Preparation of tobacco stalk hydrolysis ferment and its application in crop salt resistance

Abstract

The application provides preparation of tobacco stalk hydrolysis fermentation and application thereof in crop salt resistance, and relates to the technical field of agriculture.The tobacco stalk hydrolysis fermentation of the application is obtained by microwave-assisted hydrolysis of tobacco stalk to obtain a hydrolysis product, then the hydrolysis product is subjected to fermentation treatment by using bacillus amyloliquefaciens Cas02 to obtain a fermentation crude extract, and after freeze-drying, ethyl acetate extraction is carried out, and the residual solid phase insoluble in ethyl acetate is the tobacco stalk hydrolysis fermentation. The hydrolysis fermentation can improve the salt tolerance of crops, and can effectively alleviate the inhibition of saline-alkali stress on the growth of crops.

Description

Technical Field

[0001] This application relates to the field of agricultural technology, and in particular to the preparation of a tobacco straw hydrolysate fermentation product and its application in crop salt tolerance. Background Technology

[0002] my country is a major tobacco-producing country, and the harvest of tobacco leaves generates a large amount of waste tobacco straw. Improper disposal of this waste tobacco straw can lead to environmental pollution. Therefore, developing more feasible and higher-value tobacco straw utilization strategies has always been a research hotspot in this field.

[0003] Like other crop straws, tobacco stalks contain a large amount of lignocellulose, making them a valuable raw material for the production of bio-based chemicals and biofuels. In recent years, researchers have developed various pretreatment technologies to disrupt the anti-degradation structure of tobacco stalks, such as hydrothermal pretreatment, steam explosion pretreatment, ionic liquid pretreatment, as well as enzymatic hydrolysis and biofuel fermentation processes. The tobacco stalk products obtained using these technologies are primarily used to promote crop growth and control plant diseases.

[0004] For example, patent CN118834106A discloses a method for producing tobacco-specific fertilizer containing nicotine and humic acid from tobacco straw. This involves impregnating the residue after leaching tobacco straw with hydrochloric acid with a metal catalyst followed by hydrothermal treatment. The resulting product can promote tobacco yield. Another example is patent CN107372635A, which discloses the application of fumigating root-knot nematodes with a mixture of Artemisia annua and tobacco straw. Patent CN114875092A discloses a method for obtaining macrolide antibiotics macrolide A and macrolide W with antifungal and antiviral activities through inexpensive straw fermentation (patent CN114875092A). This method utilizes microwave-assisted degradation, Bacillus amyloliquefaciens fermentation, macroporous adsorption resin column chromatography, and liquid chromatography to obtain macrolide antibiotics macrolide A and macrolide W. This provides technical support for the large-scale production of macrolide compounds, solves the problem of straw waste disposal, and increases the added value of crop cultivation. However, the current development and utilization of tobacco straw only focuses on the enrichment and utilization of certain specific components, while the remaining products are not well utilized, resulting in significant waste. Summary of the Invention

[0005] The purpose of this application is to address the shortcomings of existing technologies by providing a method for preparing a hydrolyzed ferment of tobacco straw and its application in crop salt tolerance. This application obtains a crude fermented extract through a microwave-assisted hydrolysis and bacterial fermentation process, followed by extraction to obtain a hydrolyzed ferment. This hydrolyzed ferment can be used to improve the salt tolerance of crops and enhance the utilization value of tobacco straw.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: According to one aspect of this application, a method for preparing a hydrolyzed ferment of tobacco straw includes the following steps: (1) Microwave-assisted hydrolysis of tobacco straw was performed to obtain crude extract; (2) The crude extract was fermented using Bacillus amyloliquefaciens Cas02 to obtain fermented crude extract; (3) Extract the crude fermentation extract to obtain a liquid phase and a solid phase. The solid phase is the tobacco straw hydrolysate fermentation product.

[0007] Currently, the application of tobacco straw mainly involves enriching one or several active ingredients from fermentation extracts, but the remaining products after enrichment are not well utilized, resulting in waste. This application utilizes the remaining solid phase after extraction of fermentation extracts, realizing the full utilization of by-products, broadening the application fields of tobacco straw, and increasing its value.

