Pseudomonas fragariae producing microbial low-temperature growth-promoting protein and its application

Abstract

A strain of Pseudomonas fragariae that produces a microbial low-temperature growth-promoting protein and its application, belonging to the field of microbial technology. The strain is classified as Pseudomonas fragariae ( Pseudomonas fragi ) D12, deposited with the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with a deposit number of CCTCC NO: M2025126, and a deposit date of January 14, 2025. The cell-free culture solution of Pseudomonas fragariae D12 of the present invention can promote the growth of a variety of mesophilic bacteria and fungi functional bacteria at low temperatures, improving the cold resistance of the functional bacteria. Secondly, it can also improve the metabolic capacity of each functional bacteria and enhance the low-temperature enzymatic activity of enzymes that degrade lignocellulose. In addition, Pseudomonas fragariae D12 can use lignin as the sole carbon source for growth and metabolism, and its cell-free culture solution also contains relevant enzymes that can degrade lignin, further improving the efficiency of lignocellulose degradation at low temperatures.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Pseudomonas fragariae producing a microbial low-temperature growth-promoting protein and applications thereof. Background Art

[0002] In modern agriculture and biotechnology, microorganisms play an important role as biofertilizers, plant growth promoters, and food fermentation agents. However, low temperatures often limit the activity and growth rate of beneficial bacterial strains, thus affecting their practical application. For example, in cold climates, the activity of plant rhizosphere microbial communities decreases, leading to a decrease in plant stress resistance. During food storage, low temperatures inhibit the growth of beneficial fermentation bacteria, affecting product quality and safety.

[0003] To address these challenges, researchers have explored various methods to enhance the growth of beneficial strains under low-temperature conditions. One effective strategy is to utilize metabolites or signaling molecules secreted by the strains to promote the growth of target strains through cell-free fermentation (CFS). Cell-free culture fluids are obtained by fermenting a strain, then removing cellular components through centrifugation and filtration, leaving only the liquid secreted by the strains. These fluids contain a variety of bioactive substances, such as enzymes, metabolites, antibiotics, and signaling molecules.

[0004] Studies have shown that some Pseudomonas CFS of strains of the genus have the ability to promote the growth of other microorganisms. For example, the prior art has reported Pseudomonas fluorescens The secreted metabolites have been shown to enhance the stress resistance and growth activity of plant rhizosphere microorganisms. Pseudomonas aeruginosa Under certain conditions, CFS can promote the growth of certain lactic acid bacteria and improve fermentation efficiency. However, there are currently few studies on efficient CFS strains that widely promote the growth of a variety of microorganisms under low-temperature conditions. For Pseudomonas fragariae, CN117736931A discloses low-temperature-resistant Pseudomonas fragariae, but does not disclose its ability to promote the growth of other microorganisms at low temperatures; Patent CN113832067A discloses the use of low-temperature Pseudomonas fragariae to prepare a low-temperature growth promoter for normal-temperature bacteria, but its target of action is only bacteria, and there is no relevant record for fungi. Existing CFS mostly focuses on applications at room temperature or specific temperatures, and lacks research results that can stably function in low-temperature environments.

[0005] Furthermore, existing methods for promoting bacterial growth using CFS typically rely on the selection of specific strains and the optimization of culture conditions. However, these methods have limited applicability across different low-temperature conditions and target strains, and often require complex preparation processes, increasing the difficulty and cost of application. Therefore, there is an urgent need to develop a CFS production strain that can efficiently and broadly promote the growth of a variety of bacteria under low-temperature conditions to meet the needs of agriculture, food preservation, and biological product production. Summary of the Invention

[0006] In response to the above technical problems, the present invention provides a strain of Pseudomonas fragariae. The cell-free culture fluid (CFS) produced by this strain during the cultivation process can significantly improve the growth ability of various microorganisms whose growth is inhibited at low temperatures (below 15°C) under low temperature conditions. This solves the problem that the existing technology cannot promote the co-growth of various bacteria, fungi and other microorganisms under low temperature conditions. It has significant innovation and broad application prospects.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A strain producing microbial low-temperature growth-promoting protein, characterized in that: the strain is classified as Pseudomonas fragariae ( Pseudomonas fragi )D12, deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M2025126, and the deposit date is January 14, 2025.

