Carbon nanomaterial and application of carbon nanomaterial in high-throughput screening of strains capable of producing reducing substances

Abstract

The invention provides a carbon nanomaterial and application of the carbon nanomaterial in high-throughput screening of strains for producing reducing substances, and belongs to the technical field of high-value utilization of wastes. The invention provides a method for preparing a carbon nanomaterial by using waste residue after fruit vinegar preparation as a raw material, which comprises the following steps: carrying out primary degradation on the waste residue by a physical synergistic enzymolysis process, concentrating primary degradation liquid, inoculating probiotics for fermentation, and carrying out ultrafiltration and enrichment on small molecule degradation liquid; and compounding the micromolecule degradation liquid with an iron ion solution, and then performing irradiation to obtain the carbon nanomaterial. The carbon nanomaterial provided by the invention has high-sensitivity fluorescence response to reducing substances, and realizes high-throughput screening of strains producing the reducing substances. The method disclosed by the invention is simple, convenient and low in cost, does not need to additionally introduce large-scale equipment, and is a novel method capable of screening bacterial strains in a broad-spectrum manner in an industrialization process.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-value utilization of waste, and particularly relates to a carbon nanomaterial and its application in high-throughput screening of strains producing reducing substances. Background Art

[0002] During the fermentation process, strains producing reducing substances can generate various beneficial reducing compounds, such as vitamin C (ascorbic acid), vitamin E (tocopherol), reduced glutathione, polyphenolic compounds, and certain amino acids. These substances have various physiological activities such as antioxidant, anti-aging, and immune-enhancing effects, making them widely used in the fields of medicine, food, and environmental protection. However, there are still some problems in the screening of strains producing reducing substances at present: Firstly, traditional screening methods rely on the reduction of specific substrates by microorganisms, which require a large amount of experimental operations and reagent consumption, with high costs and low screening efficiency; Secondly, current screening methods often cannot accurately distinguish strains producing reducing substances from other strains with similar physiological characteristics, resulting in poor selectivity in strain screening. These problems have become obstacles to the practical application of strains producing reducing substances, seriously hindering the application and popularization of functional reducing components produced by fermentation in actual production.

[0003] Therefore, a new method for high-throughput screening of strains producing reducing substances is needed to make the types and yields of fermented reducing substances more extensive on the basis of improving quality and efficiency. Summary of the Invention

[0004] The present invention provides a carbon nanomaterial and its application in high-throughput screening of strains producing reducing substances, which can be used for high-value utilization of industrial waste and is applicable to high-throughput screening of strains producing reducing substances.

[0005] The present invention provides a preparation method of a carbon nanomaterial, comprising the following steps: (1) Degrading the waste residue after producing fruit vinegar by a method of physical synergistic enzymatic hydrolysis to obtain a primary degradation solution; the enzymes used in the enzymatic hydrolysis include any one or more of the following: pectinase, cellulase, β-glucosidase, lignin peroxidase, compound protease, and laccase;

[0006] (2) Concentrating the primary degradation solution obtained in step (1) and then inoculating probiotics for fermentation, and retaining the filtrate with a molecular weight cut-off less than 3 - 5 kDa to obtain a degradation solution; the probiotics include at least one of the following: Lactobacillus rhamnosus, Streptococcus thermophilus, yeast, probiotic bacillus, Clostridium butyricum, Bifidobacterium, and actinomycetes;

[0007] (3) Mixing the degradation solution obtained in step (2) with an iron ion solution and then performing irradiation to obtain the carbon nanomaterial.

[0008] In one embodiment of the present invention, the fruits used for preparing fruit vinegar in step (1) include at least one of the following: sugarcane, roselle, mulberry, pineapple, apricot, kiwifruit, and apple.

[0009] In one embodiment of the present invention, the method of physical synergistic enzymatic hydrolysis in step (1) includes at least one of the following: ultrasonic synergistic enzymatic degradation method, microwave synergistic enzymatic degradation method, airflow ultrafine grinding synergistic enzymatic degradation method, and high-pressure homogenization synergistic enzymatic degradation method.

[0010] In one embodiment of the present invention, the concentration in step (2) includes concentrating the first degradation solution to 30-80% of the original volume.

[0011] In one embodiment of the present invention, the temperature of the post-concentration fermentation is 20-60 °C, the fermentation time is 24-82 h, and the fermentation pH value is 4-7;

[0012] And the inoculation mass of the bacterial liquid during fermentation is 1-15% of the weight of the concentrated solution.

[0013] In one embodiment of the present invention, the iron ion solution in step (3) includes Fe 3+ ion solution;

[0014] The concentration of the Fe 3+ ion solution is 1-10 mol / L, and the volume ratio of the degradation solution to the Fe 3+ ion solution is (3-10):1.

[0015] In one embodiment of the present invention, the irradiation in step (3) includes X-ray irradiation, the time is 10-60 min, and the energy is 20-60 eV.

[0016] The present invention also provides a carbon nanomaterial prepared by using the above preparation method.

[0017] The present invention also provides a culture medium using the above carbon nanomaterial as a carbon source.

[0018] The present invention also provides the application of the above culture medium in high-throughput screening of strains producing reducing substances.

