Plant compound enzyme formula for confined space and preparation method thereof

Abstract

The invention discloses a plant compound enzyme formula for a confined space and a preparation method thereof, and belongs to a compound enzyme production process. The plant compound enzyme formula for a confined space is composed of the following components in parts by weight: 30-40 parts of a fermentation mixture, 4-8 parts of an enzyme protectant, and 2-6 parts of a nanocomposite, wherein the fermentation mixture is composed of bromelain, papain, catalase, peroxidase, serine protease, trypsin, hesperidinase, wormwood extract, rosemary extract, echinacea polyphenols, chamomile extract, diatomaceous earth, glucose and pure water, the enzyme protectant is prepared from corn stalks, tetramethyl piperidine oxide, kelp dry powder and ethanol, and the nanocomposite is prepared from phospholipids, cholesterol, Tween 80 and ε-polylysine. The plant compound enzyme formula obtained by the method has excellent purification efficiency and lasting purification time.

Description

Technical Field

[0001] The invention belongs to the technical field of compound enzyme formula preparation, and in particular relates to a plant compound enzyme formula special for a confined space and a preparation method thereof. Background Art

[0002] With the development of economy and the acceleration of urbanization, the problem of air pollution in confined spaces has become increasingly prominent. The presence of gaseous pollutants such as formaldehyde, benzene, nitrogen, TVOC, etc. poses a serious threat to people's health. At present, the methods for controlling air pollution in confined spaces mainly include chemical reaction methods, physical adsorption technology, nano-photocatalyst catalysis technology, plant purification and biodegradation. However, these methods have certain limitations, such as easy to cause secondary pollution, high energy consumption, unstable performance, etc. As an emerging treatment method, bioenzyme has the advantages of safety, environmental protection and high efficiency. Bioenzyme can efficiently catalyze various biochemical reactions under mild conditions and promote the metabolism of organisms. In the field of air purification, bioenzyme technology has been proven to effectively degrade pollutants in the air, such as formaldehyde, benzene, etc.

[0003] Patent CN 114904384 B discloses an air purifier containing biological enzymes and a preparation method thereof. The air purifier containing biological enzymes comprises: 15 parts of active biological enzymes, 5 parts of amino compounds, 24 parts of polymer deodorants, 4.5 parts of polysaccharides, 3 parts of whey protein peptides, and 1500 parts of water. The air purifier uses biological enzymes, polypeptides, polysaccharides, amino acids, etc. as active substances, can purify the air without photocatalysis, has no corrosive oxidation effect on metals, plants, leather and other products, and has a fast purification speed. However, when the substance is used in a confined space, there is still room for improvement in the degradation durability and purification efficiency of pollutants, and there is also room for improvement in the production efficiency of preparing purification substances by composite enzymes. Summary of the invention

[0004] The purpose of the present invention is to provide a plant complex enzyme formula for confined spaces and a preparation method thereof, so as to solve the technical problems in the prior art of using plant complex enzymes to prepare purification materials with low production efficiency and poor degradation durability and purification efficiency of pollutants by plant complex enzymes.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention provides a plant compound enzyme formula specially used for a closed space, which is composed of the following components in parts by weight: 30-40 parts of a fermentation mixture, 4-8 parts of an enzyme protectant, and 2-6 parts of a nanocomposite, wherein the fermentation mixture is composed of 2-4% of bromelain, 1-5% of papain, 2-4% of catalase, 1-3% of peroxidase, 1-3% of serine protease, 0.5-1.5% of trypsin, 1-3% of hesperidinase, 0.5-3.5% of wormwood extract, 1-3% of rosemary extract, 0.5-1.5% of echinacea polyphenols, 1-3% of chamomile extract, 5-15% of diatomaceous earth, 0.5-3.5% of glucose and 45-85% of purified water, the enzyme protectant is prepared from corn stalks, tetramethyl piperidine oxide, kelp dry powder and ethanol, and the nanocomposite is prepared from phospholipids, cholesterol, Tween 80 and ε-polylysine.

[0007] Preferably, the preparation process of the enzyme protecting agent comprises the following steps:

[0008] Q1: The corn stalks are dried, crushed, soaked, and then placed in a deflaker for deflaking, and then placed in a PFI pulper for refining to obtain fiber slurry;

[0009] Q2: Sodium carbonate and sodium bicarbonate are added to distilled water and mixed and stirred to obtain a mixed buffer solution, tetramethyl piperidine oxide and sodium bromide are added to the mixed buffer solution and stirred until completely dissolved to obtain a composite buffer solution, and then the fiber slurry is added to the composite buffer solution and stirred, and a sodium hypochlorite solution is added dropwise during the stirring period. After the stirring is completed, ethanol is added to terminate the reaction, and the reaction solution is treated by dialysis until the pH is neutral to obtain a suspension;

[0010] Q3: Weigh the dried kelp powder and put it into a reactor, then add ethanol, heat and extract, repeat the operation, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add distilled water to heat and dissolve the crystals, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent;

[0011] Q4: A freeze-drying protective agent is added to the suspension, and magnetic stirring is performed. After high-pressure homogenization, a composite aerogel is obtained. The composite aerogel is pre-frozen and placed in a freeze dryer for freeze drying to obtain an enzyme protective agent.

