Compound microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganisms, and relates to a compound microbial agent as well as a preparation method and application thereof. The invention discloses a compound microbial agent, the core component of the compound microbial agent is obtained by co-culturing bacillus cereus GX6-3 and lysinibacillus fusiformis GX7-1, and by applying the compound microbial agent, the absorptivity of plants to selenium in the environment is increased, the selenium content of the plants is increased, the yield of the plants is increased, and the yield of the plants is increased. Field test results show that the quality of fresh tea leaves can be improved by applying the compound microbial agent. In addition, by optimizing the volume ratio of all strains in co-culture strains and the inoculum size of the co-culture strains, the selenium content in thalli is increased, and the selenium conversion rate is increased; and by optimizing the components of the co-cultured culture medium, the selenium content in the thalli is further increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and relates to a composite microbial inoculum, a preparation method thereof, and an application thereof. Background Art

[0002] Selenium is one of the essential trace elements for the human body, and has functions such as anti-cancer, anti-aging, antioxidant, enhancing human immunity, and regulating the absorption and utilization of vitamins, which is very important for the human body. Selenium deficiency can cause various diseases. Approximately 51% of the population in China is affected by selenium deficiency. The selenium-deficient areas and selenium marginal areas are distributed on the geographical belt from northeast to southwest China, while the selenium-rich areas are relatively limited. Currently, the generally recognized selenium-rich areas mainly include Enshi in Hubei, Ankang in Shaanxi, etc.

[0003] The selenium element in the human body cannot be synthesized by itself and can only be obtained from the external environment. At present, the application of supplementing plant selenium-rich products is the most extensive. The simplest way to supplement selenium is to drink selenium-rich tea. The organic selenium in selenium-rich tea accounts for more than 80% of the total selenium, and at the same time, it can also supplement the functional components in ordinary tea, such as tea polyphenols, tea polysaccharides, caffeine, tea proteins, etc., which are beneficial to human absorption. Therefore, selenium-rich tea is an ideal selenium supplement resource. People often drink selenium-rich tea, which can improve the selenium nutrition of the human body and play a role in preventing diseases.

[0004] Improving the tolerance of plants to selenium, as well as the efficiency of absorbing and taking up selenium from the environment, and then converting inorganic selenium into safe and effective organic selenium or other forms of selenium is a key issue in the research of plant selenium enrichment. Microorganisms play a very important role in the migration, transformation of mineral elements in the soil environment, and biogeochemical cycles, etc. They also play an important role in the absorption and transformation of selenium in plants. For example, Long Yunchuan et al. screened a selenium-tolerant siderophore-producing strain from the rhizosphere soil of corn in Kaiyang area of Guizhou, which has strong abilities of secreting siderophores and indole acetic acid, and has the potential to increase the selenium content in crops.

[0005] At present, there are few reports on selenium-rich microorganisms in tea trees, and there are even fewer reports on related composite microbial inoculums. Screening and identifying selenium-rich microorganisms in tea trees and exploring the development of composite microbial inoculums can solve the problems that tea trees and similar crops have little absorption of soil selenium and little selenium content in products, which has important research significance for promoting the development of the selenium-rich tea industry. Summary of the Invention

[0006] To solve the above technical problems, the present invention screens endophytic strains and studies the co-culture of endophytic strains to prepare a composite microbial inoculum, aiming to improve the selenium absorption rate of plants and increase the selenium content of plants by applying the composite microbial inoculum.

[0007] On the one hand, the present invention provides a compound microbial inoculum, which is obtained by co-culturing Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1;

[0008] The preservation number of Bacillus cereus GX6-3 is CGMCC No. 33304, and the preservation number of Lysinibacillus fusiformis GX7-1 is CGMCC No. 33305.

[0009] Furthermore, in the co-culturing, the volume ratio of the seed solution of Bacillus cereus GX6-3 to the seed solution of Lysinibacillus fusiformis GX7-1 is 2:1.