[0019] Beneficial effects: The present invention provides a method for preparing carbon nanomaterials from the waste residue after fruit vinegar production. The waste residue is first degraded by a physical synergistic enzymatic hydrolysis process, and the first degradation liquid is concentrated and then inoculated with probiotics for fermentation, and the small molecule degradation liquid is enriched by ultrafiltration; the small molecule degradation liquid is compounded with an iron ion solution and then irradiated to obtain the carbon nanomaterials. The present invention performs fermentation after the first degradation, which can maximize the conversion of insoluble substances in the fruit vinegar residue into soluble substances, and can also convert macromolecular substances such as lignin in the fruit vinegar residue into small molecule substances, improving the utilization rate of the fruit vinegar residue and reducing the harm it causes to the environment. The present invention prepares carbon nanomaterials by irradiation, which has the advantages of controllable particle size, good dispersibility, high efficiency, no pollution, and no introduction of chemical reagents.

[0020] The carbon nanomaterials of the present invention have a highly sensitive fluorescence response to reducing substances. After embedding iron ions into the carbon nanomaterial structure, the present invention introduces them into the culture medium. Through the reducing ability of the reducing substances produced by the strain on trivalent iron, the embedded trivalent iron is converted into divalent iron and released from the carbon nanostructure, so that the culture medium shows the characteristic of enhanced fluorescence under a fluorescent lamp. According to the linear relationship between the enhancement ratio of the fluorescence intensity and the yield of reducing substances, the high-throughput screening of strains producing reducing substances is realized. The method of the present invention is simple, low-cost, and does not require the additional introduction of large-scale equipment, and is a new method for broad-spectrum screening of strains in the industrialization process. Description of the Drawings

[0021] Figure 1 Transmission electron micrographs of the carbon nanomaterials of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 4. Detailed Embodiments

[0022] The present invention provides a method for preparing carbon nanomaterials, comprising the following steps: (1) Degrading the waste residue after fruit vinegar production by a physical synergistic enzymatic hydrolysis method to obtain a first degradation liquid; the enzymes used in the enzymatic hydrolysis include any one or more of the following: pectinase, cellulase, β-glucosidase, lignin peroxidase, compound protease, and laccase;

[0023] (2) Concentrating the first degradation liquid obtained in step (1) and then inoculating probiotics for fermentation, and retaining the filtrate with a molecular weight cut-off less than 3-5 kDa to obtain a degradation liquid; the probiotics include at least one of the following: Lactobacillus rhamnosus, Streptococcus thermophilus, yeast, probiotic bacillus, Clostridium butyricum, Bifidobacterium, and Actinomycetes;

[0024] (3) Mixing the degradation liquid obtained in step (2) with an iron ion solution and then irradiating to obtain carbon nanomaterials.

[0025] The present invention uses the waste residue after preparing fruit vinegar as the raw material. In one embodiment, it can be the remaining residue after vinegar production from one or more of sugarcane, roselle, mulberry, pineapple, apricot, kiwifruit and apple. Before degrading the fruit vinegar residue, the present invention can also dry the fruit vinegar residue to a certain solid content, such as drying to a solid content of 30-70% in the examples, more precisely 40-65%, and further precisely 40-55%.

[0026] The present invention degrades the fruit vinegar residue with a solid content by a physical synergistic enzymatic hydrolysis method. The physical synergistic enzymatic hydrolysis method can be any one or a combination of the following: ultrasonic synergistic enzymatic degradation method, microwave synergistic enzymatic degradation method, airflow ultrafine grinding synergistic enzymatic degradation method, high-pressure homogenization synergistic enzymatic degradation method. In one embodiment, it is selected from any one of the following: ultrasonic synergistic enzymatic degradation and airflow ultrafine grinding synergistic enzymatic degradation. When performing the degradation, if ultrasound is involved in the physical method, the ultrasonic frequency is 10-30KHZ, one embodiment is 12-25KHZ, another embodiment is 15-20KHZ, the power is 2500-6000W, one embodiment is 3000-5500W, and in another embodiment is 3000-4000W. The ultrasonic time is 30-90min, one embodiment is 30-80min, and in another embodiment is 45-60min. The ultrasonic temperature is 25-45°C, one embodiment is 25-40°C, and in another embodiment is 25-35°C; if microwave is involved, the microwave frequency is 2.5-5.5GHZ, the power is 3-7.5KW, and the microwave time is 15-45min; if ultrafine grinding is involved, the crushing pressure is set to 1-2.5MPa, one embodiment is 1.2-2.4MPa, another embodiment is 1.5-2MPa, the rotation speed is 1800-3000r / min, one embodiment is 1800-2800r / min, another embodiment is 1800-2000r / min, and the time is 10-45min, one embodiment is 10-35min, another embodiment is 15-30min; if high-pressure homogenization is involved, the high-pressure homogenization pressure is set to 15-45MPa, and the homogenization time is 10-30min.