[0012] In the above process, the corn stalks are dried, crushed and soaked, and then the stalk fibers are further dispersed and refined by mechanical action to obtain fiber slurry. Then, a buffer mixture composed of sodium carbonate and sodium bicarbonate provides a stable acid-base environment for subsequent reactions. Tetramethylpiperidinyl oxide is used as an oxidant to selectively oxidize the primary alcohol hydroxyl groups in the fiber slurry to generate carboxyl groups. Then, kelp dry powder is used as a raw material, and a freeze-dried protective agent is obtained through extraction, concentration, low-temperature crystallization, repeated recrystallization and purification. The freeze-dried protective agent is added to the suspension, and after high-pressure homogenization and freeze-drying, an enzyme protective agent is obtained.

[0013] Preferably, in Q1, the drying temperature is 60-80°C, the time is 10-14h, the soaking is 4-6h, the disintegration is 10-20min, and the refining speed is 30000-35000rpm.

[0014] Preferably, in Q2, the molar ratio of sodium carbonate, sodium bicarbonate and distilled water is (6.5-7.5):(2.5-3.5):(30-35), the dosage ratio of tetramethyl piperidine oxide, sodium bromide and mixed buffer is (0.1-0.22) g:(0.5-1.5) g:(550-650) mL, the volume ratio of fiber slurry, composite buffer and ethanol is (20-40):(10-30):(50-55), the weight fraction of sodium hypochlorite solution is 5wt%, and the stirring time is 60-90 min.

[0015] Preferably, in Q3, the dosage ratio of kelp powder and ethanol is (1-2) kg: (40-80) L, the volume fraction of ethanol is 90vt%, the heating extraction temperature is 80-90°C, the time is 1-2h, the operation is repeated 3-4 times, the dosage ratio of crystals to distilled water is 1g: (0.7-0.9) mL, the heating dissolution temperature is 90-100°C, and the time is 5-10min; in Q4, the dosage ratio of suspension and lyophilization protective agent is (20-30) mL: (0.2-0.3) g, the magnetic stirring time is 2-3h, the pressure of high-pressure homogenization treatment is 50-60MPa, the pre-freezing temperature is -20 to -80°C, and the freeze-drying time is 48-52h.

[0016] Preferably, the method for preparing the nanocomposite comprises the following steps:

[0017] S1: Dissolve phospholipids and cholesterol in anhydrous ethanol, add Tween 80, dissolve by ultrasonic, evaporate by rotary evaporation, and dry to obtain a lipid film;

[0018] S2: dissolving ε-polylysine in phosphate buffer to obtain an ε-polylysine solution, adding the lipid film into the ε-polylysine solution for shaking washing, stirring, ultrasonication, and filtering to obtain a nanocomplex.

[0019] In the above process, phospholipids, cholesterol, Tween 80 and ε-polylysine were used as raw materials, and the nanocomposite was prepared by thin film dispersion combined with ultrasound method.

[0020] Preferably, in S1, the dosage ratio of phospholipid, cholesterol, anhydrous ethanol and Tween 80 is (0.1-0.3) g: (0.05-0.10) g: (30-50) mL: (0.06-0.10) g, the rotary evaporation speed is 60-80 rpm, the temperature is 50-60°C, and the time is 5-8 min; in S2, the dosage ratio of ε-polylysine to phosphate buffer is (0.2-0.3) mg: (1-2) mL, the dosage ratio of lipid film and ε-polylysine solution is (0.2-0.4) g: (5-10) mL, the temperature of oscillation washing is 60-80°C, the time is 1-3 min, the stirring time is 30-40 min, and a 0.22 μm water filter membrane is used during the filtration process.

[0021] Preferably, the method for preparing the confined space-specific plant complex enzyme formula comprises the following steps:

[0022] Step (1): placing the fermentation mixture and the enzyme protectant into a fermentation tank, stirring, heating at 39-42° C. for reaction for 34-36 hours, and then ventilating the fermentation tank with oxygen for 18-22 minutes;

[0023] Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37-38° C., and the fermentation liquid is ventilated with oxygen for 18-22 minutes at intervals of 11-12 hours, and stirred for 8-12 minutes. Before stirring, the dissolved oxygen content and pH value of the fermentation liquid are tested, and the pH value of the fermentation liquid is always maintained between 5-6.5;

[0024] Step (3): fermenting the fermented liquid for 30-32 days, and then precipitating the fermented liquid for 10-12 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. The present invention firstly uses corn stalks, sodium carbonate, sodium bicarbonate, tetramethyl piperidine oxide, sodium bromide and kelp dry powder as raw materials to prepare enzyme protectants, and then uses phospholipids, cholesterol, Tween 80 and ε-polylysine as raw materials to prepare nanocomposites. The enzyme protectants are added into the enzyme fermentation process to improve the production efficiency of the composite enzyme in producing purified substances. The addition of the nanocomposites can effectively improve the degradation durability and purification efficiency of the plant composite enzymes on pollutants.

[0027] 2. The present invention uses corn stalks, tetramethyl piperidine oxide and kelp powder as raw materials, prepares enzyme protectants by freeze drying, and adds the prepared enzyme protectants to the fermentation process, which can effectively improve enzyme activity and stability, thereby improving fermentation efficiency. The porous structure contained in the enzyme protectant can provide a stable microenvironment, so that the enzyme molecules are not easily directly affected by the external environment, and can also allow the enzyme molecules to move freely inside the protectant, reducing contact with harmful substances, reducing the risk of enzyme inactivation, and the components in the enzyme protectant can form hydrogen bonds or electrostatic effects with the enzyme molecules, stabilizing the conformation of the enzyme, and reducing its denaturation or degradation; the presence of the enzyme protectant can also stabilize the pH value of the reaction system, provide a suitable environment for the enzyme, and the presence of the porous structure helps to maintain the relative stability of the temperature, avoiding the adverse effects of too high or too low temperature on the enzyme activity; at the same time, the porous structure of the enzyme protectant allows the substrate molecules to enter freely and combine with the enzyme molecules, promotes the catalytic reaction, and also reduces the diffusion resistance of the substrate molecules in the solution, and improves the substrate utilization rate and reaction rate.

