Simple pure bacillus T3 and its culture medium, culture method and application

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a simple pure Bacillus T3, a culture medium, a culture method and application thereof. The present application provides a simple pure Bacillus T3 with a preservation number of CCTCC NO: M 2024904, a culture medium, a culture method and application thereof. The strain T3 can promote the mycelium growth and sclerotium formation of Morchella conica at the same time, and the increase of the mycelium growth speed is more than 10%, which is of great significance for improving the quality of Morchella conica, shortening the spawn period of Morchella conica and reducing the production cost. The culture medium of the strain T3 is easy to obtain, and the culture method is simple, which is conducive to realizing large-scale application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a simple pure Bacillus T3, a culture medium and culture method thereof, and application thereof. BACKGROUND

[0002] Morel (Morchella esculenta) is a rare edible and medicinal fungus belonging to Ascomycota, Pezizomycetes, Pezizales and Morchellaceae. The fruiting body of Morel is delicious and nutritious, and contains polysaccharides, phenols, protein hydrolysates and other bioactive substances. Morel is an important rare edible and medicinal fungus. Morel is initially obtained by layered culture to obtain a large number of sclerotia, and then the mature sclerotia are used to induce the growth of Morel to realize artificial cultivation. Subsequent studies have shown that the Morel varieties that can be cultivated and produced in China can form sclerotia during cultivation, and the number of sclerotia is an important standard for the quality of Morel strains and is proportional to the number of primordia formed during cultivation.

[0003] The formation of Morel sclerotia is a complex process, and the activity of the strain, the culture material, and environmental factors such as temperature, light, pH and osmotic pressure can all affect it. Edible fungus cultivation is considered as a "dialogue between bacteria and fungi". Bacteria can enhance nutrition by degrading complex polysaccharides through lignocellulose enzyme activity in fungi, and fungi can also consume bacteria and absorb carbon and nitrogen elements in bacteria as a source of nutrition. Currently, it has been found that Pseudomonas putida can stimulate the formation of Morel sclerotia, but it has no obvious effect on the growth of Morel mycelium. In addition, there are studies on the addition of Morel stem soil to obtain an improved agent to promote the growth of Morel mycelium and the formation of sclerotia. However, the improved agent material is limited in source because it can only be obtained after Morel is planted.

[0004] Since Morel can only be cultivated in soil, the microorganisms in the soil may have a very important influence on the growth of Morel. However, the interaction mechanism between bacteria in the soil and Morel is less studied at present. Continuous planting of Morel for 2 years will result in heavy land problems such as yield reduction and serious diseases and pests, so it is necessary to improve the quality of Morel strains. SUMMARY

[0005] Therefore, the present application provides a simple pure Bacillus T3, a culture medium and culture method thereof, and application thereof. The strain can significantly promote the growth of Morel mycelium and the formation of sclerotia, thereby improving the quality of the strain and shortening the spawn production period of Morel.

[0006] To solve the above technical problems, a first aspect of the present application provides a Peribacillus simplex T3, which was preserved in the China Center for Type Culture Collection on May 11, 2024, with a preservation number of CCTCC NO: M 2024904 and a preservation address of No. 299, Bayingyi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The Peribacillus simplex T3 provided by the present application is obtained by isolation and screening from the mycelium soil of Morchella esculenta, and is preliminarily determined as Peribacillus simplex according to morphological, physiological and biochemical characteristics and 16S rDNA sequencing results. The colony formed by the strain has a milky white color, a dry, raised and smooth surface, is easy to pick up, has a regular colony morphology, and is positive to Gram staining, and the bacterial cells are rod-shaped. Inoculation of the strain T3 provided by the present application into the Morchella esculenta original strain and / or cultivation medium can increase the mycelial growth rate of Morchella esculenta by more than 10%, and can also significantly promote the formation of Morchella esculenta sclerotia.

[0008] A second aspect of the present application provides a liquid culture medium of Peribacillus simplex T3 with a preservation number of CCTCC NO: M 2024904, and the composition of the liquid culture medium per liter includes: 5-15 g of a nitrogen source, 2.5-7 g of yeast powder, 2.5-10 mg of inorganic salt, and the balance is water.

[0009] In combination with the second aspect, the nitrogen source is tryptone and / or peptone; and the inorganic salt is selected from water-soluble salts containing at least one ion of Fe 2+ , Cu 2+ , Mn 2+ , K + , PO4 3- , Mg 2+ and Ca 2+ .

[0010] Preferably, the liquid culture medium per liter includes: 12 g of tryptone, 6.25 g of yeast powder, 5 mg of FeSO4, and the balance is water.