[0027] While the present invention degrades by physical means, enzymatic hydrolysis is also accompanied. The enzymes used can be at least one of the following: pectinase, cellulase, β-glucosidase, lignin peroxidase, complex protease, and laccase. For example, in one embodiment, the enzyme used is selected from one of the following: pectinase, cellulase, β-glucosidase, and lignin peroxidase. In another embodiment, the enzyme used is selected from one of the following: cellulase and β-glucosidase. The temperature of the enzymatic hydrolysis in the present invention can be 25 to 50 °C, such as 30 to 45 °C in one embodiment and 30 to 40 °C in another embodiment. The pH value of the enzymatic hydrolysis in the present invention is 3 to 6, 3 to 5.5 in one embodiment, and 3 to 4.5 in another embodiment. The time of the enzymatic hydrolysis in the present invention is 6 to 14 h, 8 to 12 h in one embodiment, and 8 to 10 h in another embodiment. When performing the enzymatic hydrolysis in the present invention, the enzyme addition amount is 2.5 to 12% of the residue mass. For example, the addition amount is 3 to 10% in one embodiment and 4 to 8% in another embodiment.

[0028] The present invention concentrates the primary degradation solution obtained by degrading through a physical synergistic enzymatic hydrolysis method, and then inoculates probiotics for fermentation to obtain a small molecule degradation solution with a high content. Before the concentration in the present invention, it also includes enzyme inactivation and centrifugation. The enzyme inactivation can be carried out with reference to the conventional methods in the art, and the present invention does not make any restrictions. The centrifugation in the present invention can remove macromolecular insoluble substances. The centrifugation is set to be centrifuged at 2000 to 6000 r / min for 10 to 60 min. In a specific embodiment, it is centrifuged at 2500 to 5000 r / min for 10 to 45 min, and in another embodiment, it is centrifuged at 2500 to 5400 r / min for 10 to 30 min.

[0029] The present invention concentrates the filtrate after centrifugation. The concentration can be carried out by heating means, such as microwave concentration. In one example, the microwave frequency is set to 1.5 to 6 GHZ, the power is 3 to 9 KW, and the microwave time is 5 to 40 min. In another example, the microwave frequency is set to 2 to 4.5 GHZ, the power is 4 to 8 KW, and the microwave time is 10 to 30 min. In another example, the microwave frequency is set to 2.5 to 4 GHZ, the power is 4 to 6 KW, and the microwave time is 10 to 25 min. Through the concentration in the present invention, the volume of the concentrated solution is microwave concentrated to 30 to 80% of the volume of the primary degradation solution. For example, it is concentrated to 40 to 70% in one example and 50 to 60% in another example.

[0030] In the present invention, probiotics are introduced for fermentation treatment after concentration. The fermented probiotics are one of Lactobacillus rhamnosus, Streptococcus thermophilus, yeast, probiotic bacillus, Clostridium butyricum, Bifidobacterium, and Actinomycetes. The fermentation temperature is 20 - 60 °C, the fermentation time is 24 - 82 h, the fermentation pH value is 4 - 7, and the added mass of the activated bacterial liquid is 1 - 15% of the weight of the concentrated solution. For example, in one embodiment, one of Lactobacillus rhamnosus, Streptococcus thermophilus, Clostridium butyricum, and Bifidobacterium is selected for fermentation, the fermentation temperature is 25 - 50 °C, the fermentation time is 26 - 68 h, the fermentation pH value is 4 - 6, and the added mass of the activated bacterial liquid is 3 - 10% of the weight of the concentrated solution. In another embodiment, one of Lactobacillus rhamnosus, Streptococcus thermophilus, and Bifidobacterium is selected for fermentation, the fermentation temperature is 25 - 45 °C, the fermentation time is 30 - 54 h, the fermentation pH value is 4 - 5.5, and the added mass of the activated bacterial liquid is 5 - 7% of the weight of the concentrated solution.

[0031] After the fermentation of the present invention, purification is carried out. The purification can be carried out by ultrafiltration to obtain a high-content small molecule degradation solution. In the ultrafiltration purification process of the present invention, the molecular weight cut-off of the ultrafiltration membrane is 3 - 5 kDa, the ultrafiltration pressure is 0.04 - 0.2 MPa, the ultrafiltration temperature is 25 - 55 °C, and the added water volume for ultrafiltration is 1 - 6 times the mass of the concentrated solution. For example, in one embodiment, the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration purification process is 3 - 5 kDa, the ultrafiltration pressure is 0.08 - 0.18 MPa, the ultrafiltration temperature is 25 - 50 °C, and the added water volume for ultrafiltration is 1 - 4.5 times the mass of the concentrated solution. In another embodiment, the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration purification process is 3 - 5 kDa, the ultrafiltration pressure is 0.1 - 0.15 MPa, the ultrafiltration temperature is 25 - 45 °C, and the added water volume for ultrafiltration is 2 - 3 times the mass of the concentrated solution.