[0028] 3. The present invention uses phospholipids, cholesterol, Tween 80 and ε-polylysine as raw materials to prepare nanocomplexes, which are added as additives to the supernatant obtained by fermentation, which can effectively improve the removal efficiency and degradation durability of the supernatant for pollutants. The nanocomplexes can encapsulate the active substances in the supernatant through a special phospholipid bilayer biomembrane structure, thereby improving the bioavailability and enabling the purification components in the supernatant to be more effectively utilized. The composite nanomaterials have good targeting and can accurately deliver the purification components to the location of the pollutants, thereby improving the purification efficiency. The nanocomplexes can be used as sustained-release carriers to slowly release the purification components into the environment, maintaining a long-term purification effect. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The following embodiments of the present invention are used:

[0031] Corn stalks were purchased from Linghang Family Farm in Xincai County;

[0032] Sodium carbonate was purchased from Henan Yingyuan Environmental Protection Equipment Manufacturing Co., Ltd., CAS number: 497-19-8;

[0033] Sodium bicarbonate was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., CAS number: 144-55-8;

[0034] Tetramethylpiperidinyl oxide was purchased from Wuhan Kemik Biopharmaceutical Technology Co., Ltd., CAS number: 2564-83-2;

[0035] Sodium bromide was purchased from Weifang Youlan Chemical Co., Ltd., CAS No.: 7647-15-6;

[0036] Sodium hypochlorite was purchased from Jinan Boyang Chemical Co., Ltd., CAS number: 7681-52-9;

[0037] Ethanol was purchased from Shandong Huayu Chemical Technology Co., Ltd., CAS No.: 64-17-5;

[0038] The kelp was purchased from the Huazhen Kelp Processing Factory in Lidao Town, Rongcheng City;

[0039] Phospholipids were purchased from Shaanxi Rankang Biotechnology Co., Ltd., CAS number: 123465-35-0;

[0040] Cholesterol was purchased from Chengdu Yuanda Chemical Co., Ltd., CAS No.: 57-88-5;

[0041] Tween 80 was purchased from Xingtai Xinlanxing Technology Co., Ltd., CAS No.: 9005-65-6;

[0042] ε-Poly-lysine was purchased from Hebei Kelongduo Biotechnology Co., Ltd., CAS number: 28211-04-3;

[0043] Phosphate buffer was purchased from Shenzhen Comma Biotechnology Co., Ltd.;

[0044] Bromelain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., CAS number: 9001-00-7;

[0045] Papain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., CAS No.: 9001-73-4;

[0046] Catalase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., CAS number: 9001-05-2;

[0047] Peroxidase was purchased from Beijing Solebow Technology Co., Ltd., CAS number: 9003-99-0;

[0048] Serine protease was purchased from Anhui Runtai Biotechnology Co., Ltd., CAS number: 37259-58-8;

[0049] Trypsin was purchased from Beijing Solebow Technology Co., Ltd., CAS number: 9002-07-7;

[0050] Hesperidinase was purchased from Yancheng Runyu Biotechnology Co., Ltd.;

[0051] Artemisia annua extract was purchased from Xi'an Anao Biotechnology Co., Ltd.;

[0052] Rosemary extract was purchased from Hunan Langlin Biological Resources Co., Ltd.;

[0053] Echinacea polyphenols were purchased from Shaanxi Snotech Biotechnology Co., Ltd.;

[0054] Chamomile extract was purchased from Xi’an Youshuo Biotechnology Co., Ltd.;

[0055] Diatomaceous earth was purchased from Lingshou County Hengxin Mineral Products Processing Plant, CAS number: 61790-53-2;

[0056] Glucose was purchased from Henan Huixin Jianjie Environmental Protection Technology Group Co., Ltd., CAS No.: 50-99-7.

[0057] Example 1

[0058] This embodiment discloses a preparation process of an enzyme protective agent, comprising the following steps:

[0059] Q1: The corn stalks were dried at 80°C for 10 hours, crushed, soaked for 6 hours, and then placed in a deflaker for 20 minutes, and then placed in a PFI pulper for refining at a refining speed of 35,000 rpm to obtain fiber slurry;

[0060] Q2: 703 g of sodium carbonate and 254 g of sodium bicarbonate were added to 625 mL of distilled water for mixing and stirring to obtain a mixed buffer solution, 0.16 g of tetramethylpiperidinium oxide and 1 g of sodium bromide were added to 600 mL of the mixed buffer solution for stirring until completely dissolved to obtain a composite buffer solution, and then 30 mL of the fiber slurry was added to 20 mL of the composite buffer solution for stirring, and a sodium hypochlorite solution with a weight fraction of 5 wt% was added dropwise during the stirring period. After stirring for 90 min, 52.5 mL of ethanol was added to terminate the reaction, and the reaction solution was treated by dialysis until the pH was neutral to obtain a suspension;

[0061] Q3: Weigh 1.5 kg of kelp powder and put it into a reactor, then add 60 L of 90% by volume ethanol, heat and extract at 90 ° C for 2 h, repeat the operation 4 times, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add 1 g of crystals to 0.8 mL of distilled water, heat and dissolve at 100 ° C for 10 min, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent;

[0062] Q4: Add 0.25 g of lyophilized protective agent to 25 mL of the suspension, stir magnetically for 3 h, and obtain the composite aerogel after 60 MPa high-pressure homogenization. Pre-freeze the composite aerogel at -80 °C and freeze-dry it in a freeze dryer for 52 h to obtain the enzyme protective agent.

