Streptomyces MB-1 and application thereof in cotton seed hull degradation

Abstract

The invention discloses streptomyces MB-1 and application thereof in cotton seed hull degradation, and belongs to the technical field of biology. The classification name of the streptomycete MB-1 is streptomycete sp, and the streptomycete MB-1 is sent to the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32420, and the preservation date is October 30, 2024. The streptomyces MB-1 disclosed by the invention is beneficial to realizing efficient bioconversion of cottonseed hull wastes rich in lignocellulose, the degradation rate of cottonseed hulls is 49.54%, the degradation rate of gossypol is 25.82%, a technical basis is provided for realizing resource application of agricultural wastes, and the development of circular agriculture is assisted.

Description

Technical Field

[0001] The invention relates to Streptomyces MB-1 and application thereof in cottonseed hull degradation, belonging to the field of biotechnology. Background Art

[0002] Cottonseed hulls are a major byproduct of cotton processing, accounting for approximately 30%-40% of the total cottonseed mass. Their composition is complex, primarily consisting of cellulose, lignin, hemicellulose, crude protein, and small amounts of trace elements such as calcium and phosphorus. They are primarily used in the development of edible fungus cultivation substrates, ruminant feed, and organic fertilizers. However, the production and application of edible fungus cultivation present several challenges, including long cultivation cycles, low yields, and low decomposition rates of fungus residue. This is primarily due to incomplete degradation of lignocellulose in the cultivation substrate, which results in insufficient utilization of the lignocellulose. Furthermore, the presence of anti-nutritional factors such as free gossypol and tannins in cottonseed hulls limits their large-scale application in ruminant feed production. While physical and chemical methods for treating cottonseed hulls are simple to operate, they are equipment-intensive and expensive. Biodegradation, however, offers advantages such as low cost and pollution-free operation, making it a research hotspot in this field. In nature, fungi that degrade lignocellulose and gossypol are primarily fungi, such as Trichoderma, Penicillium, and Aspergillus niger. However, these fungi have long growth cycles, are weak competitors for nutrients, and exhibit pathogenicity in Pleurotus ostreatus culture systems, severely reducing their quality and yield. Cellulose- and gossypol-degrading bacteria have broad potential for industrial development due to their rapid growth, rapid enzyme production, and strong adaptability. Therefore, screening for bacteria capable of degrading lignocellulose and gossypol has both theoretical and practical implications for shortening the production cycle of edible fungi and increasing the added value of cottonseed hull roughage. Summary of the Invention

[0003] In view of this, the present invention provides a Streptomyces MB-1 and its application in cottonseed hull degradation. The Streptomyces MB-1 of the present invention is screened from the culture medium of Pleurotus ostreatus, which can improve the degradation rate of cellulose and gossypol and promote the resource utilization of cottonseed hulls.

[0004] The Streptomyces MB-1 strain of the present invention is classified as Streptomyces sp. and deposited with the General Microbiology Center of the China National Committee for the Administration of Microbiological Culture Collection under the deposit number CGMCC No. 32420. The deposit date is October 30, 2024. The classification and nomenclature in the deposit certificate were based on our prior assumptions regarding the specific species. Subsequent whole-genome sequencing of the strain confirmed the species to be Streptomyces violaceoruber.

[0005] The invention discloses an application of Streptomyces MB-1 in the degradation of cottonseed hulls.

[0006] Specifically, Streptomyces MB-1 is introduced into a cottonseed hull liquid culture medium, and the pH of the system is regulated to 4-5 and the temperature to 30° C. to degrade the cottonseed hull.

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

[0008] This study addresses the technical challenge of low biodegradation rates for straw-like materials in agricultural waste by providing a highly efficient cellulose-degrading strain. This strain is highly efficient in degrading high-fiber cellulose, achieving a 49.54% degradation rate in cottonseed hulls within 15 days. It can also degrade gossypol in cottonseed hulls at a 25.82% degradation rate, providing a technical basis for the resourceful utilization of agricultural waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a diagram showing the hydrolysis effect of Congo red sodium carboxymethyl cellulose on a flat plate of Streptomyces MB-1.

[0010] Figure 2 This is the colony morphology of Streptomyces MB-1 in LB medium.

[0011] Figure 3 This is the whole genome phylogenetic tree of Streptomyces MB-1 in the examples of the present invention.

[0012] Figure 4 The optimized results of the cellulase production conditions of Streptomyces MB-1 in the examples of the present invention are shown below: A is the carbon source, B is the bran concentration, C is the nitrogen source, D is the yeast powder concentration, E is the system pH value, and F is the temperature.

