Bacillus velezensis crude protein bacteriostatic film and application thereof

Abstract

The application belongs to the technical field of bacteriostatic film and particularly relates to a bacillus velezensis crude protein bacteriostatic film and application thereof. The bacteriostatic film is prepared from bacillus velezensis T9 crude protein extract, carboxymethyl chitosan and konjac glucomannan; wherein, the taxonomic name of the bacillus velezensis T9 is bacillus velezensis (Bacillus velezensis), and the preservation number is GDMCC No:62265; the mass ratio of the bacillus velezensis T9 crude protein extract, carboxymethyl chitosan and konjac glucomannan is 0.04-0.12:1:1. The bacillus velezensis T9 crude protein extract and natural macromolecular substances carboxymethyl chitosan and konjac glucomannan are used as raw materials to prepare the bacteriostatic film, and through reasonable proportioning of the three, the bacteriostatic film has obvious inhibition effect on sugarcane arthrinium, while the thickness and tensile strength of the bacteriostatic film are ensured, which has important significance for developing safe, efficient and stable materials for preventing mold of fruit cane and controlling arthrinium.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial film technology, specifically relating to a crude protein antibacterial film of Bacillus belysaeus and its application. Background Technology

[0002] Traditional plastic packaging materials are mostly derived from fossil-derived polymers, such as polyethylene, polyvinyl chloride, and polypropylene. With increasing public concern about environmental issues and growing environmental awareness, these non-degradable plastic bags will gradually be replaced by new edible and biodegradable bio-packaging materials.

[0003] Konjac glucomannan (KGM) is a natural polysaccharide extracted from the tubers of plants in the Amorphophallus genus of the Araceae family. Due to its excellent film-forming properties, KGM can form a dense network structure, and therefore is often used in the preparation of functional food packaging materials. However, KGM films suffer from drawbacks such as poor physicochemical properties and antibacterial properties, thus limiting their widespread application in the food packaging industry.

[0004] Films formed using a single protein or polysaccharide as the film-forming matrix often exhibit poor mechanical and moisture-barrier properties, limiting their applications. However, by using two or more suitable natural macromolecules as film-forming matrices, and allowing for complementary advantages among the matrices, composite films with excellent mechanical and barrier properties can be obtained. Carboxymethyl chitosan (CM-CS) is an amphoteric derivative of chitosan, rich in COOH and NH2, and exhibits good solubility at room temperature. Furthermore, CM-CS possesses high viscosity, good biocompatibility, and biodegradability, making it a highly attractive packaging film-forming material. The unique chemical structure of its amino groups allows CM-CS to disrupt cell membrane stability, thereby producing an antibacterial effect, inhibiting Gram-positive bacteria, Gram-negative bacteria, and Candida albicans strains. However, although carboxymethyl chitosan alone has antibacterial properties, its antibacterial effect is poor. To improve the antibacterial effect of carboxymethyl chitosan, it is necessary to perform appropriate antibacterial modification to improve its antibacterial stability and efficiency, and broaden its application fields.

[0005] *Bacillus velezensis*, a new species of the genus *Bacillus*, is a Gram-positive aerobic bacterium with rod-shaped cells and is widely distributed in nature. In recent years, *Bacillus velezensis* has been primarily reported as a biocontrol bacterium, mainly for its role in promoting plant and animal growth, antagonizing pathogens, inducing systemic resistance, identifying antimicrobial substances and their gene clusters, and elucidating its antagonistic mechanisms. *Bacillus velezensis* can inhibit pathogens by synthesizing secondary metabolites, which mainly include bacteriocins, antimicrobial proteins, lipopeptides, and polyketides. The antimicrobial effect of *Bacillus velezensis* in practical applications is easily affected by the external environment. However, antimicrobial substances extracted from the fermentation broth can significantly reduce interference from temperature, humidity, and pH, improving its stability and ensuring its antimicrobial effect. Therefore, the secondary metabolites of *Bacillus* have become a research hotspot in biocontrol bacteria.

[0006] Sugarcane is rich in nutrients, containing easily absorbed sugars such as glucose, sucrose, and fructose, making it a popular food. However, sugarcane is highly susceptible to pathogen infection during transportation and storage, leading to mold growth and the production of microbial toxins. The fungus isolated from moldy sugarcane has been identified as *Arthrinium arundinis*. *Arthrinium arundinis* in moldy sugarcane produces the neurotoxin 3-nitropropionic acid. Ingestion of moldy sugarcane containing a certain amount of 3-nitropropionic acid can cause severe food poisoning, primarily manifesting as nausea, dizziness, vomiting, and visual disturbances; in severe cases, it can lead to coma, respiratory failure, and death. Therefore, given the susceptibility of sugarcane to *Arthrinium arundinis* infection and the production of 3-nitropropionic acid during storage, the development and research of safer, more efficient, and stable materials for the control of *Arthrinium arundinis* is of paramount importance.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide a crude protein antibacterial membrane of Bacillus belye and its application, which has significant antibacterial activity against Fusarium moniliforme.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] An antibacterial film is prepared from crude protein extract of Bacillus velezensis T9, carboxymethyl chitosan, and konjac glucomannan; wherein, the taxonomic name of Bacillus velezensis T9 is Bacillus velezensis, and its accession number is GDMCC No:62265; the mass ratio of crude protein extract of Bacillus velezensis T9, carboxymethyl chitosan, and konjac glucomannan is 0.04-0.12:1:1.