[0008] Furthermore, the microwave-assisted hydrolysis step is as follows: the tobacco stalks are crushed and sieved, mixed with the degradation reagent, microwave-treated, and the residual biomass is separated by centrifugation. The residual biomass is washed and dried to obtain the crude extract.

[0009] Optionally, in the microwave-assisted hydrolysis process of step (1), the degradation reagent is water or dilute sulfuric acid, the mass fraction of the dilute sulfuric acid is 0.2-0.3%, and the mass-volume ratio of tobacco straw to degradation reagent is 1g:(15-20)mL.

[0010] Optionally, in the microwave-assisted hydrolysis process of step (1), the microwave treatment conditions are: microwave power of 700-800W, temperature of 120-150℃, and time of 20-30min.

[0011] Microwave power: 700-800W, specifically 700W, 710W, 720W, 730W, 740W, 750W, 760W, 770W, 780W, 790W, 800W, or any range thereof. Temperature: 120-150℃, specifically 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, or any range thereof. Time: 20-30 minutes, specifically 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, or any range thereof.

[0012] Further, the fermentation process in step (2) includes: dispersing the crude extract in 180-200 times its weight of water to obtain a crude extract dispersion, inoculating the Bacillus amyloliquefaciens Cas02 bacterial solution into the crude extract dispersion, and culturing it at 28-30℃ and 180-200 rpm for 120 h with shaking. The OD600 value is measured every 24 h. After the culture is completed, the suspension is centrifuged, the supernatant is neutralized, and freeze-dried to obtain the fermented crude extract.

[0013] Optionally, in the fermentation process of step (2), the preparation method of the Bacillus amyloliquefaciens Cas02 bacterial solution is as follows: Bacillus amyloliquefaciens Cas02 is cultured in LB medium for 24 hours and diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution.

[0014] Optionally, in the fermentation treatment of step (2), the inoculum amount of Bacillus amyloliquefaciens Cas02 is 0.1-0.15 (v / v)%.

[0015] Optionally, in the fermentation process of step (2), the neutralization process is carried out using an alkaline solution, such as a sodium hydroxide solution.

[0016] Furthermore, in step (3), ethyl acetate is used as the extractant. The extraction process is as follows: the crude fermented extract is suspended in ethyl acetate, and after shaking and centrifugation, the residual solid phase that is insoluble in ethyl acetate is the tobacco straw hydrolysate fermented product.

[0017] Optionally, in the extraction process of step (3), the mass-to-volume ratio of the fermented crude extract to ethyl acetate is 1 g: (18-20) mL.

[0018] According to another aspect of this application, a tobacco straw hydrolysate fermented by the above-described preparation method is provided.

[0019] According to another aspect of this application, the application of the above-mentioned tobacco straw hydrolysate fermentation product in crop salt tolerance is provided. Furthermore, the application includes using straw hydrolysate fermentation products to improve the salt tolerance of crop seedlings.

[0020] Furthermore, the crops mentioned include peanuts, wheat, soybeans, etc.

[0021] Furthermore, the application includes: dispersing tobacco straw hydrolysate fermented product in water to prepare a nutrient solution for hydroponic cultivation of crops; wherein the concentration of tobacco straw hydrolysate fermented product in the nutrient solution is ≥2g / L.

[0022] Compared with the prior art, this application has the following beneficial effects: This application utilizes a microwave-assisted hydrolysis-bacterial fermentation process to obtain a crude fermentation extract. The remaining solid phase after extraction can be used to improve the salt tolerance of crops, effectively alleviate the inhibitory effect of salt-alkali stress on the growth of peanut seedlings, and fully utilize tobacco straw by-products, thereby increasing the added value of tobacco straw. Attached Figure Description

[0023] Figure 1 This is a diagram showing the growth status of peanut seedlings in this application. Detailed Implementation

[0024] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of this application, but do not limit this application in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0025] Unless otherwise specified, all chemical reagents used in the embodiments of this application were obtained through conventional commercial channels. The Bacillus amyloliquefaciens Cas02 strain used in this application is the same strain used in our prior patent application 202210428874.1, which was deposited on March 26, 2018, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.15514.

