Complex microbial inoculant for promoting growth of wormwood, growth solution and preparation method of growth solution

Abstract

The invention discloses a complex microbial inoculant for promoting the growth of wormwood, a growth solution and a preparation method of the growth solution, and relates to the technical field of microorganism application. According to the complex microbial inoculant for promoting the growth of the wormwood, the complex microbial inoculant for promoting the growth of the wormwood comprises the complex bacteria and the cyperus esculentus cakes, the complex bacteria comprise bacillus subtilis, streptococcus thermophilus and lactobacillus delbrueckii, the cyperus esculentus cakes in the complex microbial inoculant can provide more nutrition support for the wormwood, flora in soil is effectively improved, and the growth of the wormwood is promoted. The growth of effective microbial communities is promoted, pathogenic bacteria are repelled, and lower leaf litter is reduced, so that the yield of wormwood and volatile oil thereof is increased.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a composite bacterial agent for promoting the growth of wormwood, a growth liquid and a preparation method thereof. Background Art

[0002] Artemisia argyi Levl.et Van is a perennial herb or semi-shrub plant of the genus Artemisia in the Asteraceae family. It is rich in active substances such as volatile oils and flavonoids, and has the effects of warming the meridians, removing dampness, dispelling cold, stopping bleeding, inhibiting bacteria, resisting insects and resisting oxidation. Artemisia argyi has important application value in the fields of food, health care, cosmetics and animal husbandry, and the domestic demand for wormwood is huge.

[0003] However, the standardized planting procedures for mugwort are still imperfect. Under artificial cultivation conditions, it can generally be harvested about 2 months after the emergence of seedlings, that is, around the Dragon Boat Festival, mid-to-late July, and around National Day, with about 3 harvests per year. In the southern climate area, one more harvest can be made before winter. In artificial cultivation, the lower leaves of the plant begin to dry up and fall off seriously around January. By February, there are only a few leaves about 50 cm above the ground, which seriously reduces the leaf harvest. In order to avoid the lower leaves from withering, in actual cultivation and production, they are mostly harvested at a height of 1.5m or even lower. At this time, the yield and the content of effective ingredients such as volatile oil have not yet reached the optimal level, which has become a bottleneck problem in mugwort cultivation. Summary of the invention

[0004] The main purpose of the present invention is to provide a composite bacterial agent for promoting the growth of mugwort, a growth liquid and a preparation method thereof, aiming to propose a composite bacterial agent for promoting the growth of mugwort, which can provide more nutritional support to mugwort, effectively improve the bacterial flora in the soil, promote the growth of beneficial bacteria, drive away pathogens, reduce the withering of lower leaves, and thus increase the yield of mugwort and its volatile oil.

[0005] To achieve the above-mentioned purpose, the present invention provides a composite bacterial agent for promoting the growth of wormwood, wherein the composite bacterial agent for promoting the growth of wormwood comprises composite bacteria and cyperus oleifera cake, wherein the composite bacteria comprises Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii.

[0006] In some embodiments, in the composite bacteria, the mass ratio of the Bacillus subtilis, the Streptococcus thermophilus and the Lactobacillus delbrueckii is (25-30):(3-5):1.

[0007] The present invention provides a growth liquid, wherein the growth liquid comprises the composite bacterial agent for promoting the growth of wormwood as described above.

[0008] In some embodiments, a method for preparing a growth liquid is provided, the preparation steps comprising:

[0009] S10, crushing and sieving the cyperus juncea cake and mixing it with water as a culture medium, and sterilizing the culture medium for standby use;

[0010] S20, preparing Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii into an inoculation solution, inoculating and fermenting on the culture medium, post-ripening and extracting to obtain a growth solution.

[0011] In some embodiments, in step S10, the mesh size of the sieve is 10 meshes; and / or,

[0012] The mass ratio of the jatropha bean cake to the water is 1:2; and / or,

[0013] The pH value of the culture medium is 5 to 7; and / or,

[0014] The sterilization temperature is 110°C to 130°C; and / or,

[0015] The sterilization time is 15 to 25 minutes.