[0010] Furthermore, in the co-culturing, the culture medium used includes inorganic salts, a carbon source, and a nitrogen source.

[0011] Furthermore, in the co-culturing, the inorganic salt of the culture medium is one of KCl, MnSO4, CaCl2, MgSO4, KH2PO4, and NaCl.

[0012] Furthermore, in the co-culturing, the carbon source of the culture medium is one of glucose, sucrose, soluble starch, maltose, lactose, and mannitol.

[0013] Furthermore, in the co-culturing, the nitrogen source of the culture medium is composed of tryptone and yeast extract, or tryptone and yeast powder, or tryptone and beef extract, or beef extract and yeast extract.

[0014] Furthermore, in the co-culturing, the culture medium used, in terms of mass percentage, is composed of 1% glucose, 0.75% tryptone, 0.75% yeast powder, 1% CaCl2, and the balance being distilled water.

[0015] On the other hand, the present invention provides a method for increasing the selenium content in plants by applying the compound microbial inoculum.

[0016] Furthermore, the plant is a tea tree.

[0017] Third aspect, the present invention also provides a method for improving the quality of fresh tea leaves, the method comprising: applying the compound microbial inoculant to tea trees to improve the quality of fresh tea leaves; the quality of the fresh leaves includes at least one of selenium content, theanine content, tea polyphenol content, total catechin content, soluble sugar content, water extract content, free amino acid content, and phenol-ammonia ratio.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0019] (1) The present invention discloses a compound microbial inoculant, the core components of which are obtained by co-culturing Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1. Applying this compound microbial inoculant can improve the absorption rate of selenium in the environment by tea trees and increase the selenium content in fresh tea leaves. When the final Se concentration of the compound microbial inoculant is 20 mg / L - 80 mg / L, the selenium content in young leaves and old leaves of tea tree shoots reaches the standard of selenium-enriched tea. Especially when the final Se concentration of the compound microbial inoculant is 80 mg / L, the total selenium content in young leaves and old leaves reaches 0.88 mg / kg and 1.99 mg / kg respectively. Applying the compound microbial inoculant significantly increases the selenium content in fresh leaves and improves the absorption rate of selenium by tea trees; in addition, after applying the compound microbial inoculant, the contents of soluble sugar, theanine, water extract, tea polyphenols, and free amino acids in fresh tea leaves of tea trees have all changed. After comprehensive evaluation, it is confirmed that applying the compound microbial inoculant can comprehensively improve the quality of fresh tea leaves of tea trees, thereby improving the quality of selenium-enriched tea.

[0020] (2) The present invention further explores the preparation method of the compound microbial inoculant, and discloses a method for preparing the compound microbial inoculant by co-culturing. By optimizing the volume ratio of each strain in the co-cultured strains and the inoculation amount of the co-cultured strains, the selenium content in the bacterial cells is increased and the selenium conversion rate is improved; by optimizing the components of the co-cultured medium, the selenium content in the bacterial cells is further increased. Description of the Drawings

[0021] Figure 1 It is a graph showing the measurement results of the total selenium content in bacterial cells after GX6-3 and GX7-1 are mixed in different proportions and inoculated with different amounts.

[0022] Figure 2 It is the measurement result of the total selenium content in fresh tea leaves of tea trees after applying the compound microbial inoculant; A is the measurement result of the total selenium content in young leaves and B is the measurement result of the total selenium content in old leaves.

[0023] Figure 3 It is the measurement result of the theanine content in fresh tea leaves of tea trees after applying the compound microbial inoculant.

[0024] Figure 4 The determination results of the content of tea polyphenols in fresh tea leaves after applying the compound microbial inoculant.

[0025] Figure 5 The determination results of the total amount of catechins in fresh tea leaves after applying the compound microbial inoculant.

[0026] Figure 6 The determination results of the content of soluble sugar in fresh tea leaves after applying the compound microbial inoculant.

[0027] Figure 7 The determination results of the content of water extracts in fresh tea leaves after applying the compound microbial inoculant.

