Streptomyces MB-1 and application thereof in cottonseed hull degradation

Abstract

The application discloses a streptomyces MB-1 and application thereof in cottonseed shell degradation, and belongs to the technical field of biology.The classification and naming of the streptomyces MB-1 is streptomyces sp., is sent to the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.32420 and a preservation date of October 30, 2024.The streptomyces MB-1 is beneficial to realizing efficient biological conversion of cottonseed shell waste rich in lignocellulose, has a cottonseed shell degradation rate of 49.54%, has a gossypol degradation rate of 25.82%, provides a technical basis for realizing agricultural waste resource application, and helps the development of circular agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to a Streptomyces MB-1 and its application in cottonseed hull degradation, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Cottonseed hull is the main by-product in cotton processing, accounting for about 30-40% of the total mass of cottonseed, and its components are complex, including cellulose, lignin, hemicellulose, crude protein, and a small amount of trace elements such as calcium and phosphorus. It is mainly used in edible fungus cultivation substrate, ruminant feed and organic fertilizer development, etc. However, there are some problems in the production and application of edible fungus cultivation, such as long cultivation period, low yield and low decomposition rate of fungus residue, etc. The main reason is that the lignocellulose in the cultivation substrate is not completely degraded, resulting in that the lignocellulose cannot be fully utilized. In addition, the presence of free gossypol and tannin in cottonseed hull limits its large-scale application in ruminant feed production. Although physical and chemical methods for treating cottonseed hull are simple to operate, they require high equipment and high cost. The biological degradation method has the advantages of low cost and no pollution, and has become a research hotspot in this field. The microorganisms that degrade lignocellulose and gossypol in nature are mainly fungi, such as Trichoderma, Penicillium and Aspergillus niger, etc. However, due to the long growth period, weak nutritional competition and the pathogenic characteristics in the Pleurotus ostreatus cultivation system, it seriously reduces the quality and yield. The cellulose and gossypol degrading bacteria have broad industrial development potential due to their rapid growth, short enzyme production time and strong adaptability. Therefore, screening bacteria with lignocellulose and gossypol degradation has theoretical and practical significance for shortening the production period of edible fungi and improving the added value of cottonseed hull roughage. SUMMARY

[0003] Therefore, the present application provides a Streptomyces MB-1 and its application in cottonseed hull degradation. The Streptomyces MB-1 is screened from Pleurotus ostreatus cultivation substrate, and can improve the cellulose and gossypol degradation rate and promote the resource utilization of cottonseed hull.

[0004] The Streptomyces MB-1 of the present application is classified and named as Streptomyces sp., and is preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No.32420 and the preservation date of October 30, 2024. The classification and naming in the preservation certificate is a speculation when we have not determined the specific strain. Subsequently, through whole genome sequencing of the strain, it is determined that the specific species is Streptomyces violaceoruber.

[0005] The application of the Streptomyces MB-1 in cottonseed hull degradation.

[0006] Specific is to introduce the streptomyces MB-1 into cotton seed shell liquid medium, and the degradation of cotton seed shell is carried out by adjusting the pH value of the system to 4-5 and the temperature to 30 DEG C.

[0007] The components and composition of the cotton seed shell liquid medium are as follows: cotton seed shell 20 g, potassium chloride 0.5 g, ferrous sulfate heptahydrate 0.05 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, ammonium sulfate 3 g, and distilled water is added to 1 L.

[0008] The present application aims at the technical problem of low biological degradation rate of straw substances in agricultural wastes, and provides a high-efficiency cellulose degradation strain. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 It is a Congo red carboxymethyl cellulose sodium plate hydrolysis effect diagram of streptomyces MB-1.

[0010] Figure 2 It is a colony morphology diagram of streptomyces MB-1 in LB medium.

[0011] Figure 3 It is a whole genome phylogenetic tree of streptomyces MB-1 in the embodiment of the present application.

[0012] Figure 4 It is the optimization result of cellulose enzyme production conditions of streptomyces MB-1 in the embodiment of the present application.

[0013] Figure 5 It is the degradation effect of streptomyces MB-1 on different straws.

[0014] Figure 6 It is the degradation effect of streptomyces MB-1 on cellulose, hemicellulose, lignin and gossypol in cotton seed shell. DETAILED DESCRIPTION

[0015] The present application will be described in more detail below in combination with specific implementation examples, so that those skilled in the art can better understand it. The specific implementation examples of the present application listed below are intended to further illustrate and supplement the objectives, technical implementation means and effects of the present application, and are not used to limit the scope of the present application.