[0063] This embodiment discloses a method for preparing a nanocomposite, comprising the following steps:

[0064] S1: 0.2 g phospholipid and 0.075 g cholesterol were mixed and dissolved in 40 mL anhydrous ethanol, 0.08 g Tween 80 was added, ultrasonically dissolved, rotary evaporated at 60°C and 80 rpm for 8 min, and dried to obtain a lipid film;

[0065] S2: 0.25 mg of ε-polylysine was dissolved in 1.5 mL of phosphate buffer to obtain an ε-polylysine solution, 0.3 g of lipid film was added to 7.5 mL of the ε-polylysine solution, and the mixture was washed by shaking at 80° C. for 3 min, stirred for 40 min, and ultrasonicated. The mixture was filtered using a 0.22 μm water filter membrane to obtain a nanocomplex.

[0066] The present embodiment discloses a plant complex enzyme formula for confined spaces, which is composed of the following ingredients in parts by weight: 35 parts of a fermentation mixture, 6 parts of an enzyme protectant, and 4 parts of a nanocomposite, wherein the fermentation mixture is composed of 3% bromelain, 3% papain, 3% catalase, 2% peroxidase, 2% serine protease, 1% trypsin, 2% hesperidinase, 2% wormwood extract, 2% rosemary extract, 1% echinacea polyphenols, 2% chamomile extract, 10% diatomaceous earth, 2% glucose and 65% purified water.

[0067] This embodiment discloses a method for preparing a plant complex enzyme formula for a confined space, comprising the following steps:

[0068] Step (1): placing the fermentation mixture and the enzyme protectant into a fermenter, stirring, heating at 40° C. for 36 h, and then ventilating the fermenter with oxygen for 20 min;

[0069] Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37° C. The fermentation liquid is ventilated with oxygen for 20 minutes every 12 hours and stirred for 10 minutes. The dissolved oxygen content and pH value of the fermentation liquid are tested before stirring. The pH value of the fermentation liquid is always maintained between 5 and 6.5.

[0070] Step (3): fermenting the fermented liquid for 30 days, and then precipitating the fermented liquid for 10 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

[0071] Example 2

[0072] This embodiment discloses a preparation process of an enzyme protective agent, comprising the following steps:

[0073] Q1: The corn stalks were dried at 80°C for 10 hours, crushed, soaked for 6 hours, and then placed in a deflaker for 20 minutes, and then placed in a PFI pulper for refining at a refining speed of 35,000 rpm to obtain fiber slurry;

[0074] Q2: 728 g of sodium carbonate and 210 g of sodium bicarbonate were added to 610 mL of distilled water for mixing and stirring to obtain a mixed buffer solution, 0.1 g of tetramethylpiperidinium oxide and 0.5 g of sodium bromide were added to 550 mL of the mixed buffer solution for stirring until completely dissolved to obtain a composite buffer solution, and then 20 mL of the fiber slurry was added to 10 mL of the composite buffer solution for stirring, and a sodium hypochlorite solution with a weight fraction of 5 wt% was added dropwise during the stirring period. After stirring for 90 min, 50 mL of ethanol was added to terminate the reaction, and the reaction solution was treated by dialysis until the pH was neutral to obtain a suspension;

[0075] Q3: Weigh 1 kg of kelp powder and put it into a reactor, then add 40 L of 90% by volume ethanol, heat and extract at 90 ° C for 2 h, repeat the operation 4 times, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add 1 g of crystals to 0.7 mL of distilled water, heat and dissolve at 100 ° C for 10 min, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent;

[0076] Q4: Add 0.2 g of lyophilized protective agent to 20 mL of the suspension, stir magnetically for 3 h, and obtain the composite aerogel after 60 MPa high-pressure homogenization. Pre-freeze the composite aerogel at -80 °C and freeze-dry it in a freeze dryer for 52 h to obtain the enzyme protective agent.

[0077] This embodiment discloses a method for preparing a nanocomposite, comprising the following steps:

[0078] S1: 0.1 g phospholipid and 0.05 g cholesterol were mixed and dissolved in 30 mL anhydrous ethanol, 0.06 g Tween 80 was added, ultrasonically dissolved, rotary evaporated at 60°C and 80 rpm for 8 min, and dried to obtain a lipid film;

[0079] S2: 0.2 mg of ε-polylysine was dissolved in 1 mL of phosphate buffer to obtain an ε-polylysine solution, 0.2 g of lipid film was added to 5 mL of the ε-polylysine solution, and the mixture was washed by shaking at 80°C for 3 min, stirred for 40 min, and ultrasonicated. The mixture was filtered using a 0.22 μm water filter membrane to obtain a nanocomplex.