[0032] The degradation solution obtained after the above purification in the present invention is compounded with an iron ion solution and then irradiated with X-rays to prepare a novel carbon nanomaterial. Before compounding, the degradation solution can also be concentrated to 25 - 70% of the volume of the degradation solution, and the pH value is 2 - 6. For example, in one embodiment, it is concentrated to 30 - 60% of the volume of the degradation solution, and the pH value is 2 - 5. In another embodiment, it is concentrated to 35 - 50% of the volume of the degradation solution, and the pH value is 3 - 4.5. The present invention does not have special limitations on the concentration method. In one specific embodiment, the concentration can be carried out by microwave radiation. The microwave frequency is 2 - 6 GHZ, the power is 3 - 8 KW, and the microwave time is 15 - 90 min. In one embodiment, the microwave frequency is 2 - 4.5 GHZ, the power is 4 - 6.5 KW, and the microwave time is 20 - 60 min. In another embodiment, the microwave frequency is 2.5 - 3.5 GHZ, the power is 4 - 5 KW, and the microwave time is 30 - 45 min.

[0033] The ferric ion solution involved in the compounding of the present invention is a trivalent ferric ion (Fe 3+ ) solution with a concentration of 1-10 mol / L. For example, in one embodiment, the concentration is 2-8 mol / L, and in another embodiment, the concentration is 3-5 mol / L. When compounding in the present invention, the volume ratio of the concentrated degradation liquid to the ferric ion solution is (3-10):1. For example, in one embodiment, the volume ratio is set to (5-8):1, and in another embodiment, it is (5.5-6.5):1.

[0034] The present invention performs irradiation nanonization treatment on the above compounded liquid. The irradiation can be X-ray irradiation. The X-ray radiation time is 10-60 min, and the energy is 20-60 eV. For example, in one embodiment, the X-ray radiation time is 15-45 min, and the energy is 30-50 eV. In another embodiment, the X-ray radiation time is 20-30 min, and the energy is 35-45 eV.

[0035] After the irradiation, the present invention can also perform purification. The purification can be carried out through an ultrafiltration membrane. The molecular weight cut-off of the ultrafiltration membrane for ultrafiltration purification treatment is <3 kDa, the ultrafiltration pressure is 0.05-0.3 MPa, and the amount of water added for ultrafiltration is 0.2-3 times the mass of the liquid after radiation. For example, in one embodiment, the molecular weight cut-off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.1-0.25 MPa, and the amount of water added for ultrafiltration is 1-2.5 times the mass of the liquid after radiation. In another embodiment, the ultrafiltration pressure is set to 0.15-0.2 MPa, and the amount of water added for ultrafiltration is 1.5-2 times the mass of the liquid after radiation.

[0036] The present invention dries the purified liquid after irradiation and ultrafiltration purification, such as obtaining carbon nanomaterials through freeze-drying. The freeze-drying temperature of the freeze-drying in the present invention is -60 to -30 °C, and the freeze-drying time is 12-36 h. In one embodiment, the freeze-drying temperature is set to -50 to -30 °C, and the freeze-drying time is 20-30 h. In another embodiment, the freeze-drying temperature is set to -45 to -30 °C, and the freeze-drying time is 24-28 h.

[0037] The present invention also provides carbon nanomaterials prepared by the above preparation method.

[0038] After embedding trivalent iron into the carbon nanomaterial structure, the present invention introduces it into the culture medium. Through the reducing power of the reducing substances produced by the strain on trivalent iron, the embedded trivalent iron is converted into divalent iron and released from the carbon nanostructure, so that the culture medium shows the characteristic of enhanced fluorescence under a fluorescent lamp. Taking the fluorescence of the carbon nanomaterial culture medium without adding trivalent iron as the initial fluorescence intensity (F 0) The culture medium with ferric ions embedded in carbon nanomaterials shows a phenomenon of fluorescence intensity quenching. After inoculating the strain that produces reducing substances, due to the release of reducing substances by the strain, it shows a steadily increasing trend, and the fluorescence intensity gradually recovers. Taking the fluorescence intensity after 48 hours of inoculation as F, according to the fluorescence recovery rate % = F 0 -F / F 0 * 100% to observe the yield of reducing substances produced, and a high-throughput screening of strains producing reducing substances is achieved based on a recovery rate > 60%.

[0039] The present invention also provides a culture medium using the above carbon nanomaterials as a carbon source.

[0040] The culture medium of the present invention can be one of MRS culture medium, TSA culture medium, and Wilkins-Chalgren anaerobic culture medium; among them, the mass ratio of carbon nanomaterials to the culture medium is m:m = (0.05 - 0.5):1, it can be m:m = (0.1 - 0.45):1, and it can also be m:m = (0.2 - 0.4):1.

[0041] The present invention also provides the application of the above culture medium in the high-throughput screening of strains producing reducing substances.

[0042] The strains producing reducing substances referred to in the present invention can be one of Streptococcus thermophilus, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium, Pediococcus acidilactici, and Oenococcus oeni. For example, in one embodiment, it is used to screen one strain among Streptococcus thermophilus, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium, and in another embodiment, it is used to screen one strain among Streptococcus thermophilus, Lactobacillus rhamnosus, and Bifidobacterium.