[0009] The Pseudomonas fragariae D12 is a Gram-negative short rod-shaped bacterium with a length of 1.8-2.2 μm. The colony morphology is irregularly circular with a concave center and a serrated edge, and the color changes to milky yellow and translucent.

[0010] The Pseudomonas fragrans D12 can grow efficiently at a temperature below 15°C, and its cell-free culture fluid has growth-promoting activity on a variety of bacteria, fungi and other microorganisms at low temperatures.

[0011] Experiments have shown that CFS of Pseudomonas fragrans D12 can inhibit the growth of various target strains that are inhibited at low temperatures at 10℃, such as Bacillus subtilis (Bacillus subtilis), Bacillus cereus (Bacillus cereus), Bacillus megaterium (Bacillus megaterium) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), fungi such as Aspergillus niger (Aspergillus niger), Trichoderma reesei (Trichoderma reesei), Trametes versicolor (white rot fungi) and Irpex lacteusIt has a significant promoting effect on the growth and metabolism of functional strains such as (white capsule rake tooth fungus), showing wide application potential and good stability.

[0012] In addition, during the culture process, Pseudomonas fragrans D12 can use lignin as the only carbon source for growth and metabolism, thereby achieving the decomposition of lignin.

[0013] Furthermore, the growth temperature of the above-mentioned Pseudomonas fragrans D12 is 4~30°C.

[0014] Furthermore, the working temperature of the cell-free culture medium produced by the above-mentioned Pseudomonas fragariae D12 is 0-15°C, preferably 10°C.

[0015] Pseudomonas fragrans D12 and the cell-free culture medium prepared therefrom can be applied to low-temperature agricultural production to improve the growth efficiency and activity of the target strain in a low-temperature environment.

[0016] Specifically, the application of Pseudomonas fragrans D12 and its cell-free culture solution in promoting low-temperature fermentation of low-temperature-sensitive microorganisms, specifically in low-temperature composting fermentation, wherein the low-temperature fermentation temperature is 0~15°C.

[0017] Further preferably, the low-temperature fermentation temperature is 10°C.

[0018] The present invention has the following technical effects:

[0019] The CFS produced by the Pseudomonas fragariae D12 of the present invention can promote the growth of various low-temperature-sensitive normal-temperature bacteria and fungi functional bacteria at low temperatures below 15°C, thereby improving the cold resistance of the functional bacteria. Secondly, it can also improve the metabolic capacity of various functional bacteria and improve the low-temperature enzymatic activity of enzymes that degrade lignocellulose. In addition, the Pseudomonas fragariae D12 can use lignin as the sole carbon source for growth and metabolism, and the CFS of the Pseudomonas fragariae D12 also contains related enzymes that can degrade lignin, thereby further improving the degradation efficiency of lignocellulose at low temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Pulsed-field gel electrophoresis pattern of Pseudomonas fragariae D12 of the present invention.

[0021] Figure 2 : Growth curves of Pseudomonas fragrans D12 at different temperatures.

[0022] Figure 3 : OD values ​​of PfCFs against different bacteria after culture at 10℃ for 52h.

[0023] Figure 4 : Effects of PfCFs on the dry weight (DW) of different fungi after incubation at 10℃ for 96 h.

[0024] Figure 5 : PfCFs Bacillus subtilis Effect of culture temperature on cellulase and xylanase activities after 52 h at 10 °C.

[0025] Figure 6 : Growth status of Pseudomonas fragrans D12 in culture medium with lignin and alkaline lignin as carbon sources, (a) is the culture medium with lignin as the carbon source; (b) is the culture medium with alkaline lignin as the carbon source.

[0026] Figure 7 : Growth curve of Pseudomonas fragrans D12 in culture medium with lignin as carbon source and changes in lignin concentration.

[0027] Figure 8 :PfCFs with different molecular weights Bacillus subtilis OD value after culturing at 10°C for 52 h.

[0028] Figure 9 :Different substances in PfCFs Bacillus subtilis Effect of OD value after cultivation at 10℃ for 52h.