[0028] Figure 8 The determination results of the content of free amino acids in fresh tea leaves after applying the compound microbial inoculant.

[0029] Figure 9 The phenolic ammonia ratio in fresh tea leaves after applying the compound microbial inoculant.

[0030] Figure 10 The test results of the influence of different culture medium components on the selenium enrichment rate of the bacteria; A is the test result of the influence of adding different carbon sources on the selenium enrichment rate of the bacteria, B is the test result of the influence of adding different inorganic salts on the selenium enrichment rate of the bacteria, and C is the test result of the influence of adding different nitrogen sources on the selenium enrichment rate of the bacteria. Specific embodiments

[0031] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0032] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings, but the specific embodiments cited are not intended to limit the present invention.

[0033] In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials can all be purchased on the market unless otherwise specified.

[0034] The present invention uses two strains of endophytic bacteria, Bacillus cereus GX6-3 (abbreviated as GX6-3) and Lysinibacillus fusiformis GX7-1 (abbreviated as GX7-1), screened and obtained in this laboratory, to prepare a compound microbial inoculant by compounding. Among them, Bacillus cereus GX6-3 was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on January 6, 2025, with the deposit number CGMCC No. 33304; Lysinibacillus fusiformis GX7-1 was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on January 6, 2025, with the deposit number CGMCC No. 33305.

[0035] Example 1

[0036] This example is for optimizing the co-culture conditions of the endophytic bacteria Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1.

[0037] Take out the preserved strains of the endophytic bacteria Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1, streak and activate them on an LB solid medium, and incubate them in an inverted position at 28°C for 24 hours to obtain the activated Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1. Inoculate the activated Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1 into an LB liquid medium and culture them at 28°C and 180 rpm for 24 hours to prepare the primary seed liquid of Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1.

[0038] Prepare co-culture seed liquids with three ratios by mixing the primary seed liquids of Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1 at a volume ratio of 1:1, 1:2, and 2:1.

[0039] The above co-cultured seed liquid was inoculated into LB liquid medium containing 5 mM sodium selenite at inoculation amounts of 4%, 6%, and 8% by volume percentage, cultured at 28 °C and 180 rpm for 24 h, the fermentation broth was collected, the fermentation broth was placed in a centrifuge tube, centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, the bacterial cell precipitate at the bottom of the centrifuge tube was resuspended with sterile water, the resuspended bacterial cells were freeze-dried conventionally in a freeze dryer for 3 days, the dry weight of the bacterial cells was weighed, and the total selenium content was determined by the first method of GB5009.93-2017, hydride generation atomic fluorescence spectrometry, microwave digestion instrument method, and the total selenium amount was calculated. The calculation formula is as follows:

[0040] Total selenium amount = dry weight of bacterial cells * total selenium content

[0041] The measurement results are as Figure 1 shown. When the co-cultured seeds were inoculated at an inoculation amount of 8% by volume percentage, the total selenium amount of the bacterial cells was the highest, and the highest total selenium amount was close to 40 mg, which was higher than the total selenium amount of the bacterial cells cultured under the inoculation amounts of 4% and 6%. When the inoculation amount of the co-cultured seeds was 8% by volume percentage, and the volume ratio of the first-stage seed liquid of Bacillus cereus GX6-3 to Lysinibacillus fusiformis GX7-1 was 2:1, the total selenium amount in the bacterial cells was the highest. It is shown that the co-cultured seed liquid with a volume ratio of 2:1 of the first-stage seed liquid of Bacillus cereus GX6-3 to Lysinibacillus fusiformis GX7-1 inoculated at an inoculation amount of 8% by volume percentage is the optimal co-culture condition.

[0042] Example 2

[0043] This example is for the preparation of a composite microbial inoculant.

[0044] Preparation of selenium-rich fermentation broth: The first-stage seed liquid of Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1 prepared in Example 1 was mixed at a volume ratio of 2:1 to obtain the co-cultured seed liquid. The co-cultured seed liquid was inoculated into LB liquid medium containing 5 mM sodium selenite at an inoculation amount of 8% by volume percentage, cultured at 28 °C and 180 rpm for 24 h, and the fermentation broth was collected to obtain the selenium-rich fermentation broth.