[0011] Preferably, the mass ratio of the Bacillus vesiculosus T9 crude protein extract, carboxymethyl chitosan, and konjac glucomannan is 0.08:1:1.

[0012] Preferably, the method for preparing the crude protein extract of Bacillus belyssus T9 includes the following steps:

[0013] S1. Inoculate Bacillus belye T9 into LB liquid medium and culture at 28°C and 140 r / min in a shaker for 48 h to obtain seed culture;

[0014] S2. Inoculate the seed culture at a 2% inoculation rate into a culture medium containing 1% yeast powder, 1% soybean powder, and 0.05% sodium chloride; fill 200 mL of the solution into a 500 mL Erlenmeyer flask, and then culture on a shaker at 140 r / min and 28 °C for 72 h.

[0015] S3. Add 60% saturated ammonium sulfate solution to the fermentation supernatant, let stand at 4℃ for 24 h, centrifuge at 7000 r / min for 20 min, collect the precipitate, dissolve it with phosphate buffer at pH 7.2, dialyze to remove salt, and freeze dry under vacuum to obtain the crude protein extract of Bacillus vesiculosus T9.

[0016] The second objective of this invention is to provide a method for preparing the antibacterial film, specifically: dissolving crude protein extract of Bacillus vesiculosus T9 in distilled water, adding carboxymethyl chitosan and konjac glucomannan, stirring, then adding glycerol and Tween-80 dropwise, stirring again to mix thoroughly to obtain a film-forming solution; then casting the film-forming solution onto a plastic petri dish and drying it at 45°C to obtain the antibacterial film.

[0017] A third objective of this invention is to provide the application of the antibacterial film in inhibiting the growth of *Fusarium moniliforme* hyphae.

[0018] A fourth object of the present invention is to provide the application of the antibacterial film in preventing mold growth in sugarcane.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention prepares an antibacterial film using crude protein extract of Bacillus vesiculosus T9 and natural macromolecules carboxymethyl chitosan and konjac glucomannan as raw materials. Through the reasonable ratio of the three, the antibacterial film has a certain antibacterial thickness and good tensile strength, and also has a significant inhibitory effect on Fusarium moniliforme in sugarcane. This is of great significance for the development of safe, efficient and stable materials for preventing mold growth in sugarcane and controlling Fusarium moniliforme.

[0021] Preservation Information

[0022] Bacillus belye T9, with accession number GDMCC No:62265, accession date December 8, 2022, deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0023] Figure 1 The effect of the antibacterial film on the length of lesions in sugarcane after inoculation with Fusarium moniliforme on days 3, 5, 7 and 10.

[0024] Explanation of key figure labels:

[0025] Figure 1 From left to right, the numbers are CK, KC, B4, B8, B12, KCB4, KCB8, and KCB12. Detailed Implementation

[0026] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0027] The Bacillus belyss T9 of this application has been published in publication (announcement) number CN116410889A, with the patent title "A strain T9 and its application". The morphological and molecular biological identification results of Bacillus belyss T9 have been described in that patent.

[0028] All of the following experimental treatments were repeated three times.

[0029] Preparation of antibacterial film and its antibacterial properties and tensile strength testing

[0030] 1. Antibacterial film

[0031] 1.1 Obtaining crude protein extract from Bacillus belyssus T9

[0032] S1. Inoculate Bacillus belye T9 into LB liquid medium and culture at 28°C and 140 r / min in a shaker for 48 h to obtain seed culture;

[0033] S2. Inoculate the seed culture at a 2% inoculation rate into a culture medium containing 1% yeast powder, 1% soybean powder, and 0.05% sodium chloride; fill 200 mL of the solution into a 500 mL Erlenmeyer flask, and then culture on a shaker at 140 r / min and 28 °C for 72 h.

[0034] S3. Add 60% saturated ammonium sulfate solution to the fermentation supernatant, let stand at 4℃ for 24 h, centrifuge at 7000 r / min for 20 min, collect the precipitate, dissolve it with phosphate buffer at pH 7.2, dialyze to remove salt, and freeze dry under vacuum to obtain the crude protein extract of Bacillus vesiculosus T9.