[0029] In the accompanying drawings, “***” and “****” indicate the range of significant differences, ***: 0.0001<p<0.001; ****: p<0.0001. DETAILED DESCRIPTION

[0030] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0031] The low-temperature-intolerant microorganisms mentioned in the present invention refer to microorganisms whose growth and metabolism are inhibited, whose activity is poor, or even whose growth and metabolism stop at temperatures below 15°C, especially functional bacteria such as fungi and bacteria that degrade lignocellulose.

[0032] Example 1

[0033] Isolation, screening and identification of original strains

[0034] (I) Isolation of strains

[0035] (1) 5 g of soil sample was collected from the forest area of ​​Changbai Mountain Nature Reserve, dissolved in 100 mL of a prepared salt solution, and cultured at 4 °C and 180 rpm for 4 h. The salt solution consisted of 0.006 mM FeSO4·7H2O, 0.01 mM CaCO3·7H2O, 0.08 mM MgSO4·7H2O, 0.07 mM MnSO4·7H2O, and 0.006 mM ZnSO47H2O.

[0036] (2) 1 mL of the mixed soil sample salt solution was subjected to different gradients (10 -2 , 10 -3 , 10 -4 ) and 200 μL of the dilution was spread on R2a solid plates, which were placed in a 4°C constant temperature incubator for 10 days. The fixed plates contained 0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 15.0 g agar, and 1000 ml of distilled water, with a final pH of 7.2±0.2.

[0037] (3) Then, single colonies are picked based on their morphology, color, size, and glossiness.

[0038] (II) Screening of the original strain D12

[0039] (1) Dissolve the single colony in physiological saline (0.8% sodium chloride solution), use an inoculation loop to pick up an appropriate amount of bacterial liquid and streak culture on LB solid medium. After multiple passages and combined with microscopic observation, pick single colonies with uniform colony morphology and state and place them in LB liquid medium. Shake the flask at 160 rpm and 4°C for 3 days to obtain the fermentation liquid.

[0040] (2) The fermentation broth obtained from the above culture was centrifuged (6000 rpm, 15 min, 4°C) and filtered through a 0.22 μm filter membrane to obtain a cell-free culture medium.

[0041] (3) Add the above-mentioned cell-free culture medium to a liquid culture medium containing the indicator strain Bacillus subtilis, culture at 0-15°C and observe the growth. At the same time, set up a control group without cell-free culture medium.

[0042] (4) By measuring the growth curve of the indicator strain, the strain that can produce a cell-free culture solution that promotes bacterial growth was named D12. The screened strain was inoculated into LB liquid medium and cultured at 160 rpm and 4°C with shaking. The logarithmic phase bacterial solution was preserved in glycerol (400 uL of 50% glycerol was added to 600 uL of bacterial solution).

[0043] By detecting the growth curve of the indicator strain, it can be seen that the screened original strain D12 has the ability to produce bacterial growth-promoting factors under low temperature environment.

[0044] Example 2

[0045] New strains obtained by mutation of original strains and their identification

[0046] (I) ARTP mutagenesis treatment (1) Take the original strain D12 logarithmic phase bacterial solution, wash it with sterile saline and prepare it into a concentration of 1×10 8 CFU / mL bacterial suspension; (2) The bacterial suspension was treated with the ARTP mutagenesis system (power 120 W, helium flow rate 10 SLM), and the lethality curve was drawn through preliminary experiments, and the optimal treatment time was determined to be 90 s (lethality rate 85±3%); (3) The mutagenized bacterial liquid was spread on LB selective plates containing 0.1% sodium dodecyl sulfate (SDS), and after culturing at 4°C for 5 days, single morphologically variant colonies were picked (the diameter was 30%-40% larger than the original strain, and radial folds appeared on the edge); (4) The genetic stability was verified by three generations of subculture, and the phenotypically stable mutant strain D12 (ARTP mutant strain) was selected.

