Preparation method of tea saponin surfactant and water treatment agent

Abstract

The invention provides a preparation method of a tea saponin surfactant and a water treatment agent, which comprises the following steps: taking the remaining part of tea seeds and / or tea fruits after oil extraction as a tea saponin raw material, and carrying out fermentation treatment to obtain a fermentation mixture; and adding the fermentation mixture and a NaCl solution into a rotocel extractor, and separating to obtain a non-hemolytic toxin nonionic surfactant, a water treatment agent, wet meal and coagulated hemolytic toxin through a specific reaction between the tachypleus amebocyte lysate and the hemolytic toxin and a condensation phase change caused by salting-out. According to the preparation method of the tea saponin surfactant and the water treatment agent, the NaCl solution is added when the rotocel extractor works, and hemolytic toxins and other components are effectively separated while tea saponin is prepared.

Description

Technical Field

[0001] The invention belongs to the field of tea saponin preparation, and specifically relates to a method for preparing a tea saponin surfactant and a water treatment agent, and specifically relates to a continuous preparation method of the tea saponin surfactant and the water treatment agent and a double-support flat-rotating leaching device thereof. Background Art

[0002] Tea saponins are a type of glycoside compound found in the tea dregs after oil is extracted from tea seeds and fruit. They are a highly effective natural surfactant with wide applications in light industry, chemical industry, pesticides, feed, aquaculture, textiles, oil production, mining, building materials, and highway construction. They can be used to make emulsifiers, detergents, pesticide adjuvants, feed additives, crab and shrimp aquaculture protective agents, textile auxiliaries, oilfield foaming agents, mining flotation agents, foam stabilizers for aerated concrete, and concrete admixtures—antifreeze. However, there is no specific mention of surfactants and water treatment agents used in the food industry.

[0003] This is because tea saponin has a destructive effect on animal red blood cells, causing hemolysis. Its activity is measured by the maximum dilution factor at which hemolysis occurs, known as the hemolytic index. Tea saponin's hemolytic index is 100,000. Tea saponin only causes hemolysis in red blood cells (including nucleated fish blood, chicken blood, and non-nucleated human blood), but has no effect on white blood cells. Tea saponin is toxic to fish but not shrimp. The hemolytic mechanism is that tea saponin alters the permeability of cholesterol-containing cell membranes, initially disrupting the cell membrane, leading to cytoplasmic extravasation and ultimately the disintegration of the entire red blood cell. This hemolytic effect requires contact with blood, making it non-toxic to humans and animals when orally administered.

[0004] The existing tea saponin preparation methods have the following disadvantages: tea saponin surfactants and water treatment agents contain hemolytic cytotoxins and cannot be used directly in food. When used as water treatment for fish ponds, they may reduce the production of fish, earthworms, snails, etc. or cause disease (see the literature [Wang Huilin, Jin Huaiyuan, Gao Ye, Xia Sudong, Li Jie. Isolation and identification of the pathogen of Aeromonas hydrophila in American shad. Progress in Fisheries Science, 2024, 45(2): 257-266]); and the product cannot produce hemolytic toxins alone for other medicinal values ​​such as the inhibition of melanoma (see the literature [Yang Lanzhu, Li Shiyi, Zheng Wenjing, Li Jing, Bao Zhekui, Yang Jingya. Study on the inhibitory effect of heat-resistant direct hemolytic toxin produced by Vibrio parahaemolyticus on melanoma. Marine Science 2022, 46 (8): 121-128]).

[0005] Therefore, it is necessary to propose a new method for preparing tea saponin surfactant and water treatment agent. Hemolytic toxins are included in bacterial toxins. The fourth part of the 2020 edition of the Chinese Pharmacopoeia, "Bacterial Endotoxin Test Method" and "Guidelines for Validation of Analytical Methods", has established a dynamic turbidity quantitative detection method for bacterial endotoxins. That is, by injecting sodium chloride injection and reacting quantitatively with Limulus amebocyte lysate, hemolytic cytotoxins can be removed (see [Li Hui, Tang Manli, Yang Yuling, Wu Su. Establishment and Validation of Laboratory Bacterial Endotoxin Quantitative Detection Method. Quality Safety and Inspection, 2023(6):14~18]). In this way, the hemolytic toxins and other components in tea saponin can be effectively separated, making the use of tea saponin non-ionic surfactants more extensive while solving the problems of difficult filtration and purification of tea saponin. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a tea saponin surfactant and a water treatment agent, which can achieve effective separation of hemolytic toxins and other components while preparing tea saponin.

[0007] In order to achieve the above object, the present invention provides a method for preparing a tea saponin surfactant and a water treatment agent, comprising:

[0008] S1: fermenting the tea seeds and / or tea fruits as tea saponin raw materials to obtain a fermentation mixture;

[0009] S2: Fermentation mixture and NaCl solution are added to the horizontal rotary extractor, and the non-ionic surfactant and water treatment agent without hemolytic toxin, wet meal and coagulated hemolytic toxin are separated through the specific reaction of Limulus amebocyte lysate with hemolytic toxin and the coagulation phase change caused by salting out.