[0045] Preparation of the liquid preparation of the composite microbial inoculant: The selenium-rich fermentation broth prepared above was diluted with sterile water at a certain ratio until the Se content was 100 mg / g, and the diluted selenium-rich fermentation broth was aseptically filled to obtain the liquid preparation of the composite microbial inoculant.

[0046] Preparation of compound microbial inoculant powder: The selenium-rich fermentation broth obtained above was centrifuged, and the cell precipitate was retained. The cell precipitate was resuspended with 5 wt% of cryoprotectant, and then freeze-dried using a freeze dryer. The dry powder was collected and diluted with zeolite powder to a Se content of 100 mg / g, thus obtaining the compound microbial inoculant powder preparation.

[0047] Example 3

[0048] This example is a field effect verification test of the compound microbial inoculant.

[0049] The compound microbial inoculant powder prepared in Example 2 was diluted with sterile water to dilutions of 20 mg / L, 40 mg / L, and 80 mg / L based on the Se content in the compound microbial inoculant powder, serving as the compound inoculant test groups (GX7-1, GX6-3). The above dilutions were sprayed on the leaves of healthy tea seedlings. Meanwhile, the group sprayed with sterile water was used as the blank control group (CK), and the group sprayed with sodium selenite at concentrations of 20 mg / L, 40 mg / L, and 80 mg / L based on the Se content was used as the Na2SO3 control group (Na2SO3). Each treatment was sprayed three times at intervals of 5 days. Five days after the last spraying, young and old leaves of the tea seedlings were taken to measure their total selenium content, theanine content, tea polyphenol content, total catechin content, water extract, soluble sugar content, and total free amino acid content.

[0050] (1) Determination of total selenium content

[0051] The measurement results are as Figure 2 shown, where Figure 2 A in it is the measurement result of young leaves, Figure 2 B in it is the measurement result of old leaves. The results show that when applying the compound microbial inoculant powder, when the final concentration is 40 mg / L (based on the Se content, the same below) and 80 mg / L, the total selenium content in young and old leaves is significantly increased compared with the CK group and the Na2SO3 group. Among them, when the final concentration of the compound microbial inoculant powder sprayed on the leaves is 40 mg / L, the total selenium content in young and old leaves is 0.49 mg / kg and 1.48 mg / kg respectively; when the final concentration of the compound microbial inoculant powder sprayed on the leaves is 80 mg / L, the total selenium content in young and old leaves is 0.88 mg / kg and 1.99 mg / kg, both of which have reached the selenium-rich tea standard. The results show that this compound microbial inoculant has a selenium-rich effect.

[0052] (2) Theanine content

[0053] The theanine measurement results are as Figure 3As shown, when applying the compound microbial inoculant powder at final concentrations of 20 mg / L (calculated by Se content, the same below), 40 mg / L, and 80 mg / L, the theanine content in fresh tea leaves was significantly increased compared with the CK group and the Na2SO3 group. When the spraying concentration on leaves was 20 mg / L, the theanine content was 1481.63 mg / kg; when the spraying concentration on leaves was 40 mg / L, the theanine content was 1681.06 mg / kg; when the spraying concentration on leaves was 80 mg / L, the theanine content was the highest, at 2144.82 mg / kg. With the increase of the final concentration of the compound microbial inoculant powder, the theanine content in fresh tea leaves increased. It indicates that spraying the compound microbial inoculant can increase the theanine content in fresh tea leaves, thereby improving the flavor of tea and increasing its health care value.