[0047] (II) Analysis of physiological and biochemical characteristics of the strains before and after mutagenesis (1) Morphological observation: Gram staining showed that the original strain D12 was a Gram-negative short rod-shaped bacterium with a length of about 2 μm. The colony shape was round with a slight protrusion in the middle and smooth edges. The colony color was opaque white. The mutant strain D12 maintained the Gram-negative short rod shape (1.8-2.2 μm), but the colony morphology changed to an irregular circle with a concave center and serrated edges. The color changed to milky yellow and translucent.

[0048] (2) Changes in key metabolic indicators:

[0049] The strains were subjected to physiological and chemical identification before and after mutagenesis. The measured physiological and chemical indicators are shown in Table 1, where "+" represents positive and "-" represents negative.

[0050] Table 1:

[0051]

[0052] (3) Functional verification: The cell-free culture medium of the mutant strain D12 was added to the culture medium containing Bacillus subtilis under the same conditions as above. After culturing at 10°C for 48 h, the biomass increased by 42% compared with the original D12 treatment group (OD600 0.86 vs 0.61), and the lag phase of the indicator bacteria was shortened by 3.5 h.

[0053] (III) Molecular identification

[0054] (1) By using blastn to compare with all available 16S rDNA sequences in the NCBI database, the mutant strain D12 had a similarity of 99.8% with the original D12 strain, and its sequence is shown in SEQ ID NO.1. Finally, combined with morphological observations and physiological and biochemical tests, it was speculated that the mutant strain D12 was Pseudomonas fragariae, and the mutant strain D12 was finally named Pseudomonas fragariae D12 ( Pseudomonas.fragi D12);

[0055] (2) Whole-genome resequencing revealed an A214G point mutation in the gyrB gene, and the expression of the sigma factor rpoS gene, which is associated with phenotypic changes, was upregulated by 3.7-fold;

[0056] (3) Pulsed-field gel electrophoresis Figure 1 As shown, combined with whole genome sequencing comparison, the chromosome size before and after the mutation increased from 5.2 Mb to 5.4 Mb, suggesting the presence of a genomic island insertion.

[0057] The mutant strain Pseudomonas fragariae D12 obtained by mutagenesis of the present invention was deposited in the China Center for Type Culture Collection and classified as Pseudomonas fragariae ( Pseudomonas fragi )D12, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M2025126, and the deposit date is January 14, 2025.

[0058] Example 3: Growth of Pseudomonas fragrans D12 and Preparation of Cell-free Culture Medium

[0059] (1) Growth characteristics of Pseudomonas fragrans D12

[0060] (1) The mutated Pseudomonas fragariae D12 was inoculated into LB liquid culture medium and cultured at a speed of 180 rpm and different temperatures (4, 10, 15, 25, 30 °C). Samples were taken at fixed points and the OD value of the bacterial solution was measured. 600 The growth curves at different temperatures are plotted. The results are as follows Figure 2 shown.

[0061] (II) Preparation of cell-free culture medium of Pseudomonas fragrans D12

[0062] After removing Pseudomonas fragrans D12 from -80°C storage, single colonies were first streaked onto LB plates containing 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, and 2% agar. Subsequently, single colonies were picked and inoculated into LB liquid medium for rejuvenation. Subsequently, 1 mL of the rejuvenation culture was transferred to 100 mL of liquid LB medium and incubated at 160 rpm at 30°C for 5 days. After the incubation period, the cells were centrifuged at 12,000 rpm for 10 minutes at 4°C to separate the supernatant from the cells. The supernatant was then sterilized by filtration through a 0.22 μm sterile filter to prepare the cell-free culture fluid (PfCFs) of Pseudomonas fragrans D12.

[0063] Example 4

[0064] Promoting effect of cell-free culture fluids (PfCFs) on low-temperature growth of functional strains

[0065] Select functional strains (including bacteria and fungi) that have the ability to degrade cellulose but are restricted in growth at low temperatures within 15°C. Bacillus subtilis (Bacillus subtilis), Bacillus cereus (Bacillus cereus), Bacillus megaterium (Bacillus megaterium) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), fungi such as Aspergillus niger (Aspergillus niger), Trichoderma reesei (Trichoderma reesei), Trametes versicolor (white rot fungi) and Irpex lacteus (Albugo serrata), by adding 25% PfCFs and culturing at 10°C, with a control group cultured at 10°C without adding PfCFs, to evaluate the effect of PfCFs on the low-temperature growth of the above functional bacteria (the suitable growth temperature of the above functional bacteria is 25-35°C, and the growth is significantly inhibited below 15°C). The experimental results are as follows Figure 3 and Figure 4 As shown, it can be seen that under low temperature conditions, the growth rate of functional strains of various bacteria and the number of functional fungi in the experimental group with PfCFs added were significantly higher than those in the control group.