[0010] The step S1 specifically includes: using tea seeds and / or tea fruits of Camellia oleifera and Camellia sinensis as raw materials for tea saponin, dissolving the raw materials for tea saponin with deionized water in a mass ratio of 1:10 to obtain a mixed solution; and fermenting the mixed solution with basidiomycetes to obtain a fermentation mixture.

[0011] During dissolution, the stirring speed is adjusted to 60-300 r / min; the mixed liquid is fermented with basidiomycetes, specifically comprising: selecting spores and fruiting bodies of 2-3 kinds of mushrooms, boletes, gelatinous fungi, and yeast to prepare basidiomycete hyphae, and fermenting the mixed liquid with the basidiomycete hyphae at room temperature of 10-30°C.

[0012] The solvent of the NaCl solution is a water treatment agent, and the water treatment agent includes at least one of a corrosion inhibitor, a scale inhibitor, a bactericide, a flocculant, a purifier, a cleaning agent, and a pre-filming agent; and / or the mass fraction of the NaCl solution is 0.5-0.95%. The dosage of the limulus amebocyte lysate follows the law of mass action, and is determined by experiments to have a hemolytic effect of 30.0-33.3 mg of red blood cells on 1 mg of the fermentation mixture, and 1 mg of limulus amebocyte lysate is used for 1 mg of the fermentation mixture.

[0013] The horizontal rotary extractor includes a hollow rotating body, a grid bottom arranged at the bottom of the hollow rotating body, a surfactant collecting grid and a meal outlet arranged below the grid bottom and spaced apart from each other, and a hemolytic toxin coagulation plate arranged between the grid bottom and the surfactant collecting grid and the meal outlet; the hemolytic toxin coagulation plate is coated with a limulus amebocyte lysate so that the hemolytic toxin will only coagulate on the hemolytic toxin coagulation plate.

[0014] The horizontal rotary extractor also includes a rotating shaft and a speed regulating device. The rotating shaft drives the hollow rotating body to rotate through a transmission chain, and the speed regulating device is used to change the rotation speed of the hollow rotating body.

[0015] The horizontal rotary extractor also includes a NaCl solution spray head and a mixing pump arranged at the bottom of the horizontal rotary extractor. The mixing pump is used to regularly add horseshoe crab reagent and NaCl solution through the NaCl solution spray head and to change the spraying speed of the NaCl solution spray head, thereby controlling the product purity of the non-ionic surfactant and water treatment agent without hemolytic toxins; in step S2, when the horizontal rotary extractor is working, the computer uses a photobiochemical concentration sensor integrated with a fluorescence colorimeter and / or an electric focusing electrophoresis instrument to monitor the hemolytic toxin concentration of the fermentation mixture and the hemolytic toxin concentration in the non-ionic surfactant and water treatment agent without hemolytic toxins in real time as real-time data of raw material concentration and product purity; the computer adjusts the circulation pump according to the real-time data of the sensor to control the raw material concentration and product purity in real time.

[0016] The described flat-rotating extractor adopts a flat-rotating extractor with a double-layer grid bottom, and its grid bottom includes a fixed grid bottom and an movable grid bottom arranged above the fixed grid bottom. The bottom of the movable grid bottom is provided with a scraper and the whole is arranged to be rotatable or retractable, and the operation of the movable grid bottom is controlled by the computer's extension and retraction instructions of the movable grid bottom, so that the residue on the fixed grid bottom is scraped off by the movable grid bottom to prevent material jamming when the flat-rotating extractor is working.

[0017] The step S2 further includes: treating the wet meal with a vertical desolventizer to recover a water treatment agent; and / or the preparation method further includes step S3: further separating the nonionic surfactant and the water treatment agent using physical properties.

[0018] The efficiency of removing hemolytic toxins is 90.0-99.0%; surfactants without hemolytic toxins are used for food; water treatment agents without hemolytic toxins are used for fish ponds; for non-ionic surfactants, liquid products are obtained directly or powder products are obtained by drying; for water treatment agents, liquid products are obtained directly or powder products are obtained by drying.

[0019] The present invention's method for producing a tea saponin surfactant and water treatment agent effectively separates hemolytic toxins from other components while producing tea saponin by adding a NaCl solution during operation of a rotary extractor. Furthermore, the present invention utilizes an improved rotary extractor, which prevents blockage and leakage and enables continuous operation, resolving issues such as the difficulty of filtering and purifying tea saponin. This effective separation of hemolytic toxins from other components broadens the application of tea saponin nonionic surfactants and addresses the difficulties of filtering and purifying tea saponin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a flowchart of a method for preparing a tea saponin surfactant and a water treatment agent according to an embodiment of the present invention.

[0021] Figure 2 The present invention is a structural diagram of a rotary extractor used in a method for preparing a tea saponin surfactant and a water treatment agent according to an embodiment of the present invention.