[0016] In some embodiments, in step S20,

[0017] The fermentation temperature is 35-38°C; and / or,

[0018] The fermentation time is 22 to 26 hours; and / or,

[0019] The freeze-drying temperature is 3 to 5°C; and / or,

[0020] The ripening time is 22 to 26 hours.

[0021] In the technical solution of the present invention, by adding composite bacteria and cyperus rotundus cake, wherein the composite bacteria includes a combination of Bacillus subtilis, Streptococcus thermophilus, and Lactobacillus delbrueckii in the cyperus rotundus cake, the bacterial flora in the soil is effectively improved, the growth of beneficial bacterial flora is promoted, pathogens are driven away, and the withering of the lower leaves is reduced, thereby providing the yield of wormwood and its volatile oil, increasing the growth of wormwood leaves, reducing the shedding of the lower leaves of wormwood, making the growth better, the volatile oil content higher, and the yield and quality better. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a comparison picture of the size of the leaves in the six weeks in Examples 1, 2, 3 and Comparative Example 1 provided by the present invention;

[0023] Figure 2 It is a comparison picture of the leaf growth in Examples 1, 2, 3 and Comparative Example 1 at 6 weeks;

[0024] Figure 3 This is a comparison picture of the planting areas in week 6 between Example 3 and Comparative Example 1.

[0025] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0026] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0027] Artemisia argyi Levl.et Van is a perennial herb or semi-shrub plant of the genus Artemisia in the Asteraceae family. It is rich in active substances such as volatile oils and flavonoids, and has the effects of warming the meridians, removing dampness, dispelling cold, stopping bleeding, inhibiting bacteria, resisting insects and resisting oxidation. Artemisia argyi has important application value in the fields of food, health care, cosmetics and animal husbandry, and the domestic demand for wormwood is huge.

[0028] However, the standardized planting procedures for mugwort are still imperfect. Under artificial cultivation conditions, it can generally be harvested about 2 months after the emergence of seedlings, that is, around the Dragon Boat Festival, mid-to-late July, and around National Day, with about 3 harvests per year. In the southern climate area, one more harvest can be made before winter. In artificial cultivation, the lower leaves of the plant begin to dry up and fall off seriously around January. By February, there are only a few leaves about 50 cm above the ground, which seriously reduces the leaf harvest. In order to avoid the lower leaves from withering, in actual cultivation and production, they are mostly harvested at a height of 1.5m or even lower. At this time, the yield and the content of effective ingredients such as volatile oil have not yet reached the optimal level, which has become a bottleneck problem in mugwort cultivation.

[0029] The present invention provides a composite bacterial agent for promoting the growth of wormwood. The present invention provides a composite bacterial agent for promoting the growth of wormwood. The composite bacterial agent for promoting the growth of wormwood comprises composite bacteria and cyperus oleifera cake. The composite bacteria comprises Bacillus subtilis, Streptococcus thermophilus, and Lactobacillus delbrueckii.

[0030] It should be noted that the deposit numbers of the three strains in the present invention are:

[0031] Bacillus subtilis Deposit Number: CICC10089;

[0032] Streptococcus thermophilus deposit number: CICC6222;

[0033] The Lactobacillus delbrueckii collection number is CICC6103. The above three strains are all from the China Industrial Microbiological Culture Collection Center.

[0034] The present invention adds a composite bacterial agent and cyperus rotundus cake, wherein the substance contained in the cyperus rotundus cake itself is used as the raw material of the culture medium. The product obtained by mixing and fermenting the composite bacterial agent and the cyperus rotundus cake can increase the growth of the wormwood leaf surface, reduce the shedding of the lower leaves of the wormwood, make the growth better, the volatile oil content higher, and the yield and quality better.

[0035] In any embodiment, the present invention does not limit the ratio of each component in the composite bacterial agent, and can be added according to actual needs. In the composite bacteria, the mass ratio of the Bacillus subtilis, the thermophilic Streptococcus and the Lactobacillus delbrueckii is (25-30): (3-5): 1, which can be 25:3:1, 26:4:1 or 30:5:1. Within the above ratio range, it is beneficial to give full play to the advantages of each bacteria, and the fermentation can be fully carried out, so that the fermented product is sprayed on the wormwood, which reduces the withering of the lower leaves, thereby providing the yield of wormwood and its volatile oil.