[0066] Example 5

[0067] Changes in key enzyme activities in functional strains and their functional characteristics at low temperatures

[0068] Under low temperature conditions, the key enzyme activities of functional strains are often inhibited, thereby limiting their biodegradation ability. Therefore, the effects of PfCFs on the related enzyme activities of functional bacteria were tested at temperatures as low as 10°C:

[0069] (1) In the present invention, the activity changes of key enzymes, cellulase and xylanase, of the functional strains were systematically evaluated at low temperatures by adding Pseudomonas fragariae D12 cell-free culture fluid (PfCFs) to the culture fluid containing Bacillus subtilis.

[0070] The experimental results are as follows Figure 5 As shown in the results, the addition of 25% PfCFs significantly increased cellulase and xylanase activities at 10°C. At 10°C, cellulase activity increased by 283.61% and xylanase activity by 313.79% compared to the control. This enhanced enzyme activity not only improved the degradation efficiency of cellulose and xylan at low temperatures but also significantly boosted the overall metabolic activity and growth rate of the strain.

[0071] (2) Degradation of lignin by Pseudomonas fragrans D12

[0072] Prepare a culture medium with lignin as the sole carbon source. The specific formula is as follows:

[0073] Lignin degradation medium (g / L): 1.0 g lignin, 1.0 g KH2PO4, 1.0 g K2HPO4, 1 g NaNO3, 1 g (NH4)2SO4, 1 g NaCl, 0.5 g MgSO4·7H2O, 0.05 g CaCl2, distilled water to 1 L, pH adjusted to 6.8. In the above medium with lignin as the sole carbon source, the inoculum size was 1% (6.1 × 10 8 CFU / mL) was inoculated with Pseudomonas fragrans D12 and cultured with shaking at 10°C for 48 h. The growth of Pseudomonas fragrans D12 in the culture medium with lignin as the carbon source was shown in Figure 2. Figure 6 shown.

[0074] Inoculate Pseudomonas fragariae D12 in the above-mentioned culture medium with lignin as the sole carbon source, culture at 10°C with shaking for 48 hours, sample once every 12 hours, aspirate the supernatant and dilute it 10-fold with distilled water. Use distilled water as a control and measure the absorbance at 280 nm using an ultraviolet spectrophotometer. Substitute the absorbance into the regression equation to calculate the lignin degradation rate. The formula is as follows:

[0075] Lignin degradation rate (%) = A1−A2 / A1× 100%

[0076] Wherein, A1 represents the concentration of lignin in the lignin degradation medium without inoculation (control);

[0077] A2 represents the concentration of lignin in the sample to be tested and the concentration of lignin remaining in the culture medium at a certain moment.

[0078] The results are as follows Figure 7As shown in the figure, the OD value trend is a rapid increase followed by a slow increase to a peak. Pseudomonas fragrans D12 grew rapidly within the first 24 hours of culture, with the fastest OD increase reaching 1.886 at 36 hours. Growth slowed between 24 and 36 hours, reaching a peak OD of 2.117 at 36 hours, indicating a 7.0×10 increase in viable counts. 8 CFU / mL.

[0079] The trend in lignin degradation rate, as measured by lignin concentration, showed an initial lignin content of approximately 0.51 g. As the culture progressed, the lignin content gradually decreased, while the lignin degradation rate gradually increased. At the 12th hour of incubation, the lignin degradation rate was 15%, and at 24 hours, the lignin degradation rate reached 22.6%, reaching its maximum degradation rate. At this point, the lignin content in the solution was 0.397 g. Over the next 24 to 48 hours, the lignin degradation rate remained relatively stable. This suggests that Pseudomonas fragrans D12 can grow at low temperatures using lignin as its sole carbon source. It can synthesize enzymes that degrade lignin in these conditions, thereby achieving its ability to degrade lignin.