[0011] A third aspect of the present application provides a culture method of Peribacillus simplex T3 with a preservation number of CCTCC NO: M 2024904, which specifically includes: inoculating the strain T3 into the above-mentioned liquid culture medium, and performing shake flask culture at 20-40°C, with an initial pH of 6.0-8.5; wherein the liquid loading amount of the shake flask culture medium is 30-150 mL / 250 mL, and the rotation speed is 140-220 r / min.

[0012] Preferably, the strain T3 is inoculated into the liquid medium described above, and is subjected to shake flask culture at 25℃, with an initial pH of 7.5, a liquid volume of 30 mL / 250 mL, and a rotation speed of 200 r / min.

[0013] The fourth aspect of the present application provides an application of the Peribacillus simplex T3 with the preservation number of CCTCC NO: M 2024904 in promoting the growth of Morel mycelium and / or sclerotia formation.

[0014] In combination with the fourth aspect, the Morel can be any cultivable Morel variety, including but not limited to Morchella sextelata, Morchella heptelata and Morchella conica.

[0015] In combination with the fourth aspect, the strain T3 is subjected to shake flask culture in the liquid medium for 12-18 h, is diluted with sterile water to an OD 600 value of 1.0, is centrifuged to obtain the bacterial body, and is then resuspended with sterile water to a corresponding concentration to prepare a bacterial solution; the Morel spores are inoculated into a matching stock or cultivation substrate, and the T3 bacterial solution is inoculated at the same time, and is subjected to static culture.

[0016] Illustratively, first, the strain T3 is subjected to shake flask culture in the liquid medium for 16 h, is diluted with sterile water to an OD 600 value of 1.0, is centrifuged to obtain the bacterial body, and is then resuspended with sterile water to a corresponding concentration; second, a Morel stock and / or cultivation medium is prepared, for example, the culture medium formula of the Morchella sextelata strain can be: 60 wt% of wheat grains, 21 wt% of bran, 17 wt% of corn cob, 1 wt% of gypsum and 1 wt% of quicklime, and the culture medium formula of the Morchella heptelata strain can be: 58 wt% of wheat grains, 12 wt% of bran, 28 wt% of corn cob, 1 wt% of gypsum and 1 wt% of quicklime. The bagged amount is 450 g, and the water content is 60%-65%; finally, three Morchella sextelata or Morchella heptelata cakes with a diameter of 10 mm are inoculated into the matching stock and / or cultivation medium, 3 mL of the T3 bacterial solution is inoculated at the same time, and is subjected to static culture at 18℃.

[0017] The fifth aspect of the present application provides an application of the Peribacillus simplex T3 with the preservation number of CCTCC NO: M 2024904 in improving the quality of Morel spores.

[0018] The sixth aspect of the present application provides an application of the Peribacillus simplex T3 with the preservation number of CCTCC NO: M 2024904 in shortening the preparation period of Morel spores.

[0019] The present application provides a simple pure Bacillus T3 strain, a culture medium, a culture method and an application thereof, the strain T3 can significantly promote the mycelium growth and sclerotium formation of Morchella esculenta at the same time, and has important significance for improving the Morchella esculenta strain quality and shortening the Morchella esculenta seed production period. Moreover, the culture medium of the strain T3 is easy to obtain, the culture method is simple, and is beneficial to realize large-scale application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Culture photos of the mycelium growth and sclerotium formation of Morchella esculenta inoculated with no bacteria, inoculated with T3 bacteria and inoculated with T4 bacteria;

[0021] Figure 2 A colony morphology photo of the simple pure Bacillus T3;

[0022] Figure 3 A bacterial body microscopic image of the simple pure Bacillus T3 after Gram staining;

[0023] Figure 4 A phylogenetic tree of the simple pure Bacillus T3;

[0024] Figure 5 Effects of adding different types of carbon sources on the growth of the simple pure Bacillus T3;

[0025] Figure 6 Effects of adding different types of nitrogen sources on the growth of the simple pure Bacillus T3;

[0026] Figure 7 Effects of adding different types of inorganic salts on the growth of the simple pure Bacillus T3;

[0027] Figure 8 Effects of the optimized culture medium on the growth of the simple pure Bacillus T3;

[0028] Figure 9 Effects of the pH of the culture medium on the growth of the simple pure Bacillus T3;

[0029] Figure 10 Effects of the culture temperature on the growth of the simple pure Bacillus T3;

[0030] Figure 11 Effects of the liquid loading amount on the growth of the simple pure Bacillus T3;

[0031] Figure 12 Effects of the rotation speed on the growth of the simple pure Bacillus T3;