[0043] In order to further illustrate the present invention, the following combines examples to describe in detail a carbon nanomaterial provided by the present invention and its application in the high-throughput screening of strains producing reducing substances, but they cannot be understood as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Step 1. Primary degradation of fruit vinegar residue

[0046] Apple fruit vinegar dried to a solid content of 40% is subjected to primary degradation treatment under the action of ultrasonic-assisted enzymatic degradation. The ultrasonic frequency is 15 KHZ, the power is 3500 W, and the ultrasonic time is 30 min. After the ultrasonic treatment, it is cooled to room temperature, and β-glucosidase is introduced into the above solution, the pH is adjusted to 4, and the reaction is carried out at 35 °C for 10 h. The added mass of β-glucosidase during this enzymatic hydrolysis process is 5% of the dry weight of the residue.

[0047] Step 2. Post-fermentation treatment of the primary degradation solution

[0048] Centrifuge the first degradation solution prepared in Step 1 to remove insoluble substances (centrifugation speed: 2500 r / min, centrifugation time: 45 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 3 GHz, the power is 4 KW, and the microwave time is 20 min. After concentrating to 60% of the first degradation solution and cooling to room temperature, inoculate the activated probiotic Lactobacillus rhamnosus for fermentation. The fermentation broth is intercepted by an ultrafiltration membrane with a molecular weight cut-off of 3 - 5 kDa, and finally a fermentation broth with a high content of small molecules is obtained.

[0049] Among them, the fermentation temperature is 30 °C, the fermentation time is 36 h, the fermentation pH is 4.0, and the inoculum size of the strain is 5% of the weight of the concentrated solution; the ultrafiltration pressure is 0.1 MPa, the ultrafiltration temperature is 30 °C, and the amount of water added during ultrafiltration is 2 times the mass of the concentrated solution.

[0050] Step 3: Preparation of a novel carbon nanomaterial by X-ray irradiation

[0051] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 4, and concentrate it to 60% of the original volume under microwave irradiation (microwave frequency: 2 GHz, power: 4 KW, microwave time: 60 min). After concentration, add a 3 mol / L ferric ion solution, and the volume ratio between the two is 5:1. Stir for 15 min and then irradiate with 30 eV X-rays for 30 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut-off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.15 MPa, and the amount of water added during ultrafiltration is 1.5 times the mass of the irradiated liquid; the ultrafiltered liquid is processed by freeze-drying, with a freeze-drying temperature of -50 °C and a freeze-drying time of 20 h.

[0052] Step 4: Preparation of the culture medium

[0053] Re-compound the freeze-dried carbon nanomaterial powder with MRS medium (the mass ratio of carbon nanomaterial to medium is m:m = 0.2:1), and use it for high-throughput screening of Streptococcus thermophilus strains that produce reducing substances.

[0054] Example 2

[0055] Step 1: Primary degradation of fruit vinegar residue

[0056] Perform primary degradation on apple fruit vinegar dried to a solid content of 45% under the action of ultrasonic-assisted enzymatic degradation. The ultrasonic frequency is 18 KHz, the power is 3800 W, and the ultrasonic time is 45 min. After ultrasonic treatment, cool to room temperature, and introduce cellulase into the above solution, adjust the pH to 4.2 and react at 38 °C for 8.5 h. The added mass of cellulase during this enzymatic hydrolysis process is 6% of the dry weight of the residue.

[0057] Step 2: Post-fermentation treatment of the primary degradation solution

[0058] Centrifuge the first degradation solution prepared in Step 1 to remove insoluble substances (centrifugation speed: 3000 r / min, centrifugation time: 15 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 3.5 GHz, the power is 5 KW, and the microwave time is 15 min. After concentrating to 55% of the first degradation solution and cooling to room temperature, inoculate the activated probiotic Streptococcus thermophilus for fermentation. The fermentation broth is intercepted by a 3 - 5 kDa ultrafiltration membrane to finally obtain a fermentation broth with a high content of small and medium molecules.

[0059] Among them, the fermentation temperature is 40 °C, the fermentation time is 40 h, the fermentation pH is 4.5, and the inoculum size of the strain is 5.5% of the weight of the concentrated solution; the ultrafiltration pressure is 0.12 MPa, the ultrafiltration temperature is 35 °C, and the water addition amount during ultrafiltration is 2.5 times the mass of the concentrated solution.

[0060] Step 3: Preparation of a novel carbon nanomaterial by X - ray irradiation

[0061] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 3.5, and concentrate it to 35% of the original volume under microwave irradiation (microwave frequency: 3 GHz, power: 4.5 KW, microwave time: 35 min). After concentration, add a 3.5 mol / L ferric ion solution, and the volume ratio between the two is 5.8:1. Stir for 25 min and then irradiate with 35 eV X - rays for 30 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut - off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.18 MPa, and the water addition amount during ultrafiltration is 1.8 times the mass of the liquid after irradiation; the liquid after ultrafiltration is processed by freeze - drying, the freeze - drying temperature is - 45 °C, and the freeze - drying time is 24 h;

[0062] Step 4: Preparation of the culture medium

[0063] Re - compound the freeze - dried carbon nanomaterial powder with TSA medium (the mass ratio of carbon nanomaterial to medium is m:m = 0.25:1), and use it for the high - throughput screening of Lactobacillus rhamnosus strains that produce reducing substances.