[0054] (3) Total polyphenol content

[0055] The test results of the total polyphenol content are as Figure 4 shown. When applying the compound microbial inoculant powder at final concentrations of 20 mg / L (calculated by Se content, the same below), 40 mg / L, and 80 mg / L, the total polyphenol content in fresh tea leaves was significantly decreased compared with the CK group and the Na2SO3 group. Among them, when applying the compound microbial inoculant powder at a final concentration of 20 mg / L, the total polyphenol content in fresh tea leaves was 19.07%, a decrease of 3.21% compared with the CK group and a decrease of 2.63% compared with the Na2SO3 group; when applying the compound microbial inoculant powder at a final concentration of 40 mg / L, the total polyphenol content in fresh tea leaves was 18.50%, a decrease of 3.79% compared with the CK group and a decrease of 2.89% compared with the Na2SO3 group; when applying the compound microbial inoculant powder at a final concentration of 80 mg / L, the total polyphenol content in fresh tea leaves was the lowest, at 17.97%, a decrease of 4.32% compared with the CK group and a decrease of 2.42% compared with the Na2SO3 group. It indicates that spraying the compound microbial inoculant can reduce the total polyphenol content in fresh tea leaves, thereby reducing the bitterness and astringency of tea.

[0056] (4) Total catechins

[0057] The test results of catechins are as Figure 5As shown, when applying the compound microbial inoculant powder at final concentrations of 20 mg / L (calculated by Se content, the same below), 40 mg / L, and 80 mg / L, the catechin content in fresh tea leaves decreased significantly compared with the CK group and the Na2SO3 group. When the spraying concentration on the leaves was 20 mg / L, the catechin was 144.26 mg; when the spraying concentration on the leaves was 40 mg / L, the total catechin content was 133.33 mg; when the spraying concentration on the leaves was 80 mg / L, the total catechin content was the lowest, at 126.26 mg. This indicates that with the increase in the final concentration of the compound microbial inoculant powder, the catechin content in fresh tea leaves decreases, thereby reducing the bitterness of the tea leaves.

[0058] (5) Soluble sugar content

[0059] The test results of the soluble sugar content are as Figure 6 shown. When applying the compound microbial inoculant powder, different final concentrations have different effects on the soluble sugar content in fresh tea leaves. When the final concentration of the compound microbial inoculant powder is 20 mg / L (calculated by Se content, the same below) and 40 mg / L, the change in the soluble sugar content is not significant. When the final concentration of the compound microbial inoculant powder is 80 mg / L, the soluble sugar content in fresh tea leaves increases significantly. Compared with the CK group, the soluble sugar content in fresh tea leaves increases by 1.33%, and compared with the Na2SO3 group, the soluble sugar in fresh tea leaves increases by 0.69%. The increase in the soluble sugar content is beneficial to alleviating the bitter taste of the tea soup and improving the taste of the tea soup. This indicates that when the final concentration of the compound microbial inoculant powder is 80 mg / L, it can effectively improve the taste of the tea leaves.

[0060] (6) Water extract content

[0061] The test results of the water extract content in fresh tea leaves are as Figure 7 shown. When applying the compound microbial inoculant powder, the water extract content in fresh tea leaves increases compared with the CK group and the Na2SO3 group. Especially when the final concentration of the compound microbial inoculant powder is 80 mg / L (calculated by Se content), the water extract in fresh tea leaves is the highest, approaching 50%. This indicates that applying the compound microbial inoculant can increase the water extract content in fresh tea leaves and enhance the concentration of the tea soup taste.

[0062] (7) Free amino acid content

[0063] The test results of the free amino acid content in fresh tea leaves are as Figure 8As shown in the figure, when the compound microbial inoculant powder was applied at a final concentration of 20 mg / L (calculated based on the Se content, the same below), the free amino acid content in fresh tea leaves was 2.70%, which was significantly different from that of the CK group, and the free amino acid content in fresh tea leaves increased by 0.33%; when the compound microbial inoculant powder was applied at a final concentration of 40 mg / L, the free amino acid content in fresh tea leaves was 3.17%, which was significantly different from that of CK, and the free amino acid content in fresh tea leaves increased by 0.71%. Compared with the Na2SO3 group, the free amino acid content in fresh tea leaves increased by 0.52%; when the compound microbial inoculant powder was applied at a final concentration of 80 mg / L, the free amino acid content in fresh tea leaves was the highest, at 3.58%, which was significantly different from that of the CK group, and the leached free amino acid content in fresh tea leaves increased by 1.12%. Compared with Na2SO3, the free amino acid content in fresh tea leaves increased by 0.70%. It shows that applying the compound microbial inoculant can effectively increase the free amino acid content in fresh tea leaves, thereby enhancing the taste of tea and making the tea sweet, mellow and fresh.