[0080] Low-temperature enzyme activity is closely related to the functional characteristics of the strain at low temperatures. From the above results, it can be seen that the addition of CFS of Pseudomonas fragariae D12 improves the growth and metabolic ability of normal-temperature functional bacteria at temperatures below 15°C, improves the enzyme production efficiency of the strain, significantly improves the low-temperature enzyme activity of the corresponding enzyme, and improves the growth and metabolism of the functional strain at low temperatures, verifying its potential value in practical applications.

[0081] Example 6

[0082] Preliminary exploration of growth-promoting factors in cell-free culture medium of Pseudomonas fragrans D12

[0083] In order to preliminarily explore the key factors that promote the growth of functional strains in the cell-free culture of Pseudomonas fragrans D12, PfCFs were separated into components of different molecular weights and acted on Bacillus subtilis at 10°C. The experimental results are as follows: Figure 8 As shown in the figure, under the conditions of PfCFs components with a molecular weight of 5~30kDa, the OD value of Bacillus subtilis increased significantly, indicating that the components with a molecular weight of 5-30kDa have extremely significant activity in promoting the growth of mesophilic bacteria, while the OD values ​​of other molecular weight components remained basically unchanged, indicating that the PfCFs components with a molecular weight of less than 5kDa and greater than 30kDa have poor growth-promoting activity and basically have no growth-promoting effect on functional bacteria at low temperatures.

[0084] In order to investigate the growth-promoting factors of the 5-30 kDa molecular weight fraction, the growth-promoting activities of biomacromolecules such as polysaccharides, nucleic acids, and proteins in this fraction of PfCFs were further verified:

[0085] Nucleic acid extraction involves isolating genomic DNA and total RNA from PfCFs using specialized kits. The polysaccharide extraction process includes strain activation, culture expansion, bacterial removal, concentration, protein removal, alcohol precipitation, and dialysis, ultimately yielding the crude polysaccharide product.

[0086] The results were verified by experiments. Figure 9 It shows that the crude polysaccharide and nucleic acid extracts in the cell-free culture medium of Pseudomonas fragrans D12 have no promoting effect on the growth of functional strains. According to the test sequence, after first testing that the nucleic acid and extracellular polysaccharide of PfCFs have no obvious promoting effect on the low-temperature growth of functional strains, proteinase K was directly used to directly hydrolyze some proteins in PfCFs to observe the effect of proteins in PfCFs on functional bacteria. This omitted the complex protein extraction steps such as precipitation, centrifugation, and dialysis desalting. As can be seen from the figure, the growth-promoting activity of the cell-free culture medium that has been treated with proteinase K to destroy some protein structures is lower than that of the untreated cell-free culture medium, indicating that protein substances play a key role in the growth-promoting effect.

[0087] In summary, the proteins in the cell-free culture medium of Pseudomonas fragrans D12 effectively promoted the growth and metabolism of the functional strain at low temperatures and increased the activity of key enzymes. Furthermore, the CFS of Pseudomonas fragrans D12 also contains enzymes that can degrade lignin, further enhancing the efficiency of lignocellulose degradation at low temperatures. These functions provide an effective solution for biomass degradation in low-temperature environments and have broad application prospects.

[0088] Through the verification of the above functions of Pseudomonas fragrans D12 and its CFS, it can be further clarified that D12 and its CFS can be applied to agricultural production, such as microbial fermentation under low-temperature environments (such as compost fermentation, biomass fermentation to produce sugar, ethanol production and other biotransformations).

[0089] Example 7

[0090] The CFS prepared by Pseudomonas fragrans D12 was applied to straw degradation:

[0091] The cellulase and xylanase production of Bacillus subtilis was inhibited at low temperature (the optimal growth temperature was 30°C and the viable cell count was 2.0×10 8cfu / mL, which basically stops growing and metabolizing at temperatures below 6°C), was added with a mass percentage concentration of 25% PfCFs to form a composite inoculum. The volume ratio of PfCFs to bacterial solution was 1:100. The composite inoculum was sprinkled into the pretreated corn straw and fermented in a natural environment of 0-6°C.