[0064] Example 3

[0065] Step 1: Primary degradation of fruit vinegar residue

[0066] Perform primary degradation on pineapple fruit vinegar dried to a solid content of 60% under the assistance of ultrasonic - assisted enzymatic degradation. The ultrasonic frequency is 22 KHz, the power is 5000 W, and the ultrasonic time is 60 min. After ultrasonic treatment, cool to room temperature, introduce pectinase into the above solution, adjust the pH to 5, and react at 40 °C for 11 h. The added mass of pectinase during this enzymatic hydrolysis process is 8% of the dry weight of the residue.

[0067] Step 2: Post - fermentation treatment of the primary degradation solution

[0068] Centrifuge the primary degradation solution prepared in Step 1 to remove insoluble substances (centrifugation speed: 4500 r / min, centrifugation time: 20 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 4 GHz, the power is 6 KW, and the microwave time is 28 min. After concentrating to 65% of the primary degradation solution and cooling to room temperature, inoculate the activated probiotic Clostridium butyricum for fermentation. The fermentation broth is intercepted by a 3 - 5 kDa ultrafiltration membrane to finally obtain a fermentation broth with a high content of small and medium molecules.

[0069] Among them, the fermentation temperature is 45 °C, the fermentation time is 65 h, the fermentation pH is 5.0, and the inoculation amount of the strain is 8% of the weight of the concentrated solution; the ultrafiltration pressure is 0.16 MPa, the ultrafiltration temperature is 40 °C, and the water addition amount during ultrafiltration is 3 times the mass of the concentrated solution.

[0070] Step 3: Preparation of a novel carbon nanomaterial by X - ray irradiation

[0071] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 5, and concentrate it to 40% of the original volume under microwave irradiation (microwave frequency: 3 GHz, power: 6 KW, microwave time: 25 min). After concentration, add a 7 mol / L ferric ion solution, and the volume ratio between the two is 6:1. Stir for 15 min and then irradiate with 40 eV X - rays for 25 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut - off of the ultrafiltration membrane is successively <3 kDa, the ultrafiltration pressure is 0.20 MPa, and the water addition amount during ultrafiltration is 2 times the mass of the irradiated liquid; the ultrafiltered liquid is processed by freeze - drying, the freeze - drying temperature is - 45 °C, and the freeze - drying time is 28 h;

[0072] Step 4: Preparation of the culture medium

[0073] Compound the freeze - dried carbon nanomaterial powder with TSA medium (the mass ratio of carbon nanomaterial to medium is m:m = 0.25:1), and use it for the high - throughput screening of Bifidobacterium lactis strains producing reducing substances.

[0074] Example 4

[0075] Step 1: Primary degradation of fruit vinegar residue

[0076] Perform primary degradation on kiwifruit vinegar dried to a solid content of 48% under the assistance of ultrasonic - assisted enzymatic degradation. The ultrasonic frequency is 16 KHz, the power is 3200 W, and the ultrasonic time is 55 min. After ultrasonic treatment, cool to room temperature, and introduce β - glucosidase into the above solution, adjust the pH to 3.5 and react at 32 °C for 9 h. The added mass of β - glucosidase during this enzymatic hydrolysis process is 4.2% of the dry weight of the residue.

[0077] Step 2: Post - fermentation treatment of the primary degradation solution

[0078] Centrifuge the first degradation solution prepared in Step 1 to remove insoluble substances (centrifugation speed: 3600 r / min, centrifugation time: 26 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 2.7 GHz, the power is 4.5 KW, and the microwave time is 23 min. After concentrating to 52% of the first degradation solution and cooling to room temperature, inoculate the activated probiotic Bifidobacterium for fermentation. The fermentation broth is intercepted by an ultrafiltration membrane with a molecular weight cut-off of 3 - 5 kDa, and finally a fermentation broth with a high content of small and large molecules is obtained.

[0079] Among them, the fermentation temperature is 32 °C, the fermentation time is 48 h, the fermentation pH is 4.2, and the inoculum size of the strain is 6.2% of the weight of the concentrated solution; the ultrafiltration pressure is 0.13 MPa, the ultrafiltration temperature is 28 °C, and the water addition amount during ultrafiltration is 2.2 times the mass of the concentrated solution.

[0080] Step 3: Prepare a novel carbon nanomaterial by X-ray irradiation

[0081] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 3, and concentrate it to 42% of the original volume under microwave irradiation (microwave frequency: 2.6 GHz, power: 4.8 KW, microwave time: 40 min). After concentration, add a 4.6 mol / L ferric ion solution, and the volume ratio between the two is 6.2:1. Stir for 15 min and then irradiate with 42 eV X-rays for 28 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut-off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.16 MPa, and the water addition amount during ultrafiltration is 1.7 times the mass of the liquid after irradiation; the liquid after ultrafiltration is processed by freeze-drying, the freeze-drying temperature is -35 °C, and the freeze-drying time is 26 h.

[0082] Step 4: Prepare the culture medium

[0083] Re-compound the freeze-dried carbon nanomaterial powder with MRS culture medium (the mass ratio of carbon nanomaterial to culture medium is m:m = 0.3:1), and use it for high-throughput screening of Streptococcus thermophilus strains that produce reducing substances.