[0064] (8) Phenol-ammonia ratio

[0065] The determination results of the phenol-ammonia ratio in fresh tea leaves are as Figure 9 shown. After applying the compound microbial inoculant powder, the phenol-ammonia ratio in fresh tea leaves decreased significantly compared with the CK group and the Na2SO3 group. It shows that applying the compound microbial inoculant can reduce the phenol-ammonia ratio in fresh tea leaves, thereby improving the taste of the tea soup, making the tea soup more mellow, fresh and with a better flavor.

[0066] The test results of each index in this example need to be comprehensively evaluated to assess its impact on the taste of the tea soup. It is one-sided to only look at the evaluation results of the content of a certain taste substance, and there are effects such as superposition, modulation, synergy or inhibition among various taste components. Through the comparison of fresh tea leaves of tea trees applied with different concentrations of compound microbial inoculants in terms of total selenium content, soluble sugar, theanine, water extract, tea polyphenols, free amino acids, etc., it is found that applying the compound microbial inoculant can comprehensively improve the quality of fresh tea leaves. In addition, applying the compound microbial inoculant significantly increases the selenium content in fresh tea leaves. When the final concentration of the compound microbial inoculant powder is in the range of 40 mg / L - 80 mg / L (calculated based on the Se content), the young leaves and old leaves of tea trees can meet the standards of selenium-enriched tea.

[0067] Example 4

[0068] This example is about the optimization of the fermentation medium of the compound microbial inoculant

[0069] The initial fermentation medium was LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and made up to 1 L with distilled water.

[0070] Selenium enrichment rate = total selenium content absorbed by bacteria (μg / mL) / selenium addition amount × 100%

[0071] (1) Optimize the carbon source in the fermentation medium

[0072] Add six carbon sources, namely glucose, sucrose, soluble starch, maltose, lactose, and mannitol, to the LB liquid medium containing 5 mM sodium selenite at a final concentration of 10 g / L. Inoculate GX6-3 and GX7-1, and shake culture at 28 °C and 180 rmp for 24 h. Measure the total selenium content absorbed by the bacteria, calculate the selenium enrichment rates of GX6-3 and GX7-1, and use the LB liquid medium containing 5 mM sodium selenite as the control (CK). Compare the effects of different carbon source media on the selenium enrichment rates of the strains.

[0073] The test results are as Figure 10 shown in A of []. When adding glucose as the carbon source among the six added carbon sources, the selenium enrichment rate of the strain is the highest, at 42.04%. Therefore, glucose is determined as the optimal added carbon source.

[0074] (2) Optimize the inorganic salts in the fermentation medium

[0075] Use five inorganic salts, namely KCl, MnSO4, CaCl2, MgSO4, and KH2PO4, to replace NaCl in the LB medium respectively, and add them to the LB liquid medium containing 5 mM sodium selenite. Inoculate GX6-3 and GX7-1, and shake culture at 28 °C and 180 rmp for 24 h. Measure the total selenium content absorbed by the bacteria, calculate the selenium enrichment rate of the bacteria. At the same time, use the LB liquid medium containing 5 mM sodium selenite as the control (CK). Compare the effects of adding different inorganic salts on the selenium enrichment rates of the bacteria.

[0076] The test results are as Figure 10 shown in B of []. When adding CaCl2 to replace NaCl in the LB medium among these five inorganic salts, the selenium enrichment rate of the strain is the highest, at 28.29%. Therefore, CaCl2 is selected as the optimal inorganic salt.