[0022] Figure 3 This is a top view schematic diagram of the grid bottom of the horizontal rotary extractor.

[0023] The accompanying drawings are marked as follows: 1. feeding device; 2. rotating shaft; 3. shell; 4. meal discharge conveyor; 5. mixing pump; 6. surfactant collecting grid; 7. speed regulating device; 8. grid bottom; 9. transmission chain; 10. NaCl solution spray head; 11. central rotating shaft; 12. hemolytic toxin coagulation plate. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0025] The present invention is based on the following principle: Tea saponin surfactant is a nonionic surfactant that is suitable for industrial applications. However, it cannot be used directly as a nonionic surfactant for ponds such as fish farming. The hemolytic toxins contained in the tea saponin must be removed. It should be noted that not all tea saponin surfactants are hemolytic.

[0026] Hemolysin (also known as hemolytic toxin) ① refers to any substance that can lyse red blood cells and release hemoglobin. ② A sensitive, complementary, fixed antibody that specifically binds to a red blood cell antigenic type and is produced in response to stimulation with that surface antigen. This antibody causes red blood cell lysis and releases hemoglobin. Hemolysin, also known as cytotoxin, is specifically a subset of cytolysins, which are toxins secreted by bacteria that can lyse cells. Hemolysin is a perforating toxin, also known as a membrane-attacking toxin. For details, see [Baidu Encyclopedia, National Food and Drug Administration Medical Device (Entry) No. 1400523, 2012].

[0027] The hemolytic toxin contained in tea saponin is destructive to animal red blood cells, causing hemolysis. Its activity is measured by the maximum dilution factor that produces hemolysis (i.e., the hemolytic index). Tea saponin's hemolytic activity is comparable to that of tea saponin and camellia saponin. The hemolytic index for tea saponin, tea saponin, and camellia saponin is all 100,000. Tea saponin only causes hemolysis in red blood cells (including nucleated fish blood, chicken blood, and non-nucleated human blood), but has no effect on white blood cells. For details, please refer to Chapter V. Hemolytic activity and fish toxicity of tea saponins in [Zhu Quanfen, Xia Chunhua, Fan Xingtu, Liu Rongxiang, Tian Jiehua, Wang Lu, Tang Liping. Research on the fish toxicity and application of tea saponins [J]. Tea Science, 1993, 13(01): 69-78. doi: 10.13305 / j.cnki.jts.1993.01.011].

[0028] The structure of algal hemolytic toxin is an oleic amide compound. For details, see [Guo Huige, Isolation, Purification and Toxicity Research of Karenia mikimotoi Hemolytic Toxin and Cytotoxin, Xiamen University Graduate Thesis, May 2014]. The marine kadun algae hemolytic toxin consists of multiple components, which may be glycolipids, digalactose, polyoxyethylene polyethers, macrolides, porphyrin derivatives and polyunsaturated fatty acids. For details, see [Jiang Tao, Teng Deqiang, Jiang Tianjiu, Lü Songhui, Yang Weidong. Research Progress on Marine Microalgae Hemolytic Toxins. Journal of Tropical and Subtropical Botany, 2012, 20(3): 311-318].

[0029] Based on this, the hemolytic toxin in sasanian saponin may also be lipid peroxides or glycolipids. Hemolytic toxins are primarily detected using the red blood cell lysis assay, which relies on the fact that hemolytic toxins act on red blood cells, causing them to dissolve and rupture. The presence of hemolytic toxins is determined by changes in absorbance, and the hemolytic capacity is then determined by comparing the absorbance with known concentrations of hemolytic substances.

[0030] like Figure 1The figure shows a method for preparing a tea saponin surfactant and a water treatment agent according to one embodiment of the present invention, which is used to prepare a non-ionic surfactant (i.e., tea saponin surfactant) without hemolytic toxins, a water treatment agent without hemolytic toxins, and coagulated hemolytic toxins from raw materials such as oil tea and camellia. The recovered coagulated hemolytic toxins are used for medical purposes after conversion. The obtained water treatment agent without hemolytic toxins can be used for fish pond farming to prevent the lysis of fish red blood cells, as well as the ecological environment of loaches, snails, clams, frog eggs, tadpoles and some aquatic insects. In addition, the non-ionic surfactant allows the surfactant to be expanded to the food industry, with an efficiency of 95.0-99.0%. As Figure 1 As shown, the method for preparing the tea saponin surfactant and water treatment agent specifically includes:

[0031] Step S1: fermenting the tea seeds and / or tea fruits as tea saponin raw materials to obtain a fermentation mixture;

[0032] Among them, the role of fermentation is, on the one hand, to depolymerize the complex terpenes in tea saponins; on the other hand, during the fermentation process, the action of probiotics such as yeast and plant lactic acid bacteria can also inhibit the existing hemolytic toxins.