[0016] The culture medium and its formula used in the following examples are as follows:

[0017] The formula of CMC-Na medium is: 2 g of ammonium sulfate, 0.5 g of magnesium sulfate heptahydrate, 1 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, 5 g of sodium carboxymethyl cellulose into a beaker, add deionized water to 1 L, adjust pH to 7.0, sterilize at 121℃ for 20 min.

[0018] Liquid enzyme production medium: sodium carboxymethyl cellulose 5 g, potassium chloride 0.5 g, ferrous sulfate heptahydrate 0.05 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, ammonium sulfate 3 g, distilled water to 1 L.

[0019] The formula of LB medium is: 5 g of yeast powder, 10 g of tryptone, 10 g of sodium chloride, add deionized water to 1 L, sterilize at 115℃ for 30 min.

[0020] Straw liquid medium: straw 20 g, potassium chloride 0.5 g, ferrous sulfate heptahydrate 0.05 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, ammonium sulfate 3 g, distilled water to 1 L.

[0021] Example 1: Isolation and identification of MB-1

[0022] 1. Isolation and purification of cellulose-degrading bacteria

[0023] The sample was collected from the cultivation medium of P. ostreatus in the third growth period. 10 g of sample was weighed and added with 90 mL of sterile water, and shaken at 30℃ and 120 rpm for 20 min. After the end, 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Take 100 uL of each concentration and spread on CMC-Na medium plates, set 3 repeats for each dilution, and place in a constant temperature incubator at 30℃ for 3 d. After that, the single colonies grown on the CMC-Na medium were further isolated and purified. Add appropriate amount of 1.0 mg / mL Congo red dye to the culture dish of the isolated and purified single colony, dye for 30 min, then pour out the dye, add appropriate amount of 1.0 mol / L NaCl solution, soak for 20 min, pour out and observe the hydrolysis ring on the plate, select the strain with larger ratio of transparent ring diameter to colony diameter (D / d), and number the strain as MB-1. Preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC No. 32420, and the preservation time of October 30, 2024.

[0024] Figure 1The hydrolysis effect of the MB-1 strain on a Congo red sodium carboxymethylcellulose plate is shown. As can be seen from the figure, after three days of incubation at 30°C on a sodium carboxymethylcellulose plate inoculated with MB-1, Congo red staining revealed a distinct hydrolysis zone, with a ratio of hydrolysis zone diameter to colony diameter of 4.32. This result indicates that the MB-1 strain has a strong ability to degrade cellulose.

[0025] 2. Identification of MB-1

[0026] The strain MB-1 was inoculated into LB medium and cultured at 30℃ for 72 hours. The growth of the colonies on the plate was observed and recorded. The colony morphology of MB-1 in LB medium is shown in Figure 2 The colony had irregular edges, a dry, dense, powdery surface, and formed white hyphae. It also secreted pigments that diffused into the culture medium, giving the plate a brownish color. Based on its morphological characteristics, it was preliminarily identified as belonging to the genus Streptomyces.

[0027] DNA of this strain was extracted and whole genome sequencing was performed. The single copy core gene results identified in the results of common and unique gene analysis were aligned with protein multiple sequences using MUSCLE software and converted into CDS results. PhyML software was used to construct an evolutionary tree using the ML method (maximum likelihood method) (see Figure 3 ), the results showed that the isolated and purified strain MB-1 was Streptomyces violaceoruber.

[0028] Example 2: Optimization of MB-1 cellulase production conditions

[0029] Optimization of enzyme production fermentation conditions of MB-1: the effects of carbon source, carbon source concentration, nitrogen source, nitrogen source concentration, pH and temperature on the enzyme production conditions of the strain during liquid fermentation.

[0030] 1. Preparation of crude enzyme solution

[0031] The screened MB-1 strain was inoculated into LB medium and cultured at 30°C, 150 rpm for 48 h. Then, a 5% inoculum of the strain was inoculated into sodium CMC medium and cultured at 30°C, 150 rpm for 3 days. The supernatant was centrifuged and used as the crude enzyme solution.

[0032] 2. Preparation of glucose standard curve

[0033] Preparation of 1 mg / mL glucose standard solution, take 6 test tubes, in turn take glucose standard solution 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mL, respectively, add distilled water to a total volume of 2.0 mL. Add 2.0 mL of 3, 5-dinitrosalicylic acid reagent (DNS reagent) to each test tube, shake well, then react in boiling water bath for 5 min, cool to room temperature, and dilute to 25 mL with distilled water. The blank sample is adjusted to zero, and the absorbance of the solution is measured at 540 nm. The linear equation of the standard curve is established with glucose content (mg) as the horizontal coordinate and the corresponding OD value as the vertical coordinate. The OD value of the sample is measured, and the glucose production is calculated according to the standard curve.

