Application of lysozyme nanoflower composite material in inhibition of alicyclic acid bacillus

Abstract

The invention belongs to the technical field of inhibition of microorganisms in food, and discloses application of a lysozyme nanoflower composite material to inhibition of alicyclic acid bacillus, and the minimum inhibitory concentration and the minimum bactericidal concentration of the lysozyme nanoflower composite material to the alicyclic acid bacillus are 4.0 [mu] g mL <-1 > and 63.0 [mu] g mL <-1 > respectively. The lysozyme nanoflower composite material has good antibacterial activity and biological safety, and can be used for pollution control of spoilage microorganisms in fruit juice beverages, so that the shelf life of products is prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial inhibition in food and relates to application of a lysozyme nanoflower composite material in inhibiting alicyclobacillus. Background Art

[0002] Alicyclobacillus Alicyclobacillus acidocaldarius ) is a type of Gram-positive bacteria that is non-pathogenic, spore-forming, and can tolerate acidic and hot environments. This type of bacteria exhibits acidophilic and heat-resistant characteristics, and can survive conventional pasteurization in industrial production. The low pH value and nutrient-rich matrix of juice provide good growth and reproduction conditions for Alicyclobacillus. Alicyclobacillus is widely distributed, and spoilage incidents caused by this type of bacteria have been reported in many countries around the world, involving a variety of fruit juices and fruit juice drinks. Alicyclobacillus can produce metabolites with pungent odors through growth and metabolism to contaminate juice, such as guaiacol and halogenated phenols. Since these metabolites do not cause swelling of the juice, it is difficult to detect the contamination of Alicyclobacillus in the juice with the naked eye. As one of the key quality and safety factors in the juice industry, the contamination of Alicyclobacillus has seriously affected the rapid development of fruit processing and import and export trade.

[0003] Lysozyme (Ly, EC 3.2.1.17) is an alkaline enzyme widely present in biological tissues. It can hydrolyze the peptidoglycan in the bacterial cell wall to dissolve bacterial cells, so it has good antibacterial activity against Gram-positive bacteria. Lysozyme has stable chemical properties, good safety and pH adaptability, and shows good thermal stability under acidic conditions. Therefore, it can be used for the preservation of low pH foods. Because lysozyme carries a large amount of positive charge, it is easy to combine with negatively charged components in juice to produce precipitates, which also affects its antibacterial activity, limiting the application of lysozyme in juice foods.

[0004] Organic-inorganic hybrid nanoflowers are nanomaterials composed of bioactive substances as organic components and phosphate crystals as inorganic components. During the preparation of nanoflowers, bioactive substances provide nucleation sites for phosphate crystals by combining with metal ions, and at the same time, they induce phosphate crystals to present nanoflower morphology. The construction of organic-inorganic hybrid nanoflowers does not affect the structure of bioactive substances, and nanoflowers with high specific surface area are conducive to the function of bioactive substances. At present, a variety of bioactive substances including amino acids, proteins, and natural enzymes are used to construct organic-inorganic hybrid nanoflowers. Summary of the invention

[0005] The present invention aims at the technical problem of contamination of juice by Alicyclobacillus and provides an application of a lysozyme nanoflower composite material in inhibiting Alicyclobacillus. The lysozyme nanoflower composite material exhibits good antibacterial activity and biosafety and can be used for contamination control of spoilage microorganisms in juice drinks, thereby extending the shelf life of the product.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides an application of a lysozyme nanoflower composite material in inhibiting alicyclobacillus, wherein the minimum inhibitory concentration and the minimum bactericidal concentration of the lysozyme nanoflower composite material for alicyclobacillus are 4.0 μg mL -1 and 63.0 μg mL -1 .

[0007] The preparation method of the lysozyme nanoflower composite material in the technical solution of the present invention comprises the following steps: dissolving lysozyme in a phosphate buffer solution, then adding a copper sulfate aqueous solution, and fully dissolving the mixed solution at room temperature; after incubating the mixed solution at 30°C for 72 hours, centrifuging and collecting the product, washing it with deionized water for multiple times, and freeze-drying it to obtain the lysozyme nanoflower composite material.

[0008] The concentration of lysozyme in the mixed solution of the technical solution of the present invention is not less than 1.0 mg mL -1 .