[0033] Step S1 specifically includes: using tea seeds and / or tea fruits of Camellia oleifera and Camellia sinensis as raw materials for tea saponin, dissolving the raw materials for tea saponin with deionized water in a mass ratio of 1:10 to obtain a mixed solution; and fermenting the mixed solution with basidiomycetes to obtain a fermentation mixture.

[0034] During dissolution, the stirring speed is adjusted to 60-300 r / min to allow the raw materials to dissolve quickly.

[0035] The mixed liquid is fermented with basidiomycetes, specifically comprising: selecting spores and fruiting bodies of 2-3 kinds of mushrooms, boletes, gelatinous fungi and yeast to prepare basidiomycete hyphae, and fermenting the mixed liquid with the basidiomycete hyphae at room temperature of 10-30°C.

[0036] The mycelial growth temperature range for basidiomycetes is 5-32°C, with an optimum temperature of 24-27°C. Growth is poor below 10°C and above 32°C, and growth ceases at 34°C. Mycelial growth is damaged by heat at 36°C, turning yellow, and death is likely to occur above 38°C. The fruiting body develops in the temperature range of 5-24°C, with 15-20°C being the most suitable. A temperature difference of 8-10°C is optimal for the primordium to develop and form fruiting bodies in basidiomycetes.

[0037] Step S2: adding the fermentation mixture and NaCl solution to the horizontal rotary extractor to separate the non-ionic surfactant and water treatment agent without hemolytic toxin, wet meal and coagulated hemolytic toxin through the specific reaction of the limulus amebocyte lysate with the hemolytic toxin and the coagulation phase change caused by salting out.

[0038] Compared to existing technologies, the present invention adds salt (NaCl) to the fermentation mixture. The specific reaction of the limulus amebocyte lysate with the hemolytic toxin and the resulting coagulation phase transition caused by salting out remove the hemolytic toxin, separating the hemolytic toxin-free nonionic surfactant and water treatment agent. This allows the hemolytic toxin to be removed from the nonionic surfactant and water treatment agent. The recovered coagulated hemolytic toxin can be converted and used in pharmaceuticals.

[0039] Salting out is a common method for impurity removal and purification. The salting out process of the present invention involves reacting with Limulus Amebocyte Lysate (LAL) to remove hemolytic toxins. Limulus Amebocyte Lysate (LAL) is a sterile, freeze-dried product made from the blood amebocyte lysate of the marine arthropod Limulus. It contains procoagulant and coagulogen, which are activated by trace amounts of bacterial endotoxins and fungal glucans. This biological reagent, extracted from the amebocyte lysate of the blue blood of the marine arthropod Limulus, is freeze-dried at low temperatures and can accurately and rapidly qualitatively or quantitatively detect the presence of bacterial endotoxins in a sample. As a standardized laboratory reagent for removing bacterial endotoxins, LAL can reduce or avoid salting out reactions between sodium chloride and surfactants such as tea oil. See the literature [Feng Guohe, Zhu Yanchun. Diagnosis and treatment of food poisoning gastroenteritis [J]. China Practical Rural Doctors Journal, 2003, 10(003): 5-6. DOI: 10.3969 / j.issn.1672-7185.2003.03.003], [Yang Yan, Fan Hongxia. Clinical and experimental study on the treatment of bacterial vaginosis with Longdan Xiegan Pills (Decoction) [J]. Medical Theory and Practice, 2005, 18(11):3.DOI:CNKI:SUN:YXLL.0.2005-11-016.], [Wu Chunlin. Diagnosis and treatment of piglet edema [J]. Journal of Traditional Chinese Veterinary Medicine, 2016(5):2.DOI:CNKI:SUN:ZYSS.0.2016-05-050.], [Zhou Guilian. Research progress on enterotoxic substances of Vibrio parahaemolyticus (review) [J]. Chinese Journal of Food Hygiene, 1992(2):5], these documents prove that hemolytic toxins come from Gram-negative bacteria such as hemolytic Escherichia coli and Vibrio parahaemolyticus, so Limulus amebocyte lysate reagent can be used to detect hemolytic toxins.

[0040] The present invention forms a gel substance by causing the horseshoe crab reagent to undergo a specific gelation reaction with the hemolytic toxin in the presence of NaCl without reacting with the tea saponin surfactant (non-hemolytic toxin). NaCl is used to remove the gel substance, and the gel substance is further coagulated and phase-changed by salting out. The tea saponin surfactant (non-hemolytic toxin) has a lower degree of coagulation due to its greater solubility than the gel substance, thereby collecting and removing the coagulated hemolytic toxin from the coagulation plate in Figure 12.

[0041] The pH of the limulus test solution typically forms a gel at a pH of 6 to 8. When reacting the limulus test solution with a test solution such as sodium chloride, the pH of the test solution does not need to be considered. However, when testing test solutions of other preparations, such as those with strong buffering capacity or surface activity, the pH of the test solution should be considered. Therefore, the present invention further uses NaCl as a degelling agent.