[0092] At the same time, corn straw pretreated with Bacillus subtilis culture without PfCFs was fermented under the same environment as the PfCFs-free control group 1.

[0093] Comparative Example 1

[0094] A CFS was prepared by purchasing a low-temperature fragility Pseudomonas fragariae strain, and a composite bacterial agent was prepared according to the same method as in Example 7, and the same corn straw was fermented under the same conditions. The low-temperature fragility Pseudomonas fragariae strain was purchased from Beijing Biobo Biotechnology Co., Ltd., with a platform number of bio-02547 and an original number of L3-1.

[0095] Example 8

[0096] The CFS prepared by Pseudomonas fragrans D12 was applied to straw degradation:

[0097] The bacterial liquid of Bacillus subtilis (the optimal growth temperature is 30°C and the number of viable cells is 2.0×10 8 cfu / mL, and basically stopped growing and metabolizing at 6°C) and the bacterial solution of White Capsule Rake Tooth Fungus, which produces lignin-degrading enzymes at room temperature but is inhibited at low temperatures (the optimal growth temperature is 28°C, the viable cell count is 1.0×10 8 cfu / mL, which basically stops growing and metabolizing below 6°C) was prepared into a mixed bacterial solution at a volume ratio of 1:1, and then PfCFs with a mass percentage concentration of 25% was added to form a composite bacterial agent. The volume ratio of PfCFs to the mixed bacterial solution was 1:100, and the composite bacterial agent was sprinkled into the treated corn straw and fermented in a natural environment of 0-6°C.

[0098] At the same time, corn straw pretreated with solid-state fermentation of a mixed bacterial solution (composed of Bacillus subtilis and Bacillus subtilis in a volume ratio of 1:1) without the addition of PfCFs was used as the PfCFs-free control group 2, and the corn straw was fermented at room temperature of 30°C and at a temperature of 0-6°C, respectively.

[0099] After 30 days of fermentation, the degradation efficiency of cellulose, hemicellulose, and lignin in each group of straw was calculated to reflect the fermentation degree of the straw. After the corn straw was fermented, the degradation rates of lignin, cellulose, and hemicellulose were determined by chemical analysis (such as the Van's detergent method). The specific steps are as follows:

[0100] (1) Determination of neutral detergent fiber (DNF)

[0101] 1. Weigh 1g of air-dried sample, place it in a beaker, and add 100mL of neutral detergent and a few drops of n-octanol.

[0102] 2. Boil and reflux for 1 hour, and filter while hot using a glass sand core crucible of known weight.

[0103] 3. Rinse the residue with boiling water until the filtrate is neutral, and dry it together with the crucible residue at 105℃ to constant weight. Record the weight m1 (DNF + crucible weight m0).

[0104] (2) Acid detergent fiber (ADF) determination

[0105] 1. Take the DNF in (1), add 100 mL of acidic detergent and a few drops of n-octanol, and boil under reflux for 1 hour.

[0106] 2. Filter while hot using a glass crucible of known weight. Rinse the residue with boiling water until the filtrate is neutral. Dry the residue together with the crucible at 105°C to constant weight. Record the weight m2 (ADF + crucible weight m0).

[0107] (III) Determination of acid detergent lignin (ADL)

[0108] 1. Take the dry ADF from step (2), add 5 mL of 72% sulfuric acid, and digest with stirring at room temperature for 2 h to dissolve the cellulose.

[0109] 2. Dilute with water to a sulfuric acid concentration of about 3%, filter, rinse with hot water until neutral, and dry the crucible together with the residue at 105°C to constant weight. Weigh and record the weight as m3 (ADL + ash + crucible weight m0).

[0110] 3. Ash in a muffle furnace at 550℃ for 2h, weigh after cooling, and record it as m4 (acid-insoluble ash + crucible weight m0).

[0111] NDF=(m1-m0) / sample dry weight × 100%.

[0112] ADF=(m2-m0) / sample dry weight×100%.