[0036] More preferably, the mass ratio of the Bacillus subtilis, the Streptococcus thermophilus and the Lactobacillus delbrueckii is 28:4:1. Under the above ratio, there is no waste of strains and the advantages of each strain can be fully utilized.

[0037] The present invention also provides a growth liquid, which includes the composite bacterial agent for promoting the growth of wormwood as described above. First, the composite bacteria are mixed with cyperus rotundus cake, and the nutrients of the cyperus rotundus cake are used as culture to prepare for the composite bacteria to ferment the growth liquid. In actual use, since the fermented and ripened state is generally semi-solid, water can be added as needed to extract the components, and the growth liquid is prepared after all the components are in the solution, which is convenient for spraying on crops.

[0038] The present invention further provides a method for preparing the growth liquid as described above, comprising the following steps:

[0039] S10, crushing the cyperus juncea cake, sieving it and mixing it with water to obtain a culture medium, and sterilizing the culture medium for later use.

[0040] Furthermore, in step S10, the mesh number of the sieve is 10 meshes, so that the cyperus juncea cake powder can be obtained for subsequent use.

[0041] Furthermore, the mass ratio of the cyperus oleifera cake to the water is 1:2, and the pH value of the culture medium is 5-7. Within this range, the culture medium is more suitable for the growth of the composite bacterial agent.

[0042] Furthermore, the sterilization temperature is 110° C. to 130° C.; and / or the sterilization time is 15 to 25 minutes, and the sterilization is sufficient.

[0043] The specific operation of step S10 is as follows: crush the cyperus oleifera bean cake until it can pass through a 10-mesh sieve to obtain bean powder, weigh 20 g of the bean powder and put it into a 250 ml conical flask, add ultrapure water with a solid-liquid ratio of 1:2 to prepare a culture medium, adjust the pH of the culture medium to 6, seal the bottle mouth with a breathable membrane, and place the conical flask in 121° C. high-pressure steam sterilization for 20 minutes to obtain the standby culture medium.

[0044] In any embodiment, S20, Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii are prepared into an inoculation solution, and the inoculation is carried out on the culture medium for fermentation, post-ripening and extraction to obtain a growth solution.

[0045] Furthermore, in step S20, the fermentation temperature is 35-38° C.; and / or the fermentation time is 22-26 hours. Within this fermentation range, the fermentation effect of the strain is better.

[0046] The specific operation of step S20 is as follows: weigh 0.4g of bacterial powder (Bacillus subtilis, Streptococcus thermophilus, Lactobacillus delbrueckii, the total relative abundance of the three bacteria is 100%), add 3ml of sterile water and vortex evenly to prepare an inoculum solution, after the culture medium of step S10 is cooled to room temperature, add the inoculum solution, mix the bacterial solution and the culture medium evenly with a sterile spoon, place it in a 37°C biochemical incubator for 24h, and then transfer it to a 4°C refrigerator for post-ripening for 24h, that is, the fermentation is completed, and the growth liquid is extracted with water after post-ripening.

[0047] The following is the optimal fermentation conditions screened out by investigating the material-liquid ratio, culture time, inoculation amount, and pH factors during the preparation of the growth solution, combined with orthogonal experiments:

[0048] The fermented product obtained after fermentation in step S20 is mixed with 0.9% sodium chloride solution at a solid-liquid ratio of 1:12, placed at 37° C. and shaken at 200 r / min for 1 hour, and the obtained postbiotic solution is freeze-dried to obtain freeze-dried powder, and then the component content is determined.

[0049] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0050] Experimental Materials

[0051] Bacillus subtilis, accession number: CICC10089;

[0052] The deposit number of Streptococcus thermophilus is CICC6222;

[0053] The deposit number of Lactobacillus delbrueckii subspecies bulgaricus is CICC6103. The above three strains are from the China Industrial Microbiological Culture Collection Management Center;

[0054] Cyperus oleifera cake was purchased from Shihe Agricultural Technology Co., Ltd. in Dingzhou City, Hebei Province, with the item number 2022080003.