[0032] Figure 13 Effects of the addition amount of the T3 bacteria on the mycelium growth speed of Morchella esculenta;

[0033] Figure 14 Effects of the added amount of T3 bacterium on the sclerotium formation and sclerotium morphology of Morchella conica and Morchella angusticeps, wherein (a) no T3 bacterium is added, (b) the added amount of T3 bacterium is 0.0005 μL / mL, (c) the added amount of T3 bacterium is 0.005 μL / mL, (d) the added amount of T3 bacterium is 0.05 μL / mL, and (e) the added amount of T3 bacterium is 0.5 μL / mL;

[0034] Figure 15 Effects of the T3 bacterium concentration on the mycelium growth rate of Morchella conica and Morchella angusticeps, wherein (a) is the effect of the T3 bacterium concentration on the mycelium growth rate of Morchella conica, and (b) is the effect of the T3 bacterium concentration on the mycelium growth rate of Morchella angusticeps;

[0035] Figure 16 (a) is a photograph of the sclerotium formation when Morchella conica is inoculated with different concentrations of T3 bacterium, and (b) is a photograph of the sclerotium formation when Morchella angusticeps is inoculated with different concentrations of T3 bacterium. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application.

[0037] The raw materials used in the following examples of the present application are all ordinary commercially available products. In the following examples, the Morchella conica and Morchella angusticeps used are strains that can be purchased on the market, and in actual applications, the Morchella in the examples can also be replaced by Morchella angusticeps or other cultivable Morchella.

[0038] In the following examples, the formula of the PDA culture medium used is: 200 g of fresh potato, 20 g of glucose, and 12 g of agar per liter of culture medium, and the rest is water; the formula of the LB culture medium used is: 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride per liter of liquid culture medium, and the rest is water; the formula of the NYBD culture medium used is: 8 g of beef extract, 5 g of yeast powder, and 20 g of glucose per liter of liquid culture medium, and the rest is water; and the formula of the NYB culture medium used is: 8 g of beef extract and 5 g of yeast powder per liter of liquid culture medium, and the rest is water.

[0039] Example 1

[0040] This example provides a method for isolating, screening, and identifying simple pure Bacillus T3

[0041] 1. Isolation and screening of simple pure Bacillus T3

[0042] (1) Isolation: 10 g of fresh Morchella mushroom soil was collected from Hengshui City, Hebei Province, and placed in a 250 mL conical flask containing 90 mL of sterile water. The suspension was shaken for 30 min. The bacterial suspension was diluted to a concentration of 10 by 10-fold dilution using the gradient dilution method. -1 ~10 -6 , take 10 -4 ~10 -6 100 μL of the bacterial suspension with different concentrations was spread on LB culture medium plates using a spreader and cultured at a constant temperature of 28°C. Different single colonies were selected according to their morphological characteristics and purified and cultured, and finally 11 bacterial strains were isolated.

[0043] (2) Screening: Using the plate confrontation method, a 5 mm diameter Morchella cake was inoculated in the center of the PDA culture medium. At a distance of 2 cm from the cake, the isolated bacteria were inoculated onto the PDA culture medium in a triangular shape with the cake as the center. The culture medium was placed in a constant temperature incubator at 20°C for inverted culture. Two strains with obvious growth-promoting effects on the growth of Morchella hyphae and the formation of sclerotia were screened out (respectively marked as T3 and T4). Among them, strain T3 had the strongest growth-promoting effect (e.g. Figure 1 shown).

[0044] 2. Identification of simple pure Bacillus T3

[0045] (1) Morphological characteristics

[0046] Observe the colony morphology and microscopic morphology of strain T3 on LB plate: the colony formed by strain T3 is milky white, the surface of the colony is dry, raised, smooth, easy to pick up, and the colony morphology is regular (such as Figure 2 As shown); Gram staining was performed and the cells were observed under an electron microscope. The experimental results were positive and the bacterial cells were rod-shaped (as shown); Figure 3 shown).

[0047] (2) Determination of physiological and biochemical characteristics

[0048] Physiological and biochemical tests were performed on the screened bacteria T3 using microbiochemical identification tubes. The results (as shown in Table 1) showed that strain T3 could reduce nitrate and utilize lysine, but could not utilize ornithine, arginine, citrate, or hippurate, could not liquefy gelatin, and was negative for VP and MR reactions.

[0049] Table 1

[0050]

[0051] (3) 16S rDNA identification

[0052] The 16S rDNA sequence of the strain T3 was sequenced by using the universal primers 27F / 1492R to amplify the PCR product from the bacterial T3 genomic DNA. The sequencing result (the sequence is shown as SEQ ID NO. 1, and the NCBI accession number is PP767395) was subjected to BLAST comparison with the NCBI data, and the phylogenetic tree was constructed as shown in Figure 4 The results show that the bacterial T3 is most closely related to Peribacillus simplex.