[0080] The present embodiment discloses a plant complex enzyme formula for confined spaces, which is composed of the following ingredients in parts by weight: 30 parts of a fermentation mixture, 4 parts of an enzyme protectant, and 2 parts of a nanocomposite, wherein the fermentation mixture is composed of 2% bromelain, 1% papain, 2% catalase, 1% peroxidase, 1% serine protease, 0.5% trypsin, 1% hesperidinase, 0.5% wormwood extract, 1% rosemary extract, 0.5% echinacea polyphenols, 1% chamomile extract, 5% diatomaceous earth, 0.5% glucose and 83% purified water.

[0081] This embodiment discloses a method for preparing a plant complex enzyme formula for a confined space, comprising the following steps:

[0082] Step (1): placing the fermentation mixture and the enzyme protectant into a fermenter, stirring, heating at 40° C. for 36 h, and then ventilating the fermenter with oxygen for 20 min;

[0083] Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37° C. The fermentation liquid is ventilated with oxygen for 20 minutes every 12 hours and stirred for 10 minutes. The dissolved oxygen content and pH value of the fermentation liquid are tested before stirring. The pH value of the fermentation liquid is always maintained between 5 and 6.5.

[0084] Step (3): fermenting the fermented liquid for 30 days, and then precipitating the fermented liquid for 10 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

[0085] Example 3

[0086] This embodiment discloses a preparation process of an enzyme protective agent, comprising the following steps:

[0087] Q1: The corn stalks were dried at 80°C for 10 hours, crushed, soaked for 6 hours, and then placed in a deflaker for 20 minutes, and then placed in a PFI pulper for refining at a refining speed of 35,000 rpm to obtain fiber slurry;

[0088] Q2: 694 g of sodium carbonate and 294 g of sodium bicarbonate were added to 607 mL of distilled water for mixing and stirring to obtain a mixed buffer solution, 0.22 g of tetramethylpiperidinium oxide and 1.5 g of sodium bromide were added to 650 mL of the mixed buffer solution for stirring until completely dissolved to obtain a composite buffer solution, and then 40 mL of the fiber slurry was added to 30 mL of the composite buffer solution for stirring, and a sodium hypochlorite solution with a weight fraction of 5 wt% was added dropwise during the stirring period. After stirring for 90 min, 55 mL of ethanol was added to terminate the reaction, and the reaction solution was treated by dialysis until the pH was neutral to obtain a suspension;

[0089] Q3: Weigh 2 kg of kelp powder and put it into a reactor, then add 80 L of 90 vt% ethanol, heat and extract at 90 ° C for 2 h, repeat the operation 4 times, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add 1 g of crystals to 0.9 mL of distilled water, heat and dissolve at 100 ° C for 10 min, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent;

[0090] Q4: Add 0.3 g of lyophilized protective agent to 30 mL of the suspension, stir magnetically for 3 h, and obtain the composite aerogel after 60 MPa high-pressure homogenization. Pre-freeze the composite aerogel at -80 °C and freeze-dry it in a freeze dryer for 52 h to obtain the enzyme protective agent.

[0091] This embodiment discloses a method for preparing a nanocomposite, comprising the following steps:

[0092] S1: 0.3 g phospholipid and 0.10 g cholesterol were mixed and dissolved in 50 mL anhydrous ethanol, 0.10 g Tween 80 was added, ultrasonically dissolved, rotary evaporated at 60°C and 80 rpm for 8 min, and dried to obtain a lipid film;

[0093] S2: 0.3 mg of ε-polylysine was dissolved in 2 mL of phosphate buffer to obtain an ε-polylysine solution, 0.4 g of lipid film was added to 10 mL of the ε-polylysine solution, and the mixture was shaken and washed at 80°C for 3 min, stirred for 40 min, and ultrasonicated. The mixture was filtered using a 0.22 μm water filter membrane to obtain a nanocomplex.

[0094] The present embodiment discloses a plant complex enzyme formula for confined spaces, which is composed of the following ingredients in parts by weight: 40 parts of a fermentation mixture, 8 parts of an enzyme protectant, and 6 parts of a nanocomposite, wherein the fermentation mixture is composed of 2% bromelain, 1% papain, 4% catalase, 3% peroxidase, 3% serine protease, 1.5% trypsin, 3% hesperidinase, 3.5% wormwood extract, 3% rosemary extract, 1.5% echinacea polyphenols, 3% chamomile extract, 15% diatomaceous earth, 3.5% glucose and 53% purified water.

[0095] This embodiment discloses a method for preparing a plant complex enzyme formula for a confined space, comprising the following steps:

[0096] Step (1): placing the fermentation mixture and the enzyme protectant into a fermenter, stirring, heating at 40° C. for 36 h, and then ventilating the fermenter with oxygen for 20 min;

[0097] Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37° C. The fermentation liquid is ventilated with oxygen for 20 minutes every 12 hours and stirred for 10 minutes. The dissolved oxygen content and pH value of the fermentation liquid are tested before stirring. The pH value of the fermentation liquid is always maintained between 5 and 6.5.