[0084] Example 5

[0085] Step 1: Primary degradation of fruit vinegar residue

[0086] Perform primary degradation treatment on Rosa roxburghii fruit vinegar dried to a solid content of 65% under the assistance of ultrasonic wave and enzymatic degradation. The ultrasonic frequency is 25 KHz, the power is 5500 W, and the ultrasonic time is 80 min. After ultrasonic treatment, cool to room temperature, introduce lignin peroxidase into the above solution, adjust the pH to 5.5, and react at 45 °C for 12 h. The added mass of lignin peroxidase during this enzymatic hydrolysis process is 10% of the dry weight of the residue.

[0087] Step 2. Post-fermentation treatment of the primary degradation liquid

[0088] Centrifuge the primary degradation liquid prepared in Step 1 to remove insoluble substances (centrifugation speed: 3800 r / min, centrifugation time: 15 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 2 GHz, the power is 8 KW, and the microwave time is 30 min. After concentrating to 70% of the primary degradation liquid and cooling to room temperature, inoculate the activated probiotic Bifidobacterium for fermentation treatment. The fermentation broth is intercepted by a 3 - 5 kDa ultrafiltration membrane to finally obtain a fermentation broth with a high content of small and large molecules.

[0089] Among them, the fermentation temperature is 50 °C, the fermentation time is 48 h, the fermentation pH is 4.5, and the inoculation amount of the strain is 10% of the weight of the concentrated liquid; the ultrafiltration pressure is 0.08 MPa, the ultrafiltration temperature is 50 °C, and the water addition amount during ultrafiltration is 4 times the mass of the concentrated liquid.

[0090] Step 3. Preparation of novel carbon nanomaterials by X-ray irradiation

[0091] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 2, and concentrate it to 50% of the original volume under microwave irradiation (microwave frequency: 4.5 GHz, power: 6.5 KW, microwave time: 40 min). After concentration, add a 5 mol / L ferric ion solution, and the volume ratio between the two is 8:1. Stir for 15 min and then irradiate with 50 eV X-rays for 45 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut-off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.25 MPa, and the water addition amount during ultrafiltration is 2.5 times the mass of the liquid after irradiation; the liquid after ultrafiltration is processed by freeze-drying, the freeze-drying temperature is -36 °C, and the freeze-drying time is 24 h;

[0092] Step 4. Preparation of the culture medium

[0093] Re-compound the freeze-dried carbon nanomaterial powder with MRS culture medium (the mass ratio of carbon nanomaterial to culture medium is m:m = 0.35:1), and use it for high-throughput screening of Lactobacillus strains producing reducing substances.

[0094] Example 6

[0095] Step 1. Primary degradation of fruit vinegar residue

[0096] Perform primary degradation treatment on kiwifruit vinegar dried to a solid content of 52% under the action of ultrasonic-assisted enzymatic degradation. The ultrasonic frequency is 20 KHz, the power is 4000 W, and the ultrasonic time is 60 min. After ultrasonic treatment, cool to room temperature, and introduce β-glucosidase into the above solution, adjust the pH to 4 and react at 40 °C for 9.5 h. The added mass of β-glucosidase during this enzymatic hydrolysis process is 7.5% of the dry weight of the residue.

[0097] Step 2. Post-fermentation treatment of the primary degradation liquid

[0098] Centrifuge the primary degradation liquid prepared in Step 1 to remove insoluble substances (centrifugation speed: 2,800 r / min, centrifugation time: 30 min). After centrifugation, concentrate it through a microwave dehydration process, where the microwave frequency is 4 GHz, the power is 6 kW, and the microwave time is 18 min. After concentrating to 50% of the primary degradation liquid and cooling to room temperature, inoculate the activated probiotic Lactobacillus rhamnosus for fermentation treatment. The fermentation broth is intercepted by a 3 - 5 kDa ultrafiltration membrane, and finally a fermentation broth with a high content of small molecules is obtained.

[0099] Among them, the fermentation temperature is 45°C, the fermentation time is 54 h, the fermentation pH is 5.5, and the inoculation amount of the strain is 6.8% of the weight of the concentrated liquid; the ultrafiltration pressure is 0.15 MPa, the ultrafiltration temperature is 45°C, and the water addition amount during ultrafiltration is 2.8 times the mass of the concentrated liquid.

[0100] Step 3. Preparation of novel carbon nanomaterials by X-ray irradiation

[0101] Adjust the pH of the fermentation ultrafiltrate obtained in Step 2 to 3.5, and concentrate it to 48% of the original volume under microwave irradiation (microwave frequency: 3.5 GHz, power: 5 kW, microwave time: 45 min). After concentration, add a 5 mol / L ferric ion solution, and the volume ratio between the two is 6.5:1. Stir for 15 min and then irradiate with 45 eV X-rays for 20 min. After irradiation, perform ultrafiltration purification on the solution. The molecular weight cut-off of the ultrafiltration membrane is <3 kDa, the ultrafiltration pressure is 0.2 MPa, and the water addition amount during ultrafiltration is 2 times the mass of the liquid after irradiation; the liquid after ultrafiltration is processed by freeze-drying, with a freeze-drying temperature of -30°C and a freeze-drying time of 28 h;

[0102] Step 4. Preparation of the culture medium

[0103] Compound the freeze-dried carbon nanomaterial powder with TSA culture medium (mass ratio of carbon nanomaterial to culture medium m:m = 0.4:1), and use it for high-throughput screening of Streptococcus thermophilus strains that produce reducing substances.