[0113] Hemicellulose (%) = D = NDF-ADF

[0114] Lignin content = (m3– m4) / sample dry weight × 100%.

[0115] Cellulose content (%) = ADF-DNF-acid insoluble ash

[0116] The calculation formula for the degradation rate of each component is:

[0117]

[0118] The above-mentioned Bacillus subtilis and Rhacophora albopicta were both independently screened strains. The final results are shown in Table 2.

[0119] Table 2: Effect of PfCFs on the fermentation efficiency of mesophilic bacteria at low temperature

[0120]

[0121] From the results of the degradation of lignocellulose in corn straw, it can be seen that in the control group 1 without PfCFs, the growth and metabolism of Bacillus subtilis completely stopped in an environment below 6°C, and the degradation efficiency of lignocellulose in the straw was extremely low, all within 10%. After the PfCFs of the present invention were further added in Example 7, the degradation rates of lignin, hemicellulose and cellulose were significantly improved, which shows that the addition of PfCFs improved the growth and metabolism of Bacillus subtilis in a low temperature environment, thereby achieving the effect of degrading cellulose and hemicellulose. The CFS in Comparative Example 1 also improved the degradation efficiency of cellulose and hemicellulose in the straw, but the overall efficiency was lower than that in Example 7, and it did not have any promoting effect on the degradation efficiency of lignin. In Example 7, in addition to the higher degradation efficiency of cellulose and hemicellulose, the degradation rate of lignin also reached 16.16%, which shows that the PfCFs of the present invention contain relevant enzymes that can degrade lignin at low temperatures, thereby further improving the degradation efficiency of lignin in the straw.

[0122] The degradation efficiency of lignin, hemicellulose and cellulose bacteria in corn straw in the control group 2 without the addition of PfCFs was reduced to less than 10% at a temperature as low as 6°C, indicating that at this temperature, both the white sac rake tooth fungus and the Bacillus subtilis were in a state of stopped growth and metabolism and could not effectively participate in the degradation of lignocellulose. Example 8 is based on the control group 2, and PfCFs were further added. It can be seen that at low temperatures, PfCFs simultaneously promoted the co-growth of the bacteria Bacillus subtilis and the fungus white sac rake tooth fungus, thereby improving the degradation efficiency of functional bacteria on lignocellulose in corn straw. Since PfCFs contain related enzymes that can degrade lignin at low temperatures, in Example 8, they work together with the white sac rake tooth fungus that produces enzymes related to lignin degradation, thereby increasing the concentration and enzyme activity of the lignin degrading enzyme in the degradation system, further improving the degradation efficiency of lignin, and the lignin degradation rate reached 27.37%, thereby changing the overall degradation effect of the composite bacterial agent on lignocellulose in corn straw.

Claims

1. A strain producing a microbial low-temperature growth-promoting protein, characterized in that: The strain is classified as Pseudomonas fragariae ( Pseudomonas fragi )D12, deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M2025126, and the deposit date is January 14, 2025.

2. A cell-free culture solution for promoting low-temperature growth of microorganisms, characterized in that: The cell-free culture solution is obtained by culturing Pseudomonas fragariae, and the Pseudomonas fragariae is Pseudomonas fragariae ( Pseudomonas fragi )D12, deposited in the China Center for Type Culture Collection, the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M2025126, and the deposit date is January 14, 2025.

3. The use of Pseudomonas fragariae D12 as claimed in claim 1 in promoting low-temperature composting fermentation of low-temperature-tolerant microorganisms, wherein the low-temperature-tolerant microorganism is at least one of Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus amyloliquefaciens, Aspergillus niger, Trichoderma reesei, white rot fungi and white capsule rake tooth fungi, and the low-temperature fermentation temperature is 0~15°C.

4. Use of the cell-free culture medium according to claim 2 in promoting low-temperature composting fermentation of low-temperature-sensitive microorganisms, wherein the low-temperature-sensitive microorganism is at least one of Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus amyloliquefaciens, Aspergillus niger, Trichoderma reesei, white rot fungi, and white cysts, and the low-temperature fermentation temperature is 0-15°C.

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