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

[0014] Figure 6 This is the degradation effect of Streptomyces MB-1 on cellulose, hemicellulose, lignin and gossypol in cottonseed hulls. DETAILED DESCRIPTION

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

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

[0017] The formula of CMC sodium medium is as follows: 2 g ammonium sulfate, 0.5 g magnesium sulfate heptahydrate, 1 g potassium dihydrogen phosphate, 0.5 g sodium chloride, and 5 g sodium carboxymethyl cellulose are placed in a beaker, and the volume is adjusted to 1 L with deionized water. The pH is adjusted to 7.0, and the medium is sterilized at 121°C for 20 min.

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

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

[0020] Straw liquid culture medium: 20 g straw, 0.5 g potassium chloride, 0.05 g ferrous sulfate heptahydrate, 0.5 g potassium dihydrogen phosphate, 0.3 g magnesium sulfate heptahydrate, 3 g ammonium sulfate, and 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 samples were collected from the culture medium of Pleurotus ostreatus during the third growing period. Weigh 10 g of sample, add 90 mL of sterile water, shake at 30°C and 120 rpm for 20 min. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 100 μL of each concentration was spread on a sodium carboxymethyl cellulose (CMC-Na) medium plate, with three replicates for each dilution. After incubation at 30°C for 3 days, the resulting single colonies were further isolated and purified on sodium carboxymethyl cellulose medium. An appropriate amount of 1.0 mg / mL Congo red stain was added to the culture dish containing the isolated and purified single colony and stained for 30 minutes. The stain was then poured out and an appropriate amount of 1.0 mol / L NaCl solution was added and soaked for 20 minutes. After pouring out, the hydrolysis zone on the plate was observed. The strain with the largest ratio of the transparent zone diameter to the colony diameter (D / d) was selected and designated MB-1. The strain was deposited at the General Microbiology Center of the China General Microbiology Culture Collection (CGMCC) with the deposit number CGMCC No. 32420, and the deposit date was 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] Prepare a 1 mg / mL glucose standard solution. Take 6 test tubes and, in order, measure 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of the glucose standard solution. Add distilled water to each tube to a total volume of 2.0 mL. Add 2.0 mL of 3,5-dinitrosalicylic acid reagent (DNS reagent) to each tube, shake thoroughly, and react in a boiling water bath for 5 minutes. Cool to room temperature and dilute to 25 mL with distilled water. Zero the blank sample and measure the absorbance of the solution at 540 nm. Construct a linear equation for the standard curve using the glucose content (mg) as the horizontal axis and the corresponding OD value as the vertical axis. Calculate the amount of glucose produced using the standard curve using the OD values ​​of the samples.

[0034] 3. Determination of cellulase activity

[0035] CMC enzyme activity assay: 1 mL of crude enzyme solution was added to 1 mL of 1% sodium carboxymethylcellulose solution (pH 4.8, prepared with 0.05 mol / L citric acid). The solution was incubated in a 50°C water bath for 30 min. Then, 1.0 mL of DNS solution was added and the reaction was incubated in a boiling water bath for 5 min. After cooling to room temperature, the absorbance was measured at 540 nm. A high-temperature inactivated enzyme solution was used as a negative control. One unit of activity (U / mL) was defined as the amount of enzyme required to catalyze the production of 1 μg of glucose per minute per 1 mL of crude enzyme solution.

[0036] Filter paper enzyme activity assay: 1 mL of crude enzyme solution was added to a 5 mL EP tube containing a filter paper strip (60 mm × 10 mm). 1 mL of pre-prepared citrate buffer (pH 4.8, 0.05 mol / L) was added and incubated at 50°C for 30 min. 1 mL of DNS reagent was added, mixed thoroughly, and the mixture was allowed to react in a boiling water bath for 5 min. The absorbance was measured at 540 nm. An equal amount of high-temperature inactivated enzyme solution (treated at 100°C for 10 min) was added as a negative control. Filter paper enzyme activity was calculated using a standard curve. One unit of activity (U / mL) was defined as the amount of enzyme required to catalyze the production of 1 μg of glucose per minute per mL of crude enzyme solution.

[0037]

[0038] in:

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

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

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

[0042] T is the enzymatic 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 Streptomyces MB-1 is classified and named Streptomyces sp. and deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.32420 and the deposit date of October 30, 2024.

2. Use of the Streptomyces MB-1 according to claim 1 in the degradation of cottonseed hulls.

3. The use according to claim 2, characterized in that: Streptomyces MB-1 was inoculated into a cottonseed hull liquid culture medium, and the pH of the system was regulated to be 4-5 and the temperature to be 30° C. to degrade the cottonseed hull.

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

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