[0026] This application provides an application of straw hydrolysate fermentation product in crop salt tolerance, and the preparation method of the straw hydrolysate fermentation product includes the following steps: (1) Microwave-assisted hydrolysis: After crushing and sieving the tobacco stalks, mix them with water or dilute sulfuric acid at a ratio of 1g:(15-20)mL. Microwave the mixture for 20-30 minutes at a microwave power of 700-800W and a temperature of 120-150℃. Centrifuge to separate the residual biomass. After washing, dry the residual biomass to obtain the crude extract.

[0027] (2) Fermentation treatment: The crude extract was dispersed in 180-200 times its weight of water to obtain a crude extract dispersion; Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h and diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution; Bacillus amyloliquefaciens Cas02 bacterial solution was inoculated into the crude extract dispersion at an inoculation rate of 0.1-0.15 (v / v)% and cultured at 28-30℃ and 180-200 rpm for 120 h with shaking. The OD600 value was measured every 24 h. After the culture was completed, the suspension was centrifuged, the supernatant was neutralized, and freeze-dried to obtain the fermented crude extract.

[0028] (3) Ethyl acetate extraction: The crude fermented extract is suspended in ethyl acetate at a mass-volume ratio of 1g:(18-20)mL. After shaking and centrifugation, the extract liquid phase and solid phase are obtained. The extract liquid phase is an antibacterial product, and the solid phase is the hydrolyzed fermented tobacco straw product, which can be used to improve the salt tolerance of crops.

[0029] The present application will be further described below by way of specific embodiments.

[0030] Example 1 The preparation method of straw hydrolysis fermentation product includes the following steps: (1) Crush and sieve 10g of tobacco stalks, mix with 20mL of distilled water, microwave at 800W and 120℃ for 20min, centrifuge to separate the residual biomass, wash and dry the residual biomass to obtain crude extract.

[0031] (2) Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h. After the culture was completed, it was diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution. 5 g of crude extract was dispersed in 1 mL of water to obtain crude extract dispersion. 100 μL of Bacillus amyloliquefaciens Cas02 bacterial solution was inoculated into 100 mL of crude extract dispersion and cultured at 28 °C and 180 rpm for 120 h with shaking. The OD600 value was measured every 24 h. After the culture was completed, the bacterial solution was centrifuged and the supernatant was freeze-dried to obtain fermented crude extract.

[0032] (3) 1g of the fermented crude extract was suspended in 18mL of ethyl acetate. After shaking and centrifugation, the solid phase was washed and dried to obtain the tobacco straw hydrolysate fermentation product.

[0033] Example 2 The preparation method of straw hydrolysis fermentation product includes the following steps: (1) Crush and sieve 10g of tobacco stalks, mix with 15mL of water, microwave at 700W and 150℃ for 30min, centrifuge to separate the residual biomass, wash and dry the residual biomass to obtain crude extract.

[0034] (2) Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h. After the culture was completed, it was diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution. 5 g of crude extract was dispersed in 1 L of water to obtain crude extract dispersion. 100 μL of Bacillus amyloliquefaciens Cas02 bacterial solution was inoculated into 100 mL of crude extract dispersion and cultured at 28 °C and 200 rpm for 120 h with shaking. The OD600 value was measured every 24 h. After the culture was completed, the bacterial solution was centrifuged and the supernatant was freeze-dried to obtain fermented crude extract.

[0035] (3) 1g of the fermented crude extract was suspended in 20mL of ethyl acetate. After shaking and centrifugation, the solid phase was washed and dried to obtain the tobacco straw hydrolysate fermentation product.

[0036] Example 3 The preparation method of straw hydrolysis fermentation product includes the following steps: (1) 10g of tobacco stalks were crushed and sieved, mixed with 20mL of 0.2% dilute sulfuric acid, and microwaved at 800W and 120℃ for 20min. The residual biomass was separated by centrifugation, and the residual biomass was washed and dried to obtain crude extract.

[0037] (2) Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h. After the culture was completed, it was diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution. 5 g of crude extract was dispersed in 1 L of water to obtain crude extract dispersion. 100 μL of Bacillus amyloliquefaciens Cas02 bacterial solution was inoculated into 100 mL of crude extract dispersion and cultured at 28 °C and 180 rpm for 120 h with shaking. The OD600 value was measured every 24 h. After the culture was completed, the bacterial solution was centrifuged and the supernatant was freeze-dried to obtain fermented crude extract.