[0104] Comparative Example 1

[0105] Compared with Example 1, except that ferric ions were not added during irradiation, the other operations were the same as those in Example 1.

[0106] Comparative Example 2

[0107] Compared with Example 1, except that the post-fermentation treatment of the primary degradation liquid was not carried out in Step 2, the other operations were the same as those in Example 1.

[0108] Comparative Example 3

[0109] Compared with Example 1, except that the strain in Step 4 is Bacillus subtilis, the remaining operations are the same as those in Example 1.

[0110] Comparative Example 4

[0111] Compared with Example 1, except that the hydrothermal method at 100 °C and a pressure of 0.3 MPa is used to replace the X-ray irradiation treatment, the remaining operations are the same as those in Example 1.

[0112] The results are as Figure 1 shown in Table 1. The particle size of the carbon nanomaterials obtained in each example is about 4 - 10.5 nm. The small-particle-size nanomaterials have the advantage of being more easily penetrated into the interior of the bacterial cells compared with the large-particle size in the comparative examples, and are more suitable for the high-throughput screening of strains. The fruit vinegar residue is rich in various substances such as polyphenols, aromatic rings, and heterocycles. After X-ray irradiation, the above substances will polymerize to form a spherical structure in the shape of a circle, and the spherical structure shows that it contains a large number of active functional groups such as carboxyl, hydroxyl, and amino groups, and has significant fluorescence characteristics. The introduction of trivalent iron will be fixed on the surface of the nanospheres by these active functional groups during the formation process of the nanospheres, resulting in the phenomenon of fluorescence quenching. As can be seen from Table 1, with the increase of the fermentation time, the release amount of reducing substances increases steadily, which is beneficial to the reduction of trivalent iron inside the carbon nanomaterials, thereby restoring the fluorescence of the carbon nanomaterials. The more the fluorescence intensity is restored, the higher the content of reducing substances produced by the strains. According to this property, the high-throughput screening of strains producing reducing substances can be realized (the fluorescence recovery rate can reach more than 60%).

[0113] Table 1 Changes in the particle size of carbon nanomaterials and fluorescence intensity before and after inoculation

[0114]

[0115]

[0116] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to these embodiments without creative work, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for preparing a carbon nanomaterial, characterized in that: The following steps are involved: (1) degrading the waste residue after preparing fruit vinegar by a physical synergistic enzymatic hydrolysis method to obtain a first degradation liquid; the enzyme used in the enzymatic hydrolysis comprises any one or more of the following: pectinase, cellulase, β-glucosidase, lignin peroxidase, composite protease and laccase; (2) concentrating the first degradation liquid of step (1) and inoculating probiotics for fermentation, and retaining the filtrate with a molecular weight of less than 3 to 5 kDa to obtain a degradation liquid; the probiotics include at least one of the following: Lactobacillus rhamnosus, Streptococcus thermophilus, yeast, probiotic spore bacteria, Clostridium butyricum, Bifidobacterium and Actinomycetes; (3) The degradation solution obtained in step (2) is mixed with the iron ion solution and then irradiated to obtain a carbon nanomaterial.

2. The preparation method according to claim 1, characterized in that: The fruit used for preparing the fruit vinegar in step (1) comprises at least one of the following: sugar cane, roxburgh, mulberry, pineapple, apricot, kiwi and apple.

3. The preparation method according to claim 1, characterized in that: The physical synergistic enzymatic degradation method in step (1) includes at least one of the following: ultrasound synergistic enzymatic degradation method, microwave synergistic enzymatic degradation method, airflow ultrafine grinding synergistic enzymatic degradation method and high-pressure homogenization synergistic enzymatic degradation method.

4. The preparation method according to claim 1, characterized in that: The concentration in step (2) includes concentrating the first degradation liquid to 30-80% of the original volume.

5. The preparation method according to claim 4, characterized in that: The post-concentration fermentation temperature is 20-60°C, the fermentation time is 24-82h, and the fermentation pH value is 4-7; During fermentation, the inoculation mass of the bacterial liquid is 1 to 15% of the weight of the concentrated liquid.

6. The preparation method according to claim 1, characterized in that: The iron ion solution in step (3) includes Fe 3+ Ionic solution; The Fe 3+ The concentration of the ion solution is 1-10 mol / L, and the degradation solution is 3+ The volume ratio of the ion solution is (3-10):

1.

7. The preparation method according to claim 1 or 6, characterized in that: The irradiation in step (3) includes X-ray irradiation, the time is 10 to 60 minutes, and the energy is 20 to 60 eV.

8. The carbon nanomaterial prepared by the preparation method according to any one of claims 1 to 7.

9. A culture medium using the carbon nanomaterial according to claim 8 as a carbon source.

10. Use of the culture medium according to claim 9 in high-throughput screening of reducing substance-producing strains.

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