[0035] 1.2 Preparation of film-forming solution

[0036] 1.2.1 Effect of the amount of crude protein extract of Bacillus belyss T9 added on antibacterial effect and tensile strength

[0037] 0 g, 0.04 g, 0.08 g and 0.12 g of crude protein extract of Bacillus belye T9 were dissolved in 100 mL of distilled water, and 1 g of carboxymethyl chitosan and 1 g of konjac glucomannan were added. After stirring for 2 h, 100 μL of glycerol and 50 μL of Tween-80 were added dropwise and stirred for 5 min to obtain film-forming solutions, which were labeled as KC, KCB4, KCB8 and KCB12, respectively.

[0038] 0 g, 0.04 g, 0.08 g and 0.12 g of crude protein extract of Bacillus belye T9 were dissolved in 100 mL of distilled water, and 1 g of carboxymethyl chitosan was added. After stirring for 2 h, 100 μL of glycerol and 50 μL of Tween-80 were added dropwise and stirred for 5 min to obtain film-forming solutions, which were labeled as C, CB4, CB8 and CB12, respectively.

[0039] 0g, 0.04g, 0.08g and 0.12g of crude protein extract of Bacillus vesiculosus T9 were dissolved in 100mL of distilled water, and 1g of konjac glucomannan was added. After stirring for 2h, 100μL of glycerol and 50μL of Tween-80 were added dropwise and stirred for 5min to obtain film-forming solutions, which were labeled as K, KB4, KB8 and KB12, respectively.

[0040] 0.04 g, 0.08 g, and 0.12 g of crude protein extract of Bacillus belyss T9 were dissolved in 100 mL of distilled water and labeled as B4, B8, and B12, respectively.

[0041] 1.2.2 Effect of the mass ratio of konjac glucomannan to carboxymethyl chitosan on antibacterial effect and tensile strength

[0042] Dissolve 0.08 g of crude protein extract of Bacillus belye T9 in 100 mL of distilled water. Add the corresponding mass of methyl chitosan and konjac glucomannan (total amount of konjac glucomannan and carboxymethyl chitosan is 2 g) according to the mass ratio of konjac glucomannan to carboxymethyl chitosan of 0.2:1.8, 0.4:1.6, 0.8:1.2, 1.0:1.0, 1.2:0.8, 1.6:0.4, and 1.8:0.2. After mixing and stirring for 2 h, add 100 μL of glycerol and 50 μL of Tween-80 dropwise and stir for 5 min to obtain film-forming solutions, which are labeled as KCB19, KCB14, KCB23, KCB8, KCB32, KCB41, and KCB91, respectively.

[0043] 1.3 Preparation of antibacterial film

[0044] 25 mL of film-forming solution was poured onto a 9 cm diameter plastic petri dish and dried at 45 °C for 12 h to obtain the corresponding antibacterial film. After being placed in a constant temperature and humidity incubator at 50% relative humidity and 25 °C for 72 h, the antibacterial properties and tensile strength of the antibacterial film were tested.

[0045] 2. Determination of inhibition zones

[0046] Fusarium moniliforme was activated using a suitable slant culture medium to prepare 1×10⁻⁶ slurries. 6 Spore / mL suspension. 200 μL of spore suspension was evenly spread on the surface of PDA medium. Wells (5 mm × 5 mm) were punched in the plates, and the film-forming solution was added dropwise into each well. All plates were incubated at 28℃ for 48 h to observe the antibacterial effect.

[0047] 3. Measure the thickness of the antibacterial film.

[0048] The thickness of the antibacterial film was measured using a micrometer.

[0049] 4. Determine the tensile strength of the antibacterial film.

[0050] The tensile strength of the antibacterial film was determined using a texture analyzer with a sample size of 10 mm × 50 mm.

[0051] 5. Effect of antibacterial film on the length of lesions in sugarcane after inoculation with Fusarium moniliforme

[0052] Wash sugarcane with sterile water and air dry, then disinfect with 75% alcohol. Drill holes (3 mm deep, 5 mm in diameter) in the sugarcane surface using a sterile inoculation needle and add 150 μL of KC, KCB4, KCB8, KCB12, B4, B8, and B12 antibacterial film solutions. This sterile treatment serves as a control group. After standing for 24 hours, add 100 μL of *Fusarium moniliforme* spore suspension to the drilled holes in the sugarcane. Place the sugarcane in an incubator and store at 20°C. Measure the length of lesions on days 3, 5, 7, and 10.

[0053] 6. Results and Analysis

[0054] 6.1 Antibacterial properties, thickness, and tensile strength of different antibacterial films

[0055] The measurement results are shown in Table 1.