[0042] Wherein, the solvent of the NaCl solution is a water treatment agent, and the water treatment agent includes a corrosion inhibitor, a scale inhibitor, a bactericide, a flocculant, a purifier, a cleaning agent, a pre-filming agent, etc. In practical applications, a water treatment agent with a composite formula is often used, or various water treatment agents are used in combination. In the present embodiment, the mass fraction of the NaCl solution is 0.5-0.95%. In addition, the dosage of the limulus amebocyte lysate follows the law of mass action and is carried out in equimolar or equivalent amounts. It is determined by experiments that 1 mg of tea saponin has the hemolytic performance of 30.0-33.3 mg of red blood cells. Specifically, the dosage of the limulus amebocyte lysate can be determined by detecting the composition of the reaction product to determine whether it is excessive or insufficient, and then through repeated experiments, the dosage of the limulus amebocyte lysate in equimolar or equivalent amounts is finally determined.

[0043] In this embodiment, the horizontal rotary extractor includes a shell 3, a feeding device 1 and a NaCl solution spray head 10 arranged above the shell 3, a hollow rotor 2 arranged inside the shell 3, a rotating shaft 11 arranged at the center of the hollow rotor 2 to drive the hollow rotor 2 to rotate, a grid bottom 8 arranged at the bottom of the hollow rotor 2, a surfactant collecting grid 6 and a meal outlet arranged below the grid bottom 8 and spaced apart from each other, a hemolytic toxin coagulation plate 12 arranged between the grid bottom 8 and the surfactant collecting grid 6 and the meal outlet, a meal outlet conveyor 4 arranged below the meal outlet, and a mixing pump 5 arranged at the bottom of the horizontal rotary extractor, the mixing pump 5 is used to regularly add horseshoe crab reagent and NaCl solution through the NaCl solution spray head 10 and to change the spraying speed of the NaCl solution spray head, thereby controlling the product purity of the non-ionic surfactant and water treatment agent without hemolytic toxins. Under the condition of ensuring the total flow rate, the limulus amebocyte lysate and the NaCl solution are regularly added into the mixing pump 5 at an interval of 2.5:1 to ensure that the addition speed ratio of the limulus amebocyte lysate and the NaCl solution is 1:2.5.

[0044] Thus, the hollow rotor 2 is divided into several sector-shaped leaching compartments by numerous radial partitions, each used to hold the fermentation mixture. Circulating NaCl solution is sprayed from a NaCl solution spray head 10 via a mixing pump 5, countercurrently spraying and soaking the fermentation mixture within each sector-shaped leaching compartment. The solution then percolates through the material layer, passes through the screen bottom 8, and flows through the hemolytic toxin coagulation plate 12 into several surfactant collection compartments 6 below. After multiple cycles of spraying and soaking, the high-concentration nonionic surfactant is filtered through a filter to remove fine meal debris and then deposited into a temporary active agent storage tank. The remaining wet meal is transferred to the meal outlet at the screen bottom 8 and delivered via the meal outlet conveyor 4 to a vertical desolventizer. Therefore, step S2 further includes treating the wet meal in the vertical desolventizer to recover a water treatment agent.

[0045] The rotating shaft 11 drives the hollow rotating body 2 to rotate via the transmission chain 9, and the speed of the hollow rotating body 2 is changed via the speed regulating device 7. The NaCl solution spray head 10 is used to add NaCl solution to remove hemolytic toxins.

[0046] The hemolytic toxin coagulation plate refers to the gel method for bacterial endotoxin testing in Part II of the 2010 edition of the Chinese Pharmacopoeia. On the basis of using NaCl solution in the present invention, the hemolytic toxin coagulation plate is coated with a limulus amebocyte lysate, so that the hemolytic toxin will only coagulate on the hemolytic toxin coagulation plate and will not coagulate in other locations such as the surfactant collection grid.

[0047] In this embodiment, the flat-rotating extractor adopts a flat-rotating extractor with a double-layer grid bottom, and its grid bottom 8 includes a fixed grid bottom 81 and an active grid bottom 82 arranged above the fixed grid bottom 81. The bottom of the active grid bottom 82 is provided with a scraper and the whole is arranged to be rotatable and / or retractable, and the operation of the active grid bottom 82 is controlled by the computer's extension and retraction instructions of the active grid bottom 82, so that the residue on the fixed grid bottom 81 is scraped off by the active grid bottom 82 to prevent material jamming when the flat-rotating extractor is working.

[0048] When the movable grid bottom 82 is configured to be rotatable, it can be configured to rotate in a circular motion along with the rotating shaft 11. In addition, the movable grid bottom 82 is configured to be retractable, and its extension and retraction directions are as follows: Figure 3 As shown, it can be directed toward the rotation axis 11 or away from the rotation axis.

[0049] The active grid bottom 82 uses a sieve plate with 50 nanometer sieve holes, and the fixed grid bottom 81 uses a fixed sieve plate with 10 nanometer sieve holes.