[0077] (3) Optimize the nitrogen source in the fermentation medium

[0078] Use five nitrogen sources to replace the nitrogen source in the LB medium respectively, and add them to the sterilized LB liquid medium containing 5 mM sodium selenite. Inoculate GX6-3 and GX7-1, and shake culture at 28 °C and 180 rmp for 24 h. Measure the total selenium content absorbed by the bacteria, calculate the selenium enrichment rate, and use the LB liquid medium containing 5 mM sodium selenite as the control (CK). Compare the effects of adding different nitrogen sources on the selenium enrichment rates of the bacteria.

[0079] The five nitrogen sources are as follows: a nitrogen source with yeast extract paste:tryptone at a mass ratio of 1:1, a nitrogen source with yeast extract powder:tryptone at a mass ratio of 1:1, a nitrogen source with beef extract:tryptone at a mass ratio of 1:1, a nitrogen source with beef extract:yeast extract at a mass ratio of 1:1, and a nitrogen source of tryptone by mass.

[0080] The test results are as Figure 10 shown in C of []. Among the five added nitrogen sources, for the nitrogen source with tryptone:yeast extract powder at a mass ratio of 1:1, the selenium absorption amount by the bacterial cells is the highest and the selenium enrichment rate reaches 35.61%. The nitrogen source with tryptone:yeast extract powder at a mass ratio of 1:1 is the optimal nitrogen source.

[0081] Based on the test results of this example, the optimal fermentation medium for the GX6-3 and GX7-1 composite microbial agents is screened as: 1% glucose, 0.75% tryptone, 0.75% yeast extract powder, 1% CaCl2, and made up to 1 L with distilled water.

[0082] As described above, the basic principle, main features and advantages of the present invention are preferably described. The above examples and descriptions are only for describing the preferred embodiments of the present invention. The present invention is not limited by the above examples. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A composite microbial inoculum, characterized in that, The composite microbial inoculum is obtained by co-culturing Bacillus cereus GX6-3 and Lysinibacillus fusiformis GX7-1; The preservation number of Bacillus cereus GX6-3 is CGMCC No. 33304, and the preservation number of Lysinibacillus fusiformis GX7-1 is CGMCC No. 33305.

2. The compound microbial inoculum according to claim 1, characterized in that In the co-culture, the volume ratio of the seed liquid of Bacillus cereus GX6-3 to the seed liquid of Lysinibacillus fusiformis GX7-1 is 2:

1.

3. The composite microbial inoculum according to claim 1, wherein In the co-culture, the culture medium used includes inorganic salts, carbon sources and nitrogen sources.

4. The composite microbial inoculum according to claim 3, in the co-culture, the inorganic salt of the culture medium used is one of KCl, MnSO4, CaCl2, MgSO4, KH2PO4, NaCl.

5. The composite microbial inoculum according to claim 3, in the co-culture, the carbon source of the culture medium used is one of glucose, sucrose, soluble starch, maltose, lactose, mannitol.

6. The composite microbial inoculum according to claim 3, in the co-culture, the nitrogen source of the culture medium used is composed of tryptone and yeast extract, or tryptone and yeast powder, or tryptone and beef extract, or beef extract and yeast extract.

7. The composite microbial inoculum according to claim 3, in the co-culture, the culture medium used is composed of 1% glucose, 0.75% tryptone, 0.75% yeast powder, 1% CaCl2, and the balance is distilled water by mass percentage.

8. A method for increasing the selenium content in plants, characterized in that, Including: Applying the composite microbial inoculum according to any one of claims 1 to 7.

9. The method according to claim 8, wherein The plant is a tea tree.

10. A method for improving the quality of fresh tea leaves, characterized in that, Including: Applying the composite microbial inoculum according to any one of claims 1 to 7 to the tea tree to improve the quality of fresh tea leaves of the tea tree; The quality of the fresh tea leaves of the tea tree includes at least one of selenium content, theanine content, tea polyphenol content, total catechin content, soluble sugar content, water extract content, free amino acid content, and phenol-ammonia ratio.

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