[0098] Step (3): fermenting the fermented liquid for 30 days, and then precipitating the fermented liquid for 10 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

[0099] Example 4

[0100] This embodiment discloses a preparation process of an enzyme protective agent, comprising the following steps:

[0101] Q1: The corn stalks were dried at 80°C for 10 hours, crushed, soaked for 6 hours, and then placed in a deflaker for 20 minutes, and then placed in a PFI pulper for refining at a refining speed of 35,000 rpm to obtain fiber slurry;

[0102] Q2: 715 g of sodium carbonate and 218 g of sodium bicarbonate were added to 588 mL of distilled water for mixing and stirring to obtain a mixed buffer solution, 0.14 g of tetramethylpiperidinium oxide and 0.75 g of sodium bromide were added to 580 mL of the mixed buffer solution for stirring until completely dissolved to obtain a composite buffer solution, and then 25 mL of the fiber slurry was added to 15 mL of the composite buffer solution for stirring, and a sodium hypochlorite solution with a weight fraction of 5 wt% was added dropwise during the stirring period. After stirring for 90 min, 51 mL of ethanol was added to terminate the reaction, and the reaction solution was treated by dialysis until the pH was neutral to obtain a suspension;

[0103] Q3: Weigh 1.2 kg of kelp powder and put it into a reactor, then add 50 L of 90% by volume ethanol, heat and extract at 90 ° C for 2 h, repeat the operation 4 times, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add 1 g of crystals to 0.75 mL of distilled water, heat and dissolve at 100 ° C for 10 min, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent;

[0104] Q4: Add 0.22 g of lyophilized protective agent to 28 mL of the suspension, stir magnetically for 3 h, and obtain the composite aerogel after 60 MPa high-pressure homogenization. Pre-freeze the composite aerogel at -80 °C and freeze-dry it in a freeze dryer for 52 h to obtain the enzyme protective agent.

[0105] This embodiment discloses a method for preparing a nanocomposite, comprising the following steps:

[0106] S1: 0.15 g phospholipid and 0.08 g cholesterol were mixed and dissolved in 45 mL anhydrous ethanol, 0.07 g Tween 80 was added, ultrasonically dissolved, rotary evaporated at 60°C and 80 rpm for 8 min, and dried to obtain a lipid film;

[0107] S2: 0.1 mg of ε-polylysine was dissolved in 1.4 mL of phosphate buffer to obtain an ε-polylysine solution, 0.25 g of lipid film was added to 8 mL of the ε-polylysine solution, and the mixture was washed by shaking at 80° C. for 3 min, stirred for 40 min, and ultrasonicated. The mixture was filtered using a 0.22 μm water filter membrane to obtain a nanocomplex.

[0108] The present embodiment discloses a plant complex enzyme formula for confined spaces, which is composed of the following ingredients in parts by weight: 32 parts of a fermentation mixture, 5 parts of an enzyme protectant, and 3 parts of a nanocomposite, wherein the fermentation mixture is composed of 2.5% bromelain, 4.5% papain, 2.5% catalase, 2% peroxidase, 1% serine protease, 0.5% trypsin, 2% hesperidinase, 0.5% wormwood extract, 1% rosemary extract, 1.5% echinacea polyphenols, 2% chamomile extract, 12% diatomaceous earth, 2.5% glucose and 65.5% purified water.

[0109] This embodiment discloses a method for preparing a plant complex enzyme formula for a confined space, comprising the following steps:

[0110] Step (1): placing the fermentation mixture and the enzyme protectant into a fermenter, stirring, heating at 40° C. for 36 h, and then ventilating the fermenter with oxygen for 20 min;

[0111] Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37° C. The fermentation liquid is ventilated with oxygen for 20 minutes every 12 hours and stirred for 10 minutes. The dissolved oxygen content and pH value of the fermentation liquid are tested before stirring. The pH value of the fermentation liquid is always maintained between 5 and 6.5.

[0112] Step (3): fermenting the fermented liquid for 30 days, and then precipitating the fermented liquid for 10 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

[0113] Comparative Example 1

[0114] Comparative Example 1 Compared with Example 1, in the preparation process of the enzyme protective agent in Comparative Example 1, no corn stalks were added, and other conditions remained unchanged.

[0115] Comparative Example 2

[0116] Comparative Example 2 Compared with Example 1, in the preparation process of the enzyme protecting agent in Comparative Example 2, no tetramethylpiperidinyl oxide was added, and other conditions remained unchanged.

[0117] Comparative Example 3

[0118] Comparative Example 3 Compared with Example 1, in the preparation process of the enzyme protective agent in Comparative Example 3, no kelp powder was added, and other conditions remained unchanged.

[0119] Comparative Example 4

[0120] Comparative Example 4 Compared with Example 1, in the preparation process of the nanocomposite in Comparative Example 4, no phospholipid was added, and other conditions remained unchanged.

[0121] Comparative Example 5

[0122] Comparative Example 5 Compared with Example 1, in the preparation process of the nanocomposite in Comparative Example 5, no cholesterol was added, and other conditions remained unchanged.

[0123] Comparative Example 6

[0124] Comparative Example 6 Compared with Example 1, in the preparation process of the nanocomposite in Comparative Example 6, no ε-polylysine was added, and other conditions remained unchanged.

[0125] Comparative Example 7

[0126] Comparative Example 7 Compared with Example 1, in the process of preparing the plant complex enzyme in Comparative Example 7, no enzyme protective agent was added, and other conditions remained unchanged.

[0127] Comparative Example 8

[0128] Comparative Example 8 Compared with Example 1, in the process of preparing the plant complex enzyme in Comparative Example 8, no nanocomposite was added, and other conditions remained unchanged.