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses lysozyme as an organic component and copper phosphate as an inorganic component, and adopts a green and mild method to construct a lysozyme nanoflower composite material, and uses it to inhibit the growth of Alicyclobacillus, showing good antibacterial activity and biosafety, and can be used for the contamination control of spoilage microorganisms in fruit juice beverages, thereby extending the shelf life of the product. The present invention evaluates the inhibitory activity of the lysozyme nanoflower composite material on Alicyclobacillus acidoterrestris by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), providing a new idea and method for the pollution control of Alicyclobacillus acidoterrestris. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The microscopic morphology and elemental mapping of lysozyme nanoflowers prepared at different lysozyme concentrations.

[0011] Figure 2 This is the X-ray diffraction pattern of lysozyme nanoflowers.

[0012] Figure 3 This is the full X-ray photoelectron spectrum and partial spectrum of lysozyme nanoflowers.

[0013] Figure 4 This is the inhibitory activity diagram and antibacterial kinetic curve of lysozyme nanoflowers against Alicyclobacillus.

[0014] Figure 5 These are scanning electron microscope images and laser confocal images of Alicyclobacillus after treatment with lysozyme nanoflowers.

[0015] Figure 6 is the hemolysis rate of lysozyme nanoflowers on rabbit red blood cells. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

[0017] Test materials: Alicyclobacillus ( Alicyclobacillus acidocaldarius ) was purchased from the German Collection of Microorganisms, with the collection number: DSM No. 3923; lysozyme was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; copper sulfate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 2% rabbit red blood cells were purchased from Shanghai Yuanye Biotechnology Co., Ltd. AAM medium formula: 2.0 g glucose, 2.00 g yeast extract powder, 1.2 g potassium dihydrogen phosphate, 1.0 g magnesium sulfate heptahydrate, 0.2 g ammonium sulfate, 0.25 g anhydrous calcium chloride, and 1 L distilled water.

[0018] Example 1 Synthesis of lysozyme nanoflower composite material Weigh 6.0 mg, 30.0 mg, and 60.0 mg of lysozyme, respectively, and dissolve them in 30 mL of phosphate buffer solution (10 mM, pH = 7.4) to prepare a concentration of 0.2 mg L -1 , 1.0 mg L -1 and 2.0 mg L -1 After the lysozyme solution was evenly dissolved, 0.2 mL of CuSO 4 (120 mM) aqueous solution and incubated at 30 °C for 72 h. After the incubation, the product was collected by centrifugation and washed several times with deionized water, and freeze-dried to obtain lysozyme nanoflowers Ly@Cu 3 (PO 4 ) 2 The microscopic morphology of lysozyme nanoflowers was observed by scanning electron microscopy, and the qualitative analysis of lysozyme nanoflowers was performed by X-ray photoelectron spectrometer and infrared spectrometer.

[0019] Figure 1 The microscopic morphology and element mapping of lysozyme nanoflowers prepared at different lysozyme concentrations, among which: Figure 1 The lysozyme concentrations corresponding to A and 1D were 0.2 mg L -1 SEM and magnified images; Figure 1 B and 1E correspond to lysozyme concentrations of 1 mg / L -1 SEM and magnified images; Figure 1 The lysozyme concentrations corresponding to C and 1F were 0.2 mg L -1 SEM and magnified images; Figure 1 G is the lysozyme concentration of 0.2 mg L -1 Transmission electron microscopy image of lysozyme nanoflowers; Figure 1 H is the lysozyme concentration of 0.2 mg L -1 Elemental mapping of lysozyme nanoflowers. Figure 1 Disordered nanosheet structures can be observed in A and 1D, indicating that the inorganic component Cu 3 (PO 4 ) 2 The crystal growth rate is slow and cannot form a flower-like structure. Figure 1 As shown in B and 1C, with the continuous increase of lysozyme concentration, the generation of nano-petals can be clearly observed. Figure 1 The lysozyme concentration in C and 1F was 2.0 mg L -1 When Cu 3 (PO 4 ) 2 The crystal nucleation sites are further increased, and a highly ordered and denser flower-like structure can be observed. Figure 1 G shows that the edge of the lysozyme nanoflowers presents an obvious layered structure. Figure 1 It can be seen that C, N, O, S, P and Cu elements are evenly distributed in Ly@Cu 3 (PO 4 ) 2 In, the successful synthesis of lysozyme nanoflowers was demonstrated.

[0020] Figure 2 This is the X-ray diffraction pattern of lysozyme nanoflowers. As shown in the figure, the X-ray diffraction pattern of lysozyme nanoflowers shows Cu at 8.9°, 12.8° and 29.5°. 3 (PO 4 ) 2 •3H 2 O (JCPDS 22-0548), indicating that the Cu 3 (PO 4 ) 2 In contrast, the characteristic peak of lysozyme does not appear in Ly@Cu 3 (PO 4 )2 The X-ray diffraction pattern shows that the introduction of lysozyme has no effect on Cu 3 (PO 4 ) 2 of the crystal structure.