[0055] Example 1 Preparation of Growth Liquid

[0056] Step S10, grinding the cyperus oleifera bean cake to obtain bean powder that can pass through a 10-mesh sieve, weighing 20 g of the bean powder and placing it in a 250 ml conical flask, adding ultrapure water with a solid-liquid ratio of 1:2 to prepare a culture medium, adjusting the pH of the culture medium to 6 by dissolving it in dilute hydrochloric acid, sealing the bottle mouth with a breathable membrane, and placing the conical flask in a 121° C. high-pressure steam sterilization for 20 min to obtain a standby culture medium;

[0057] Step S20, weigh 0.4g of the composite bacterial agent, including 0.34g of Bacillus subtilis, 0.05g of Streptococcus thermophilus, and 0.01g of Lactobacillus delbrueckii, and the total relative abundance of the three bacteria is 100%, add 3ml of sterile water and vortex evenly to prepare an inoculum solution, after the culture medium of step S10 is cooled to room temperature, add the inoculum solution, mix the bacterial solution and the culture medium evenly with a sterile medicine spoon, place it in a 37°C biochemical incubator for 24h, and then transfer it to a 4°C refrigerator for post-ripening for 24h, that is, the fermentation is completed, the fermentation product is called, 0.9% sodium chloride solution (solid-liquid ratio 1:12) is added, and it is placed at 37°C for oscillation extraction for 1h, and the particles are filtered to remove the particles to obtain the growth liquid.

[0058] Example 2

[0059] Except that the mass ratio of Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii added in the composite bacterial agent is 25:3:1, other components and preparation steps are the same as those in Example 1.

[0060] Example 3

[0061] Except that the mass ratio of Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii added in the composite bacterial agent is 30:5:1, other components and preparation steps are the same as those in Example 1.

[0062] Comparative Example 1

[0063] Blank control (just spray water).

[0064] Performance Testing

[0065] 1. Determination of the components of the growth solution

[0066] Take the fermented product of Example 1 and continue to add 0.9% sodium chloride solution at a solid-liquid ratio of 1:12 to obtain a mixture, place it at 37°C, shake and extract it at 200r / min for 1h, and obtain a cyperus juncea postbiotic solution. The cyperus juncea postbiotic solution is freeze-dried to obtain a freeze-dried powder, and then the component content of the freeze-dried powder is determined. The identification result is: every 100g of cyperus juncea postbiotic freeze-dried powder contains 28.66±5.74g protein, 1.91±0.12g fat, 12.62±1.57g total sugar, 16.01±0.06g water,

[0067] (2) The present invention uses proteomics analysis to compare blank matrix with fermentation samples, and uses labelfree detection technology to detect a total of 389 proteins. Using logFC>2 or logFC<-0.5, and FDR<0.05 as screening conditions, a total of 266 differentially expressed proteins were screened out, of which 260 proteins were significantly upregulated and 6 proteins were significantly downregulated. Table 4 shows some of the significantly upregulated differentially expressed protein information.

[0068] Table 4

[0069]

[0070]

[0071]

[0072]

[0073] (3) The present invention uses metabolomics technology to analyze and finds that 23 representative small molecule compounds can be screened out from the up-regulated metabolites of the fermented cyperus oleiferus cake postbiotics, including polyunsaturated fatty acids, steroids, flavonoids and flavonoids, alkaloids, terpenes, etc. Specifically: a total of 11,085 differential metabolites were detected in the postbiotic samples and blank samples, of which 6,413 were up-regulated and 4,672 were down-regulated. Table 5 shows some of the up-regulated metabolites in the cyperus oleiferus cake postbiotics. It can be seen from Table 5 that the up-regulated metabolites involve fatty acids, steroids, amino acids, flavonoids, lignans, alkaloids, terpenes, etc.