[0129] 1. Sterilization effect

[0130] Test group: Take 1 portion of the supernatant prepared in Example 1 and 10 portions of purified water, stir evenly and then add 30 ml 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The control group was left to stand, and the sterilization effect on Staphylococcus aureus was measured at different times. The results are shown in Table 1:

[0131] Table 1

[0132]

[0133] It can be seen from the test in Table 1 that the plant complex enzyme formula specially prepared for confined spaces has an excellent removal effect on Staphylococcus aureus, can inhibit the proliferation of pathogens and bacteria, and achieve the effect of controlling pathogens and bacteria.

[0134] 2. Effect of removing trimethylamine

[0135] Test group: Take 1 part and 0.5 part of the supernatant prepared in Example 1, take 10 parts of pure water, stir evenly and put into 10 ml 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The concentration of trimethylamine was measured at different times. The results are shown in Table 2:

[0136] Table 2

[0137]

[0138]

[0139] It can be seen from the test in Table 2 that the plant complex enzyme formula specially prepared for confined spaces has an excellent removal effect on trimethylamine, and the higher the concentration, the higher the removal rate in the same time.

[0140] 3. Effect of removing formaldehyde

[0141] Test group: Take 1 portion of the supernatant prepared in Example 1 and 10 portions of purified water, stir evenly and then add 10 ml of 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The formaldehyde concentration at different times was measured. The results are shown in Table 3:

[0142] Table 3

[0143] Test time / min <![CDATA[Formaldehyde concentration / mg·m -3 > Removal rate / % 0 1.010 0 30 0.439 56.53 60 0.273 72.97 120 0.239 76.34 180 0.152 84.95 240 0.120 88.12 300 0.090 91.09 360 0.065 93.56 720 0.054 94.65 1440 0.041 95.94

[0144] From the test in Table 3, it can be seen that the plant complex enzyme formula specially prepared for confined spaces has an excellent formaldehyde removal effect, with a removal rate of up to 95%.

[0145] 4. Effect of removing organic matter

[0146] Test group: Take 1 portion of the supernatant prepared in Example 1 and 10 portions of purified water, stir evenly and then add into 3 ml 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The concentrations of n-undecane and butyl acetate were measured at different times. The results are shown in Table 4:

[0147] Table 4

[0148] Test time / min <![CDATA[n-Undecane concentration / mg·m -3 > Removal rate / % <![CDATA[Butyl acetate concentration / mg·m -3 > Removal rate / % 0 0.223 0 0.605 0 60 0.149 33.38 0.171 71.76 360 0.059 73.71 0.082 86.44 720 0.036 83.94 0.063 89.55 1080 0.022 90.31 0.058 90.36 1440 0.011 95.15 0.040 93.32

[0149] It can be seen from the test in Table 4 that the plant complex enzyme formula specially prepared for confined spaces has excellent removal effects on n-undecane and butyl acetate.

[0150] 5. Effect of removing ammonia

[0151] Test group: Take 1 part and 0.5 part of the supernatant prepared in Example 1, take 10 parts of pure water, stir evenly and put into 10 ml 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The ammonia concentration at different times was measured. The results are shown in Table 5:

[0152] Table 5

[0153]

[0154]

[0155] It can be seen from the test in Table 5 that the plant complex enzyme formula specially prepared for confined spaces has an excellent removal effect on ammonia, and the higher the concentration, the higher the removal rate in the same time.

[0156] 6. Effect of removing hydrogen sulfide

[0157] Test group: Take 1 part and 0.5 part of the supernatant prepared in Example 1, take 10 parts of pure water, stir evenly and put into 10 ml 3 In the test chamber, the spraying amount was 1 level, 1min on / 5min off, and the test was continuous for 24 hours. The concentration of hydrogen sulfide at different times was measured. The results are shown in Table 6:

[0158] Table 6

[0159]

[0160] It can be seen from the test in Table 6 that the plant complex enzyme formula specially prepared for confined spaces has an excellent removal effect on hydrogen sulfide, and the higher the concentration, the higher the removal rate in the same time.

[0161] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0162] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A plant compound enzyme formula specially formulated for confined spaces, characterized in that: The invention is composed of the following components in parts by weight: 30-40 parts of a fermentation mixture, 4-8 parts of an enzyme protectant, and 2-6 parts of a nanocomposite, wherein the fermentation mixture is composed of 2-4% bromelain, 1-5% papain, 2-4% catalase, 1-3% horseradish peroxidase, 1-3% serine protease, 0.5-1.5% trypsin, 1-3% hesperidinase, 0.5-3.5% wormwood extract, 1-3% rosemary extract, 0.5-1.5% echinacea polyphenols, 1-3% chamomile extract, 5-15% diatomaceous earth, 0.5-3.5% glucose and 45-85% purified water, the enzyme protectant is prepared from corn stalks, tetramethyl piperidine oxide, kelp dry powder and ethanol, and the nanocomposite is prepared from phospholipids, cholesterol, Tween 80 and ε-polylysine; The method for preparing a plant compound enzyme formula for a confined space comprises the following steps: Step (1): placing the fermentation mixture and the enzyme protectant into a fermentation tank, stirring, heating at 39-42° C. for reaction for 34-36 hours, and then ventilating the fermentation tank with oxygen for 18-22 minutes; Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37-38° C., and the fermentation liquid is ventilated with oxygen for 18-22 minutes at intervals of 11-12 hours, and stirred for 8-12 minutes. Before stirring, the dissolved oxygen content and pH value of the fermentation liquid are tested, and the pH value of the fermentation liquid is always maintained between 5-6.5; Step (3): fermenting the fermented liquid for 30-32 days, and then precipitating the fermented liquid for 10-12 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