[0021] Figure 3 The X-ray photoelectron full spectrum and sub-spectrum of lysozyme nanoflowers. Figure 3 It can be seen from the full spectrum shown in A that lysozyme nanoflowers contain C, N, O, P and Cu elements, which is consistent with the results given in the transmission electron microscope element mapping diagram. The signal of the S element was not detected, which may be due to the low content of the element. Figure 3 C and Figure 3 F shows the O 1s and Cu 2p spectra of lysozyme nanoflowers, where the two peaks at 530.8 eV and 532.0 eV are attributed to CO / PO and C=O, respectively, indicating that the Cu 3 (PO 4 ) 2 existence. Figure 3 D shows the N 1s spectrum. The peaks at 399.5 eV and 400.0 eV are attributed to CN / NH and NC=O, respectively, indicating that Ly@Cu 3 (PO 4 ) 2 The successful introduction of lysozyme into

[0022] Example 2 Determination of the antibacterial activity of lysozyme nanoflowers against Alicyclobacillus Weigh 2.0 mg of lysozyme nanoflowers and disperse them in AAM medium. Prepare the concentration to 4.0 μg mL by two-fold dilution method. -1 , 8.0 μg mL -1 , 16.0 μg mL -1 , 32.0 μg mL -1 , 63.0 μg mL -1 , 125.0 μg mL -1 , 250.0 μgmL -1 , 500.0 μg mL -1 , 1000.0 μg mL -1 Lysozyme nanoflower solution.

[0023] The lysozyme nanoflower solutions of different concentrations were mixed with 10 4 CFUmL -1The cells of Bacillus alicycloides were mixed and cultured at 45 °C for 24 h. The lowest concentration at which no obvious bacterial growth occurred in the culture medium was the MIC of lysozyme nanoflowers against Bacillus alicycloides. The minimum concentration of lysozyme nanoflowers that inhibited the growth of Bacillus alicycloides was determined to be 4.0 μg mL -1 The samples without turbidity were spread on AAM solid medium and cultured at 45 °C for 24 h. The lowest concentration without obvious colony growth was the MBC of lysozyme nanoflowers against Alicyclobacillus.

[0024] The overnight cultured Alicyclobacillus cells were adjusted to a concentration of about 10 4 CFUmL -1 The samples were incubated with lysozyme nanoflowers at concentrations of 0.5 MIC, MIC, and MBC at 45 °C for 12 h. Samples were taken every 2 h, and the absorbance values ​​(OD) of each group of samples at 600 nm were measured using an ELISA reader. 600 ), not treated with lysozyme nanoflowers A. acidoterrestris As the control group, the antibacterial kinetic curve of lysozyme nanoflowers against Alicyclobacillus was drawn.

[0025] Figure 4 The figure shows the inhibitory activity of lysozyme nanoflowers against Alicyclobacillus and the antibacterial kinetic curve. Figure 4 As shown in A, after 24 h of culture, the concentration of lysozyme nanoflowers was 4.0 μg mL -1 , 8.0 μg mL -1 , 16.0 μg mL -1 and 32.0 μg mL -1 There was obvious bacterial growth in the treatment group. When the treatment concentration of lysozyme nanoflowers was greater than 32.0 μg mL -1 When the concentration of lysozyme nanoflowers was 0.5477 μg / mL, no colonies appeared on the AAM solid medium of each group, indicating that the MBC of lysozyme nanoflowers against Alicyclobacillus was 63.0 μg mL -1 .like Figure 4 As shown in B, the OD of Alicyclobacillus without lysozyme nanoflower treatment 600 The growth trend was shown within 12 h, indicating that the bacterial cells grew normally. In contrast, when the alicyclic acid Bacillus was exposed to 0.5 MIC of Ly@Cu 3 (PO 4 ) 2 After treatment, its OD 600 The value did not increase significantly within 6 h. When the incubation time exceeded 6 h, the OD 600The values ​​increased slightly, but were significantly lower than those of the control group, indicating that the growth of Alicyclobacillus was inhibited at this concentration. When the concentration of lysozyme nanoflowers was further increased to MIC and MBC, the growth of bacterial cells was completely inhibited within 12 h, indicating that Ly@Cu 3 (PO 4 ) 2 It can effectively inhibit the growth of Alicyclobacillus.