[0038] (3) 1g of the fermented crude extract was suspended in 20mL of ethyl acetate. After shaking and centrifugation, the solid phase was washed and dried to obtain the tobacco straw hydrolysate fermentation product.

[0039] Example 4 The preparation method of straw hydrolysis fermentation product includes the following steps: (1) 10g of tobacco stalks were crushed and sieved, mixed with 15mL of 0.2% dilute sulfuric acid, and microwaved at 700W and 150℃ for 30min. The residual biomass was separated by centrifugation, washed and dried to obtain crude extract.

[0040] (2) Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h. After the culture was completed, it was diluted with LB medium to an OD600 value of 0.3 to obtain Bacillus amyloliquefaciens Cas02 bacterial solution. 5 g of crude extract was dispersed in 1 L of water to obtain crude extract dispersion. 100 μL of Bacillus amyloliquefaciens Cas02 bacterial solution was inoculated into 100 mL of crude extract dispersion and cultured at 28 °C and 200 rpm for 120 h with shaking. The OD600 value was measured every 24 h. After the culture was completed, the bacterial solution was centrifuged and the supernatant was freeze-dried to obtain fermented crude extract.

[0041] (3) 1g of the fermented crude extract was suspended in 20mL of ethyl acetate. After shaking and centrifugation, the solid phase was washed and dried to obtain the tobacco straw hydrolysate fermentation product.

[0042] Comparative Example 1 The difference from Example 2 is that the temperature is 180°C during the microwave treatment in step (1).

[0043] Comparative Example 2 The difference from Example 2 is that no fermentation treatment was performed. Instead, the crude extract was directly suspended in 20 mL of ethyl acetate, shaken, centrifuged, and the solid phase was washed and dried to obtain the tobacco straw hydrolysate fermentation product.

[0044] Comparative Example 3 The difference from Example 2 is that the fermented extract is used instead of the tobacco straw hydrolysate.

[0045] Comparative Example 4 The difference from Example 2 is that the extract obtained after extraction in step (3) is concentrated using a rotary evaporator and then used instead of the tobacco straw hydrolysate fermentation product.

[0046] Comparative Example 5 The difference from Example 2 is that the fermentation process is as follows: Bacillus amyloliquefaciens with accession number CICC 10888 (purchased from China Industrial Microbial Culture Collection Center) is cultured in nutrient broth agar medium for 24 hours. After the culture is completed, it is diluted with medium to an OD600 value of 0.3 to obtain a bacterial solution. 5g of crude extract is dispersed in 1L of water to obtain a crude extract dispersion. 100μL of bacterial solution is inoculated into 100mL of crude extract dispersion and cultured at 28℃ and 200rpm with shaking for 120 hours. The OD600 value is measured every 24 hours. After the culture is completed, the bacterial solution is centrifuged and the supernatant is freeze-dried to obtain the fermented crude extract.

[0047] Experimental Example 1 This experiment uses peanut as an example to verify the effect of the tobacco straw hydrolysate prepared in the above-described examples and comparative examples on crop salt tolerance. Specifically, peanut seeds were soaked in deionized water for 5 hours, transferred to petri dishes lined with moist gauze, and germinated at 25°C in the dark for 24 hours. The tobacco straw hydrolysate was dispersed in water to obtain a nutrient solution with a concentration of 2 g / L, and sodium chloride (concentration 0-50 mmol / L) was added to the nutrient solution. The germinated seeds were sown in petri dishes with nylon mesh and cultured in the nutrient solution. Hoagland nutrient solution (commercially available water-soluble fertilizer) was used as a positive control, and water was used as a blank control. The nutrient solution was changed every two days. After 14 days of culture, seedlings were harvested, and growth indicators were measured to investigate the effect of salt stress on peanut seedling growth, including seedling height, root length, fresh weight, and dry weight. At least 5 parallel experiments were performed for each group, and the final results were expressed as the mean.

[0048] The results are as follows. Figure 1 See Tables 1 to 3.

[0049] Table 1. Plant height and root length of peanut seedlings under different salt concentrations

[0050] Table 2. Fresh weight of peanut seedlings under different salt concentrations

[0051] Table 3. Dry weight of peanut seedlings at different salt concentrations

[0052] Figure 1 In the text, CK represents the blank control group, HN represents the positive control group, CH 120 represents Example 1, CH 150 represents Example 2, CS 120 represents Example 3, and CS 150 represents Example 4.