[0034] 3. Determination of cellulase activity

[0035] CMC enzyme activity determination: take 1 mL of crude enzyme solution, add 1 mL of 1% carboxymethyl cellulose sodium solution (pH 4.8, 0.05 mol / L citric acid solution), incubate in 50°C water bath for 30 min, then add 1.0 mL DNS solution, react in boiling water bath for 5 min, cool at room temperature, and measure the absorbance at 540 nm. The high-temperature inactivated enzyme solution is used as the negative control. 1 mL of crude enzyme solution is used as the enzyme amount required to catalyze the generation of 1 μg of glucose per minute, and the unit is U / mL.

[0036] Filter paper enzyme activity determination: take 1 mL of crude enzyme solution, add to a 5 mL EP tube containing a filter paper strip (60 mm x 10 mm), add 1 mL of previously prepared citric acid buffer (pH 4.8, 0.05 mol / L), incubate at 50°C for 30 min, add 1 mL of DNS reagent, mix well, and react in boiling water bath for 5 min. Measure the absorbance at 540 nm, and use the same amount of high-temperature inactivated enzyme solution (100°C for 10 min) as the negative control. Calculate the filter paper enzyme activity according to the standard curve. 1 mL of crude enzyme solution is used as the enzyme amount required to catalyze the generation of 1 μg of glucose per minute, and the unit is U / mL.

[0037]

[0038] Wherein:

[0039] A is the glucose content calculated according to the standard curve, mg;

[0040] V is the volume of the added crude enzyme solution, mL;

[0041] N is the dilution multiple of the crude enzyme solution;

[0042] T is the enzyme reaction time, min.

[0043] By optimizing the enzyme production conditions of MB-1, such as Figure 4 As shown in the figure, when Streptomyces MB-1 used 20 g / L bran as carbon source, 0.3% soybean meal as nitrogen source, pH=5 and system temperature at 30℃, the CMC enzyme activity was the highest, which was 96.88 U / mL. At this time, the filter paper enzyme activity was 26.35 U / mL.

[0044] Example 3: Degradation of straw by MB-1

[0045] This example provides a straw degradation experiment using Streptomyces MB-1. Cottonseed hulls, corn, rice, and wheat straw were pre-ground and passed through a 40-mesh sieve. Streptomyces MB-1 was cultured in LB medium at 30°C and 150 rpm for 48 hours. Then, a 5% (v / v) inoculum of Streptomyces MB-1 was transferred to cottonseed hull, rice, corn, and wheat straw culture media, respectively, and shaken. An equal amount of sterile water was added as a control group, with three replicates for each treatment. After 15 days, the straw was rinsed repeatedly with clean water 2-3 times and dried at 80°C to constant weight. The straw degradation rate was calculated. The relative degradation rate of rice straw was also calculated using an equal amount of sterile water as a control group.

[0046]

[0047] Wherein: W0 is the dry weight of straw before degradation, and W1 is the dry weight of straw after degradation.

[0048] The results are as follows Figure 5 As shown, in the experiment without the addition of Streptomyces MB-1, the degradation rates of cottonseed hulls, rice, corn, and wheat were 9.49%, 11.72%, 13.32%, and 21.18%, respectively, after 15 days. After the addition of MB-1, the degradation rates reached 49.54%, 18.78%, 21.98%, and 41.75%, respectively, at 15 days. Compared with the treatment without the addition of MB-1, the degradation rate of cottonseed hulls increased by 4.22 times, indicating that the addition of MB-1 is highly effective in degrading cottonseed hulls. Further testing of the degradation of cellulose, hemicellulose, lignin, and gossypol in cottonseed hulls revealed that the addition of MB-1 effectively degraded lignocellulose and gossypol in cottonseed hulls, with degradation rates of lignin, cellulose, hemicellulose, and gossypol of 8.48%, 53.33%, 63.91%, and 25.82%, respectively.

Claims

1. A Streptomyces MB-1, characterized in that: The taxonomic name of the Streptomyces MB-1 is Streptomyces sp., and it is preserved in the China General Microbiological Culture Collection Center on October 30, 2024, with the preservation number CGMCC No. 32420.

2. The Streptomyces MB-1 of claim 1 is applied in the degradation of cottonseed hulls.

3. The application of claim 2, characterized in that: The Streptomyces MB-1 is inoculated into a cottonseed hull liquid medium, the pH of the system is controlled to be 4-5, and the temperature is 30°C, and the cottonseed hulls are degraded.

4. The application of claim 3, characterized in that: The components and composition of the cottonseed hull liquid medium are: cottonseed hulls 20 g, potassium chloride 0.5 g, ferrous sulfate heptahydrate 0.05 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, ammonium sulfate 3 g, and distilled water to 1 L.

Images