[0050] In step S2, when the horizontal rotary extractor is working, the computer adjusts the mixing pump 5 according to the real-time data of the sensor to control the raw material concentration of the fermentation mixture, the product purity of the non-ionic surfactant without hemolytic toxins and the water treatment agent in real time, which is suitable for the sustainable utilization of large-scale camellia oil and camellia saponin.

[0051] Among them, the effectiveness of hemolytic toxin can be detected by one of the following methods: fluorescence colorimetry, isoelectric focusing electrophoresis, and red blood cell counting in fish determination applications.

[0052] In step S2, the computer uses an optical biomass concentration sensor integrated with a fluorescence colorimeter and / or an electric focusing electrophoresis instrument to monitor in real time the hemolytic toxin concentration in the fermentation mixture, as well as the hemolytic toxin concentration in the hemolytic toxin-free non-ionic surfactant and water treatment agent, as real-time data of raw material concentration and product purity.

[0053] The optical bioconcentration sensor uses nucleic acid aptamers as the recognition element of the sensor, and is constructed by combining the two methods of cyclic voltammetry and AC impedance in electrochemical analysis, realizing the step-by-step construction and characterization of the optical bioconcentration sensor. Secondly, it is not interfered with by bovine albumin in the process of detecting endotoxins, has high sensitivity, and the detection limit can reach 0.001EU / ml and shows a good linear relationship in the endotoxin concentration range of 0.001 to 0.1EU / ml. In this embodiment, the optical bioconcentration sensor uses a portable biotoxicity detector UTOX-100-Shenzhen Youwei Environmental Technology Co., Ltd. The detection results of the optical bioconcentration sensor can be, for example: the raw material detects 0.12% of hemolytic toxins, the product detects 0.01%, and the removal rate is 91.67%.

[0054] In this embodiment, the computer is a DCS (distributed control system) computer, which is also pre-installed with the online software ClustalW2; data processing, statistics and analysis are performed using the software Rv2.15.3, and structure prediction is performed using the software PSIPRED v4.0; and codon preference analysis is performed using the NCBI GenBank database.

[0055] Step S3: further separating the nonionic surfactant and the water treatment agent by utilizing physical properties.

[0056] Nonionic surfactants and water treatment agents can be separated from each other by utilizing physical properties such as specific gravity, boiling point, and viscosity. When utilizing specific gravity, separation can be performed using a centrifuge; when utilizing boiling point, separation can be performed using distillation equipment.

[0057] Therefore, the present invention removes hemolytic toxins in surfactants and water treatment agents, so that the surfactants that remove hemolytic toxins can be used in food, and the efficiency of removing hemolytic toxins (that is, the proportion of removed hemolytic toxins to all hemolytic toxins) is 90.0-99.0%; and the water treatment agent that removes hemolytic toxins can be used in fish ponds to prevent the lysis of fish red blood cells, and optimize the ecological environment of loaches, snails, clams, frog eggs, tadpoles and some aquatic insects.

[0058] Step S3 includes: for nonionic surfactants (i.e., tea saponin surfactants), directly obtaining a liquid product or drying it to obtain a powder product; for water treatment agents, directly obtaining a liquid product or drying it to obtain a powder product. The liquid product is convenient to use, and the solid powder converted from the liquid product by methods such as spray drying is packaged for easier storage and transportation.

[0059] Example 1:

[0060] The tea saponins remaining after oil extraction from tea seeds and tea fruits are fermented into mycelium using spores and fruiting bodies of three types of basidiomycetes, namely boletus, gelatinous bacteria, and yeast. The fermentation is carried out at room temperature of 30°C with a water-to-material ratio of 1:10. After the fermentation treatment, the fermentation mixture and NaCl solution are added into a fixed and active anti-clogging double-bottomed horizontal rotary extractor. The non-ionic surfactant and water treatment agent without hemolytic toxin, wet meal and coagulated hemolytic toxin are separated through the specific reaction of Limulus amebocyte lysate with hemolytic toxin and the coagulation phase transition caused by salting out.

[0061] The dosage of the limulus amebocyte lysate (LA) follows the law of mass action and is determined experimentally, using equimolar or equivalent amounts. The specific experimental procedure is as follows: Before the fermentation mixture and NaCl solution enter the rotary extractor, 0.90% NaCl is added to 100g of the fermentation mixture, and limulus amebocyte lysate is continuously added. The transparency of the liquid is observed based on colorimetric analysis, and the feed rate of the fermentation product to the rotary extractor and the replenishment rate of the limulus amebocyte lysate are adjusted accordingly. Staff can use a concentration sensor to replenish the limulus amebocyte lysate before the transmittance in the colorimetric analysis approaches 100%. The feed rate of the fermentation product to the rotary extractor and the replenishment rate of the limulus amebocyte lysate are determined when A = Lg(1 / T), the transmittance (T) is infinite, and the absorbance (A) is zero. This confirms that 1mg of the fermentation mixture has the hemolytic capacity of 30.0mg of red blood cells, and that 1mg of limulus amebocyte lysate is sufficient to remove all hemolytic toxins from 1mg of the fermentation mixture. Subsequently, the fermentation mixture is fed into the rotary extractor at a rate of 1.0 ml / min. Based on the fermentation mixture's entry rate into the rotary extractor, a salting-out rate of 2.5 ml / min of NaCl is added. At the same time, the limulus amebocyte lysate is regularly replenished based on the dynamic turbidity changes of the limulus amebocyte lysate. The limulus amebocyte lysate and NaCl solution are also replenished via the mixing pump 5 and the NaCl solution spray head 10. The limulus amebocyte lysate addition rate is set at 1.0 ml / min to ensure the removal rate of hemolytic toxins in the tea saponin. While maintaining the total flow rate, the limulus amebocyte lysate and NaCl solution are regularly replenished into the mixing pump 5 at a ratio of 2.5:1.