2. The plant complex enzyme formula for confined space according to claim 1, characterized in that: The preparation process of the enzyme protecting agent comprises the following steps: Q1: The corn stalks are dried, crushed, soaked, and then placed in a deflaker for deflaking, and then placed in a PFI pulper for refining to obtain fiber slurry; Q2: Sodium carbonate and sodium bicarbonate are added to distilled water and mixed and stirred to obtain a mixed buffer solution, tetramethyl piperidine oxide and sodium bromide are added to the mixed buffer solution and stirred until completely dissolved to obtain a composite buffer solution, and then the fiber slurry is added to the composite buffer solution and stirred, and a sodium hypochlorite solution is added dropwise during the stirring period. After the stirring is completed, ethanol is added to terminate the reaction, and the reaction solution is treated by dialysis until the pH is neutral to obtain a suspension; Q3: Weigh the dried kelp powder and put it into a reactor, then add ethanol, heat and extract, repeat the operation, combine the extracts, concentrate under reduced pressure, crystallize at low temperature, add distilled water to heat and dissolve the crystals, cool and crystallize, centrifuge to obtain recrystallized material, repeat the operation, wash the obtained material with ethanol, centrifuge and dry, and obtain a freeze-dried protective agent; Q4: A freeze-drying protective agent is added to the suspension, and magnetic stirring is performed. After high-pressure homogenization, a composite aerogel is obtained. The composite aerogel is pre-frozen and placed in a freeze dryer for freeze drying to obtain an enzyme protective agent.

3. The plant complex enzyme formula for confined space according to claim 2, characterized in that: In the Q1, the drying temperature is 60-80°C, the time is 10-14h, the soaking is 4-6h, the disintegration is 10-20min, and the refining speed is 30000-35000rpm.

4. The plant complex enzyme formula for confined space according to claim 2, characterized in that: In the Q2, the molar ratio of sodium carbonate, sodium bicarbonate and distilled water is (6.5-7.5): (2.5-3.5): (30-35), the dosage ratio of tetramethyl piperidine oxide, sodium bromide and mixed buffer is (0.1-0.22) g: (0.5-1.5) g: (550-650) mL, the volume ratio of fiber slurry, composite buffer and ethanol is (20-40): (10-30): (50-55), the weight fraction of sodium hypochlorite solution is 5wt%, and the stirring time is 60-90 min.

5. The plant complex enzyme formula for confined space according to claim 2, characterized in that: In the Q3, the dosage ratio of kelp powder and ethanol is (1-2) kg: (40-80) L, the volume fraction of ethanol is 90vt%, the heating extraction temperature is 80-90°C, the time is 1-2h, the operation is repeated 3-4 times, the dosage ratio of crystals to distilled water is 1g: (0.7-0.9) mL, the heating dissolution temperature is 90-100°C, and the time is 5-10min; in the Q4, the dosage ratio of the suspension and the lyophilization protective agent is (20-30) mL: (0.2-0.3) g, the magnetic stirring time is 2-3h, the pressure of high-pressure homogenization treatment is 50-60MPa, the pre-freezing temperature is -20 to -80°C, and the freeze-drying time is 48-52h.

6. The plant complex enzyme formula for confined space according to claim 1, characterized in that: The method for preparing the nanocomposite comprises the following steps: S1: Dissolve phospholipids and cholesterol in anhydrous ethanol, add Tween 80, dissolve by ultrasonic, evaporate by rotary evaporation, and dry to obtain a lipid film; S2: dissolving ε-polylysine in phosphate buffer to obtain an ε-polylysine solution, adding the lipid film into the ε-polylysine solution for shaking washing, stirring, ultrasonication, and filtering to obtain a nanocomplex.

7. The plant complex enzyme formula for confined space according to claim 6, characterized in that: In the S1, the dosage ratio of phospholipid, cholesterol, anhydrous ethanol and Tween 80 is (0.1-0.3) g: (0.05-0.10) g: (30-50) mL: (0.06-0.10) g, the rotary evaporation speed is 60-80 rpm, the temperature is 50-60° C., and the time is 5-8 min. In the S2, the dosage ratio of ε-polylysine and phosphate buffer is (0.2-0.3) mg: (1-2) mL, the dosage ratio of lipid film and ε-polylysine solution is (0.2-0.4) g: (5-10) mL, the temperature of oscillation washing is 60-80° C., the time is 1-3 min, the stirring time is 30-40 min, and a 0.22 μm water filter membrane is used during the filtration process.

8. The method for preparing a plant complex enzyme formula for a confined space according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step (1): placing the fermentation mixture and the enzyme protectant into a fermentation tank, stirring, heating at 39-42° C. for reaction for 34-36 hours, and then ventilating the fermentation tank with oxygen for 18-22 minutes; Step (2): After the ventilation is completed, the temperature of the fermentation liquid in the fermenter is controlled to be 37-38° C., and the fermentation liquid is ventilated with oxygen for 18-22 minutes at intervals of 11-12 hours, and stirred for 8-12 minutes. Before stirring, the dissolved oxygen content and pH value of the fermentation liquid are tested, and the pH value of the fermentation liquid is always maintained between 5-6.5; Step (3): fermenting the fermented liquid for 30-32 days, and then precipitating the fermented liquid for 10-12 days, taking the supernatant, adding the nanocomposite, stirring and mixing evenly, and then deodorizing and sterilizing by ozone method to obtain a plant complex enzyme formula for enclosed space.

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