[0053] According to the morphological, physiological and biochemical characteristics and the 16S rDNA sequencing result, the strain T3 is preliminarily determined as Peribacillus simplex.

[0054] The Peribacillus simplex T3 provided in the present application was preserved in the China Center for Type Culture Collection on May 11, 2024, and the preservation number is CCTCC NO: M2024904, and the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0055] Example 2

[0056] The present embodiment provides the culture process optimization experiment of the Peribacillus simplex T3

[0057] The T3 strain was inoculated in the LB culture medium, and cultured at 25℃ for 24h. A single colony was picked and inoculated in the beef extract yeast glucose culture medium (NYBD), and cultured at 25℃ for 16h, as a seed liquid for standby.

[0058] (1) Carbon source screening in the culture medium: maltose, galactose, rhamnose, lactose, raffinose, citric acid and sucrose were respectively used to replace the carbon source (glucose) in the culture medium, and the results show that, compared with the culture medium without carbon source (NYB), the culture medium with the added carbon source does not obviously promote the growth of the T3 strain (as shown in Figure 5 Therefore, the NYB is selected as the basic culture medium for the subsequent experiment.

[0059] (2) Nitrogen source screening in the culture medium: the trypsin, the protein peptone, the ammonium sulfate, the potassium nitrate and the urea were respectively used to replace the nitrogen source (beef extract) in the NYB. The results show that, when the trypsin of 8g / L is used, the concentration of the T3 strain is the highest, and the OD 600 value is 1.56 (as shown in Figure 6 Therefore, the trypsin is selected as the best nitrogen source of the culture medium.

[0060] (3) Inorganic salt screening in the culture medium: the Fe 2+ , Cu 2+ , Mn 2+ , K+ PO4 3- Mg 2+ , Ca 2+ 、Na + The results showed that when 5 mg / L ferrous sulfate was used as a culture medium component, the concentration of T3 bacterial solution was the highest, and the OD 600 The value is 1.778 (such as Figure 7 As shown), ferrous sulfate was selected as the most suitable inorganic salt component.

[0061] (4) The optimal component concentration ratio was obtained through orthogonal experiments. A4B4C2 (tryptone 12 g / L, yeast powder 6.25 g / L, FeSO4 5 mg / L) was compared with the basic medium NYB (beef extract 8 g / L, yeast powder 5 g / L) in fermentation experiments. The OD of the fermentation liquid was measured. 600 The result is as follows Figure 8 As shown in the figure, it can be seen that the OD value of the culture medium after 16 hours of culture in the combination A4B4C2 medium after bacterial fermentation optimization is 600 The value was 1.54, while the OD value of the culture medium after 16 hours of culturing in NYB basic medium was 600 The value was 1.38. The growth rate in the optimized fermentation medium was 11.59% higher than that in the basic medium. Therefore, A4B4C2 (tryptone 12 g / L, yeast powder 6.25 g / L, FeSO4 5 mg / L) was selected as the optimal medium.

[0062] (5) After confirming the optimal culture medium, we continued to explore the culture conditions suitable for the growth of T3 strains by changing the initial pH of the culture medium, culture temperature, liquid volume (250 mL conical flask) and shaking speed (the results were as follows Figures 9-12 According to the experimental results, the most suitable culture conditions for strain T3 were determined as follows: initial pH: 7.5, culture temperature: 25°C, liquid volume: 30mL / 250mL, and rotation speed: 200r / min.

[0063] Example 3

[0064] This example provides an experiment on the promotion of mycelial growth and sclerotia formation of Morchella by strain T3.

[0065] (1) After culturing strain T3 for 16 h under the optimal culture process determined in Example 2, the strain was centrifuged at 4000 rpm for 10 min and resuspended in sterile water to an OD of 600 is 1.0. Use a peristaltic pump to quantitatively pour a double-layer culture medium, with 10 mL of agar medium in the lower layer and 10 mL of PDA medium containing different concentrations of T3 bacteria in the upper layer. Take a 5 mm diameter Qimei Morel KS5 cake and inoculate it into the center of the plate. The results show (as shown inFigure 13 As shown in the figure, the mycelial growth rate and sclerotia number of Morchella KS5 are closely related to the amount of T3 bacteria added: with the increase of T3 bacteria addition, the mycelial growth rate of Morchella KS5 shows a trend of first increasing and then decreasing. When the T3 bacteria addition amount is 0.005μL / mL, the mycelial growth rate of Morchella KS5 is the fastest. Figure 14 It can be seen that with the increase in the amount of T3 bacteria added, the sclerotia density of Morchella KS5 showed a trend of first increasing and then decreasing, and the sclerotia were all white. Among them, the sclerotia density was the highest when the T3 bacteria addition amount was 0.005 μL / mL.