[0053] Combination Figure 1 As shown in Table 1, when the nutrient solution was salt-free, all four types of tobacco straw fermentation hydrolysates in Examples 1-4 promoted peanut seedling growth, and there was no significant difference among the four treatments. Furthermore, the growth-promoting effect of the four tobacco straw fermentation hydrolysates was comparable to that of Hogrange nutrient solution, indicating their great potential as a water-soluble fertilizer for plants. However, when 50 mmol / L was added, the peanut seedlings in the water control group (blank control group) could not survive, while the seedlings in the four fermentation residue treatment groups could all survive, although their growth-promoting effect was slightly lower than that of the Hogrange nutrient solution group. Compared to the examples, the five products obtained in Comparative Examples 1-5 also promoted peanut seedling growth under salt-free conditions, but seedling growth significantly decreased under salt stress conditions. The results in Tables 2 and 3 show that peanut seedlings in the blank control group could not survive under 50 mmol / L sodium chloride salt conditions, while peanut seedlings with the four types of tobacco straw fermentation hydrolysates from Examples 1-4 could survive. This indicates that the tobacco straw hydrolysate fermentation product of this application can effectively alleviate the inhibitory effect of salt-alkali stress on the growth of peanut seedlings and can improve the salt resistance of corn seedlings.

[0054] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, this application is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of this application without departing from the scope of this application should be within the protection scope of this application.

Claims

1. A method for preparing a hydrolyzed fermentation product of tobacco straw, characterized in that, Includes the following steps: (1) Microwave-assisted hydrolysis of tobacco straw was performed to obtain crude extract; (2) The crude extract was fermented using Bacillus amyloliquefaciens Cas02 to obtain fermented crude extract; (3) Extract the crude fermentation extract to obtain a liquid phase and a solid phase. The solid phase is the tobacco straw hydrolysate fermentation product.

2. The preparation method according to claim 1, characterized in that, The microwave-assisted hydrolysis step in step (1) is as follows: after the tobacco stalks are crushed and sieved, they are mixed with the degradation reagent, microwaved, and centrifuged to separate the residual biomass. The residual biomass is washed and dried to obtain the crude extract.

3. The preparation method according to claim 2, characterized in that, The degradation reagent is water or dilute sulfuric acid, and the mass fraction of the dilute sulfuric acid is 0.2-0.3%; the mass-volume ratio of tobacco straw to degradation reagent is 1g:(15-20)mL.

4. The preparation method according to claim 3, characterized in that, The microwave processing conditions are: microwave power of 700-800W, temperature of 120-150℃, and time of 20-30min.

5. The preparation method according to claim 1, characterized in that, The fermentation process in step (2) includes: dispersing the crude extract in 180-200 times its weight of water to obtain a crude extract dispersion; inoculating the crude extract dispersion with Bacillus amyloliquefaciens Cas02 bacterial solution; culturing at 28-30℃ for 120h; measuring the OD600 value every 24h; centrifuging the suspension after the culture is completed; neutralizing the supernatant; and freeze-drying to obtain the fermented crude extract.

6. The preparation method according to claim 5, characterized in that, The preparation method of the Bacillus amyloliquefaciens Cas02 bacterial suspension is as follows: Bacillus amyloliquefaciens Cas02 is cultured in LB medium for 24 h, and then diluted with LB medium to an OD600 value of 0.3 to obtain the Bacillus amyloliquefaciens Cas02 bacterial suspension; The inoculum size of Bacillus amyloliquefaciens Cas02 is 0.1-0.15 (v / v)%.

7. The preparation method according to claim 1, characterized in that, In step (3), ethyl acetate is used as the extractant. The extraction process is as follows: the crude fermented extract is suspended in ethyl acetate, shaken and centrifuged, and the solid phase is taken as the tobacco straw hydrolysis fermentation product.

8. The preparation method according to claim 1, characterized in that, During the extraction process, the mass-to-volume ratio of the fermented crude extract to ethyl acetate was 1 g: (18-20) mL.

9. A hydrolyzed fermented product of tobacco straw, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The application of the tobacco straw hydrolysate fermentation product according to claim 9 in crop salt tolerance.

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