[0026] Example 3 Damage of Lysozyme Nanoflowers to Alicyclobacillus Cell Membrane The concentration of activated Alicyclobacillus cells was adjusted to 10 with sterile phosphate buffer solution (PBS, 10 mM, pH 7.4). 6 CFUmL -1 , incubated with lysozyme nanoflowers at concentrations of MIC and MBC at 45 °C for 8 h, and untreated bacterial cells were used as the control group. After incubation, the bacterial cells were collected by centrifugation and dropped on a silicon wafer dedicated to the electron microscope, and placed in a 2.5% glutaraldehyde solution at 4 °C for fixation overnight. Subsequently, the fixed samples were washed three times in PBS (10 min / time), and the samples were washed with gradient concentrations of ethanol solution (30%, 50%, 70%, 80%, 90%). The samples were washed twice with anhydrous ethanol, and then washed once with anhydrous ethanol / tert-butanol mixture (v:v=1:1) and tert-butanol solution (100%), and then the samples were pre-frozen at -20 °C and freeze-dried. The dried bacterial samples were glued to the sample stage, and after gold spraying, they were observed and photographed with a scanning electron microscope. In addition, the bacterial cells in each group of samples were incubated with fluorescent dyes SYTO9 and PI at room temperature in the dark for 15 min, and fluorescent images were taken by laser confocal microscopy.

[0027] Figure 5 The following are scanning electron microscopy and laser confocal microscopy images of Alicyclobacillus treated with lysozyme nanoflowers. Figure 5 As shown in A, the untreated Alicyclobacillus cells are intact, with a smooth surface and no obvious depressions. In contrast, after being treated with lysozyme nanoflowers at a concentration of MIC ( Figure 5 B), the surface of bacterial cells showed obvious depression and shrinkage. When the treatment concentration was further increased to MBC ( Figure 5 C), Alicyclobacillus lost its cell morphology, the bacteria were broken and a large number of holes appeared on the surface, indicating that the bacteria completely lost their vitality. Figure 5 D shows the fluorescence staining images of each group of bacterial samples. It can be seen that the control group presents strong green fluorescence and almost no red fluorescence can be observed, indicating that the bacterial cell membrane has good integrity. 3 (PO 4 )2 As the concentration increases, the red fluorescence intensity gradually increases, indicating that the bacterial cell membrane is severely damaged.

[0028] Example 4 Hemolysis rate of lysozyme nanoflowers on red blood cells Lysozyme nanoflowers with concentrations of MIC, 2MIC, MBC, and 2MBC were mixed with 2% rabbit erythrocytes and incubated at 37°C for 4 h. In addition, phosphate buffer solution (10 mM, pH=7.4) and deionized water were used as negative and positive controls, respectively. After incubation, each group of samples was centrifuged and 100 μL of supernatant was placed in a 96-well plate. The absorbance of each group of samples at 540 nm was measured by an ELISA instrument. Hemolysis rate (%) = (OD value of treatment group - OD value of negative control) / (OD value of positive control - OD value of negative control) × 100.

[0029] Figure 6 is the hemolysis rate of lysozyme nanoflowers on rabbit red blood cells. Figure 6 As shown in the figure, rabbit erythrocytes incubated with deionized water showed obvious damage. In contrast, after 2h and 4h of incubation, the erythrocytes in the MIC, 2MIC, MBC and 2MBC treatment groups showed only slight hemolysis, and the hemolysis effect of erythrocytes in each group was not significantly different from that in the negative control, and the hemolysis rate was less than 5%, indicating that the lysozyme nanoflowers had negligible hemolysis at the treatment concentration and had good biocompatibility.

[0030] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Application of a lysozyme nanoflower composite material in inhibiting alicyclobacillus, characterized in that: The minimum inhibitory concentration and minimum bactericidal concentration of the lysozyme nanoflower composite material against alicyclobacillus were 4.0 μg mL -1 and 63.0 μg mL -1 .

2. The use according to claim 1, characterized in that: The preparation method of the lysozyme nanoflower composite material comprises the following steps: dissolving lysozyme in a phosphate buffer solution, then adding a copper sulfate aqueous solution, and fully dissolving the mixture at room temperature to obtain a mixed solution; incubating the mixed solution at 30° C. for 72 h, collecting the product by centrifugation, washing it with deionized water for multiple times, and freeze-drying it to obtain the lysozyme nanoflower composite material.

3. The use according to claim 2, characterized in that: The concentration of lysozyme in the mixed solution is not less than 1.0 mg mL -1 .

Images