[0074] Table 5

[0075]

[0076]

[0077] 2. Mugwort growth performance test

[0078] The growth liquid prepared in Examples 1 to 3 and Comparative Example 1 was sprayed on the mugwort, and the leaves were sprayed with the growth liquid prepared in Examples 1 to 3 and Comparative Example 1 respectively when the seedlings emerged for 6 weeks, and each was sprayed continuously once a day. The leaf state and shedding condition were observed for 6 weeks. Figures 1 to 3 , and the volatile oil yield, eucalyptol content and borneol content thereof at the time of harvest were determined to obtain Table 1, wherein the volatile oil yield was determined by the volatile oil determination method according to the general rules of volatile oil in Part IV of the Chinese Pharmacopoeia 2020 edition, and the eucalyptol content and borneol content determination standard were determined by the content determination method under the item of Artemisia argyi in Part I of the Chinese Pharmacopoeia 2020 edition to determine the eucalyptol and borneol contents, and the determination results are shown in Table 1.

[0079] Table 1

[0080]

[0081] The results of the test are as follows Figures 1 to 3 As shown in Table 1, Figure 1 It can be seen that Figure 1 a, b, c and d correspond to Example 1, Example 2, Example 3 and Comparative Example 1, respectively. The leaf growth in 6 weeks is as follows: Figure 1 As shown: the leaf growth of Examples 1, 2, and 3 is larger than that of Comparative Example 1.

[0082] Figure 2 This is the growth of leaves at 6 weeks. Figure 2 a, b, c and d in the above correspond to Example 1, Example 2, Example 3 and Comparative Example 1, respectively. Figure 2 It can be seen that the lower leaves of Examples 1, 2, and 3 grow well, while the lower leaves of Comparative Example 1 fall off significantly. The growth of leaves in Examples 1, 2, and 3 is much improved compared with the falling condition in Comparative Example 1.

[0083] Figure 3 a is Example 3, b is Comparative Example 1, Figure 3 1 is a comparison diagram of the planting areas of Example 3 and Comparative Example 1, with Comparative Example 1 on the left and Example 3 on the right. Figure 3 It can be seen that the mugwort leaves in the entire area of ​​Example 3 grow more luxuriantly, the plants are taller, the leaves are larger, and the yield is higher than that of Comparative Example 1.

[0084] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A composite bacterial agent for promoting the growth of wormwood, characterized in that: The composite bacterial agent for promoting the growth of wormwood comprises composite bacteria and cyperus oleifera cake, wherein the composite bacteria comprises bacillus subtilis, thermophilic streptococcus, and lactobacillus delbrueckii.

2. The composite bacterial agent for promoting the growth of wormwood according to claim 1, characterized in that: In the composite bacteria, the mass ratio of the Bacillus subtilis, the Streptococcus thermophilus and the Lactobacillus delbrueckii is (25-30):(3-5):

1.

3. A growth liquid, characterized in that: The growth liquid includes the composite bacterial agent for promoting the growth of wormwood as described in claim 1 or 2.

4. A method for preparing a growth liquid as claimed in claim 3, characterized in that: The preparation steps include: S10, crushing and sieving the cyperus juncea cake and mixing it with water as a culture medium, and sterilizing the culture medium for standby use; S20, preparing Bacillus subtilis, Streptococcus thermophilus and Lactobacillus delbrueckii into an inoculation solution, inoculating and fermenting on the culture medium, post-ripening and extracting to obtain a growth solution.

5. The method for preparing the growth liquid according to claim 4, characterized in that: In step S10, The mesh size of the sieve is 10 mesh; and / or, The mass ratio of the jatropha bean cake to the water is 1:2; and / or, The pH value of the culture medium is 5 to 7; and / or, The sterilization temperature is 110°C to 130°C; and / or, The sterilization time is 15 to 25 minutes.

6. The method for preparing the growth liquid according to claim 3, characterized in that: In step S20, The fermentation temperature is 35-38°C; and / or, The fermentation time is 22 to 26 hours; and / or, The freeze-drying temperature is 3 to 5°C; and / or, The ripening time is 22 to 26 hours.

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