrophobicum* (…). Gordonia hydrophobica ), Streptomyces montana ( streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any combination of one or more of the following.

4. The application of a brown sugar culture medium suitable for salt-tolerant compound microbial agents as described in any one of claims 1 to 3 in promoting wheat growth under salt stress conditions.

5. The application of the brown sugar culture medium suitable for salt-tolerant compound bacterial agents as described in claim 4 in promoting wheat growth under salt stress conditions, characterized in that... The application uses hydrophobic Gordon's bacteria ( Gordonia hydrophobica ), Streptomyces montana ( streptomyces monticola ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis Any one or more of the following are cultured in brown sugar medium suitable for salt-tolerant compound bacterial agents until the bacterial solution OD reaches 0.02, and then wheat seeds are soaked in the solution.

6. The application of the brown sugar culture medium suitable for salt-tolerant compound bacterial agents as described in claim 4 in promoting wheat growth under salt stress, characterized in that... The wheat seeds selected were Xinchun 26 and Xindong 18 wheat seeds.

7. The application of a brown sugar culture medium suitable for salt-tolerant compound microbial agents as described in any one of claims 1 to 3 in the improvement of saline-alkali land or the development of bio-fertilizers.

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