[0066] This shows that adding 0.0005-0.05 μL / mL of T3 bacteria to the culture medium can simultaneously promote the hyphal growth and sclerotia formation of Morchella KS5, and the effect of promoting the hyphal growth and sclerotia formation of Morchella KS5 is most obvious when the addition amount is 0.005 μL / mL.

[0067] (2) After culturing strain T3 for 16 h under the optimal culture process determined in Example 2, the strain was centrifuged at 4000 rpm for 10 min and resuspended in sterile water to an OD of 600 The value is 1.0, and then the cells are resuspended to 10 6 , 10 7 , 10 8 , 10 9 Concentration. Secondly, prepare the culture medium of the original species and / or cultivated species of Morchella, wherein the culture medium formula of the six-sister Morchella strain is: 60wt% of wheat grains, 21wt% of bran, 17wt% of corn cobs, 1wt% of gypsum and 1wt% of quicklime, and the culture medium formula of the seven-sister Morchella strain is: 58wt% of wheat grains, 12wt% of bran, 28wt% of corn cobs, 1wt% of gypsum and 1wt% of quicklime. The culture bag volume is 450g, and the water content is 60% to 65%. Finally, 3 six-sister Morchella or seven-sister Morchella cakes with a diameter of 10mm are inoculated in the matching culture medium, and 3mL of T3 bacteria with different concentration gradients are inoculated at the same time, and cultured at 18°C.

[0068] The experimental results show that ( Figure 15 (a) and Figure 15 (b)) When T3 bacteria were co-cultured with Morchella serrata, the inoculation concentration was 10 8 The T3 bacteria had a significant effect on promoting the growth rate of the mycelium of the sixth sister morel compared with the control group (no T3 bacteria), which increased by 10.77% compared with the control group; when the T3 bacteria were co-cultured with the seventh sister morel, the inoculation concentration was 10 7The T3 mycelium has a significant promoting effect on the growth rate of the Morchella sextelata and Morchella septelata compared with the control group (without inoculating T3 mycelium), and the growth rate is increased by 10.53%.

[0069] The photos of the spherules of the Morchella sextelata and the Morchella septelata are shown in Figs. Figure 16 (a) and Figure 16 (b), respectively.It can be seen that the spherule formation quantity of the Morchella sextelata and the Morchella septelata inoculated with the T3 mycelium is obviously increased compared with the spherule formation quantity of the Morchella sextelata and the Morchella septelata without inoculating the T3 mycelium when the inoculation concentration is 10 6 ~ 10 9

[0070] The above merely describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can make equivalent replacements or changes according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, and all of them should be covered in the protection scope of the present application.

Claims

1. A simple pure strain of Bacillus ( Peribacillus simplex ) T3, characterized in that, The deposit number is CCTCCNO: M 2024904.

2. A method for culturing the simple pure Bacillus T3 according to claim 1, characterized in that: Simple pure Bacillus T3 was inoculated into liquid culture medium and cultured in shake flasks at 20-40°C with an initial pH of 6.0-8.5; The liquid culture medium has a liquid volume of 30-150 mL / 250 mL and a rotation speed of 140-220 r / min.

3. The culture method according to claim 2, wherein The simple pure Bacillus T3 was inoculated into the liquid culture medium and cultured in a shake flask at 25° C. with an initial pH of 7.5; wherein the liquid volume of the liquid culture medium was 30 mL / 250 mL and the rotation speed was 200 r / min.

4. Use of the simple pure Bacillus T3 according to claim 1 in promoting the mycelial growth and / or sclerotia formation of Morchella.

5. The use according to claim 4, characterized in that Simple pure Bacillus T3 was placed in liquid culture medium and shaken for 12-18 hours, and diluted with sterile water to OD 600 When the value is 1.0, centrifuge and obtain the bacteria, then resuspend it with sterile water to the corresponding concentration to prepare the bacterial solution; inoculate the Morchella strain into a matching original species or cultivated species matrix for culture, and at the same time inoculate a simple pure Bacillus T3 bacterial solution and culture it statically.

6. Use of the simple pure Bacillus T3 according to claim 1 in improving the quality of Morchella spp.

7. Use of the simple pure Bacillus T3 according to claim 1 in shortening the preparation cycle of Morchella spp.

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