[0062] The separation yielded a nonionic surfactant and water treatment agent free of hemolytic toxins, along with the coagulated hemolytic toxin. During the separation process, a DCS computer controlled the raw material concentration and product purity in real time, making it suitable for the sustainable utilization of camellia oleifera and camellia saponins on a large scale. The temperature was maintained at 30°C and the pressure at 0.10 MPa. The computer was controlled by a DCS-YN system. The system used a pre-programmed multiple sequence alignment using the online software ClustalW2 to compare the hemolytic toxin information of the raw material and the product to determine the removal rate. Data processing was performed using R v2.15.3, and the protein secondary structure of the hemolytic toxin was predicted using PSIPRED v4.0. Codon bias analysis was performed using the NCBI GenBank database, using the root directory information of the database. The information bias was within 0.10%, resulting in a separation and purification efficiency of 90.0%.

[0063] Example 2:

[0064] The tea saponins left after oil extraction from tea seeds and tea fruits are fermented into mycelium using spores and fruiting bodies of three types of basidiomycetes, namely mushrooms, colloid fungi, and yeasts. After fermentation at room temperature of 25°C and a water-to-material ratio of 1:15, the fermentation mixture and NaCl solution are added into a fixed and active anti-clogging double-bottomed horizontal rotary extractor. The non-ionic surfactant and water treatment agent without hemolytic toxins, wet meal and coagulated hemolytic toxins are separated through the specific reaction of Limulus amebocyte lysate with hemolytic toxins and the coagulation phase transition caused by salting out.

[0065] The dosage of the limulus amebocyte lysate follows the law of mass action and is carried out on an equimolar or equivalent basis, determined by experiment. The specific experimental process is as follows: before the fermentation mixture and the NaCl solution enter the rotary extractor, the fermentation mixture is added with 0.95% NaCl based on 100g of tea saponin and limulus amebocyte lysate is continuously added. The transparency of the liquid is observed according to the colorimetric analysis test to adjust the feed rate of the fermentation product of the rotary extractor and the replenishment rate of the limulus amebocyte lysate. The staff can replenish the limulus amebocyte lysate by combining it with a concentration sensor. Before the transmittance in the colorimetric analysis approaches 100%, the limulus amebocyte lysate is replenished until A=Lg(1 / T), the transmittance (T) is infinite, and the absorbance (A) is zero. It is determined that 1mg of tea saponin has 33.3mg of hemolytic performance, and that 1mg of the fermentation mixture uses 1mg of limulus amebocyte lysate to remove all hemolytic toxins. Subsequently, the speed at which the fermentation mixture enters the horizontal rotary extractor is set to 1.2 ml / min. Based on the speed at which the fermentation mixture enters the horizontal rotary extractor, the salting-out rate of NaCl is set at 3.0 ml / min. At the same time, based on the dynamic turbidity changes of the limulus amebocyte lysate, the limulus amebocyte lysate is regularly replenished. The limulus amebocyte lysate and NaCl solution are also replenished through the mixing pump 5 and the NaCl solution spray head 10. The addition rate of the limulus amebocyte lysate is set to 1.2 ml / min to ensure the removal rate of the hemolytic toxin in the tea saponin. While ensuring the total flow rate, the limulus amebocyte lysate and NaCl solution are regularly replenished into the mixing pump 5 at an interval of 2.5:1.

[0066] Using both tea saponin and blood volume as variables and blood volume as a constant, the separation yields a nonionic surfactant and water treatment agent free of hemolytic toxins, as well as a coagulated hemolytic toxin. The converted hemolytic toxin is then used as a medical anti-skin tumor drug. A DCS computer controls raw material concentration and product purification steps in real time, making this suitable for large-scale, sustainable utilization of camellia oleifera and camellia saponins. The computer is controlled by a DCS-YN, with temperature maintained at 50°C and pressure maintained at 0.11 MPa. Sequence multiple alignments are performed using the online software ClustalW2; data processing is performed using R v2.15.3; and structure prediction is performed using PSIPRED v4.0. Codon bias analysis is performed using the NCBI GenBank database, with an information bias of ±0.10%. The isolation and purification efficiency reaches 99.0%.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.