[0056] Table 1. Results of antibacterial properties, film thickness, and tensile strength tests for different antibacterial films.

[0057]

[0058]

[0059] As shown in Table 1, compared with the antibacterial films made of carboxymethyl chitosan and konjac glucomannan alone, the Bacillus vesiculosus crude protein antibacterial film of the present invention has better antifungal activity against Fusarium moniliforme. Moreover, with the increase of crude protein content in the antibacterial film, the antifungal activity of the composite film against Fusarium moniliforme increases significantly. However, the addition of Bacillus vesiculosus T9 crude protein extract increases the thickness of the antibacterial film and also has a certain inhibitory effect on the tensile strength of the antibacterial film.

[0060] When the crude protein content of Bacillus belye was 0.08%, the mass ratio of carboxymethyl chitosan to konjac glucomannan was changed. It was found that when the mass ratio of carboxymethyl chitosan to konjac glucomannan was 1:1, the antibacterial film prepared had the best antifungal activity against Fusarium moniliforme and the best tensile strength.

[0061] 6.2 Effect of antibacterial film on the length of lesions in sugarcane after inoculation with Fusarium moniliforme

[0062] See results Figure 1 And Table 2.

[0063] Table 2. Effect of antibacterial film on the length of lesions in sugarcane after inoculation with *Fusarium moniliforme*.

[0064]

[0065] from Figure 1As shown in Table 2, KCB4, KCB8, and KCB12 treatments significantly inhibited the growth of *Fusarium moniliformes* inoculated with artificially damaged tissue. On days 3, 5, 7, and 10 after KCB12 treatment, the lesion lengths of sugarcane were 10.0, 14.0, 20.0, and 23.5 mm, respectively, representing reductions of 59.2%, 65.0%, 54.0%, and 51.5% compared to the control group (CK). Treatment with *Bacillus vesiculosus* T9 crude protein extract alone showed some inhibitory effect on the lesion length compared to the control group (CK), but the inhibitory effect was significantly lower than that of the antibacterial film composed of carboxymethyl chitosan, konjac glucomannan, and *Bacillus vesiculosus* T9 crude protein. On day 10, the lesion lengths of sugarcane treated with KCB4, KCB8, and KCB12 were reduced by 28.8%, 32.5%, and 28.8% compared to those treated with B4, B8, and B12, respectively. The reason may be that the synergistic effect of carboxymethyl chitosan, konjac glucomannan and Bacillus vesiculosus T9 crude protein increases the antibacterial properties of the composite antibacterial film.

[0066] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An antibacterial film, characterized in that, The antibacterial membrane is prepared from crude protein extract of Bacillus vesiculus T9, carboxymethyl chitosan, and konjac glucomannan; wherein, the taxonomic name of Bacillus vesiculus T9 is Bacillus vesiculus (… Bacillus velezensis (), with accession number GDMCC No. 62265; The mass ratio of the crude protein extract of Bacillus vesiculus T9, carboxymethyl chitosan, and konjac glucomannan is 0.04-0.12:1:1; wherein, the preparation method of the crude protein extract of Bacillus vesiculus T9 includes the following steps: S1. Inoculate Bacillus belye T9 into LB liquid medium and culture at 28°C and 140 r / min in a shaker for 48 h to obtain seed culture; S2. Inoculate the seed culture at a 2% inoculation rate into a culture medium containing 1% yeast powder, 1% soybean powder, and 0.05% sodium chloride; fill 200 mL of the solution into a 500 mL Erlenmeyer flask, and then culture on a shaker at 140 r / min and 28 °C for 72 h. S3. Add 60% saturated ammonium sulfate solution to the fermentation supernatant, let stand at 4℃ for 24 h, centrifuge at 7000 r / min for 20 min, collect the precipitate, dissolve it with phosphate buffer at pH 7.2, dialyze to remove salt, and freeze dry under vacuum to obtain the crude protein extract of Bacillus belye T9.

2. The antibacterial film according to claim 1, characterized in that, The mass ratio of the crude protein extract of Bacillus vesiculosus T9, carboxymethyl chitosan, and konjac glucomannan was 0.08:1:

1.

3. A method for preparing the antibacterial film according to any one of claims 1-2, characterized in that, Specifically, the crude protein extract of Bacillus vesiculosus T9 was dissolved in distilled water, and carboxymethyl chitosan and konjac glucomannan were added. After stirring, glycerol and Tween-80 were added dropwise, and the mixture was stirred again to obtain a film-forming solution. The film-forming solution was then cast onto a plastic petri dish and dried at 45°C to obtain the antibacterial film.

4. The antibacterial film according to any one of claims 1-2 is used in preventing mold growth on sugarcane.

Images