Claims

1. A method for preparing a tea saponin surfactant and a water treatment agent, characterized in that: include: Step S1: fermenting the tea seeds and / or tea fruits as tea saponin raw materials to obtain a fermentation mixture; Step S2: adding the fermentation mixture and NaCl solution to the horizontal rotary extractor to separate the non-ionic surfactant and water treatment agent without hemolytic toxin, wet meal and coagulated hemolytic toxin through the specific reaction of the limulus amebocyte lysate with the hemolytic toxin and the coagulation phase change caused by salting out.

2. The method for preparing the tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The step S1 specifically includes: using tea seeds and / or tea fruits of Camellia oleifera and Camellia sinensis as raw materials for tea saponin, dissolving the raw materials for tea saponin with deionized water in a mass ratio of 1:10 to obtain a mixed solution; and fermenting the mixed solution with basidiomycetes to obtain a fermentation mixture.

3. The method for preparing the tea saponin surfactant and water treatment agent according to claim 2, characterized in that: During dissolution, adjust the stirring speed to 60-300r / min; The mixed liquid is fermented with basidiomycetes, specifically comprising: selecting spores and fruiting bodies of 2-3 kinds of mushrooms, boletes, gelatinous fungi and yeast to prepare basidiomycete hyphae, and fermenting the mixed liquid with the basidiomycete hyphae at room temperature of 10-30°C.

4. The method for preparing tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The solvent of the NaCl solution is a water treatment agent, and the water treatment agent includes at least one of a corrosion inhibitor, a scale inhibitor, a bactericide, a flocculant, a purifier, a cleaning agent, and a pre-filming agent; and / or The mass fraction of the NaCl solution is 0.5-0.95%; the dosage of the limulus amebocyte lysate follows the law of mass action, and it is determined by experiments that 1 mg of the fermentation mixture has the hemolytic property of 30.0-33.3 mg of red blood cells. 1 mg of the fermentation mixture is used to remove all hemolytic toxins using 1 mg of limulus amebocyte lysate.

5. The method for preparing tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The horizontal rotary extractor includes a hollow rotating body, a grid bottom arranged at the bottom of the hollow rotating body, a surfactant collecting grid and a meal outlet arranged below the grid bottom and spaced apart from each other, and a hemolytic toxin coagulation plate arranged between the grid bottom and the surfactant collecting grid and the meal outlet; the hemolytic toxin coagulation plate is coated with a limulus amebocyte lysate so that the hemolytic toxin will only coagulate on the hemolytic toxin coagulation plate.

6. The method for preparing the tea saponin surfactant and water treatment agent according to claim 5, characterized in that: The horizontal rotary extractor also includes a rotating shaft and a speed regulating device. The rotating shaft drives the hollow rotating body to rotate through a transmission chain, and the speed regulating device is used to change the rotation speed of the hollow rotating body.

7. The method for preparing the tea saponin surfactant and water treatment agent according to claim 5, characterized in that: The horizontal rotary extractor further includes a NaCl solution spray head and a mixing pump provided at the bottom of the horizontal rotary extractor, wherein the mixing pump is used to regularly add limulus amebocyte lysate and NaCl solution through the NaCl solution spray head and to change the spraying speed of the NaCl solution spray head, thereby controlling the product purity of the non-ionic surfactant without hemolytic toxins and the water treatment agent; In step S2, when the horizontal rotary extractor is working, the computer uses a photobioconcentration sensor integrated with a fluorescence colorimeter and / or an electric focusing electrophoresis instrument to monitor in real time the hemolytic toxin concentration of the fermentation mixture, as well as the hemolytic toxin concentration in the hemolytic toxin-free non-ionic surfactant and water treatment agent, as real-time data of raw material concentration and product purity; the computer adjusts the circulation pump according to the real-time data of the sensor to control the raw material concentration and product purity in real time.

8. The method for preparing tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The described flat rotary extractor adopts a flat rotary extractor with a double-layer grid bottom, and its grid bottom includes a fixed grid bottom and an active grid bottom arranged above the fixed grid bottom. The bottom of the active grid bottom is provided with a scraper and the whole is arranged to be rotatable and / or retractable, so that the residue on the fixed grid bottom is scraped off by the active grid bottom to prevent material jamming when the flat rotary extractor is working.

9. The method for preparing tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The step S2 further comprises: treating the wet meal with a vertical desolventizer to recover a water treatment agent; and / or The preparation method further comprises step S3: further separating the nonionic surfactant and the water treatment agent by utilizing physical properties.

10. The method for preparing tea saponin surfactant and water treatment agent according to claim 1, characterized in that: The efficiency of removing hemolytic toxins is 90.0-99.0%; Surfactants without hemolytic toxins are used in food; water treatment agents without hemolytic toxins are used in fish ponds; For nonionic surfactants, liquid products can be obtained directly or powder products can be obtained by drying; for water treatment agents, liquid products can be obtained directly or powder products can be obtained by drying.

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