A mother liquor for a six-carbon easy-to-use biopolymerization auxiliary, its preparation method and application

Abstract

This invention discloses a six-carbon-compatible biopolymerization auxiliary mother liquor, its preparation method, and its applications, belonging to the field of biopolymerization auxiliary technology. Using water, acrylic acid monomer, and caustic soda as basic raw materials, and combined with a six-carbon-compatible biopolymerization auxiliary prepared through microbial metabolic engineering, a 40% concentration mother liquor is obtained through bottom pretreatment, constant-rate dropwise addition reaction, heat-preserved polymerization, reaction termination, and neutralization / dilution steps. The core auxiliary is obtained by constructing recombinant strains by introducing hexokinase and phosphofructokinase genes into *Saccharomyces cerevisiae* or *Lactobacillus delbrueckii*, and directionally synthesizing a six-carbon structural intermediate, which is then purified at high temperature and subjected to sulfonation. This intermediate possesses a biaxial reduction mechanism and high efficiency. The mother liquor is odorless, environmentally friendly, and exhibits excellent compatibility with bio-based raw materials and organic / inorganic systems. It can be widely used in building materials, coatings, chemicals, and polymer polymerization. The preparation process is simple and low-cost, and its performance is significantly superior to traditional chemical synthesis auxiliaries, driving the industry towards biosynthesis.

Description

Technical Field

[0001] This invention relates to the field of biopolymerization auxiliaries, and more specifically, to a six-carbon easy-to-polymerize biopolymerization auxiliary mother liquor, its preparation method, and its application. Background Technology

[0002] In various fields such as materials processing, building materials, and coatings chemicals, polymerization auxiliaries are increasingly in demand as key components for regulating product performance. Traditional polymerization auxiliaries are mostly prepared using chemical synthesis processes, which generally suffer from the following technical shortcomings:

[0003] Poor environmental performance: The production process is prone to generating toxic and harmful byproducts, and the finished products often have an irritating odor. Their use poses potential hazards to the environment and the health of operators, which does not meet the requirements of current green production and environmental protection policies.

[0004] Limited Performance: Most traditional additives can only achieve single functions such as dispersion, stabilization, or polymerization promotion, making it difficult to simultaneously satisfy high activity and high resistance (anti-interference and anti-degradation ability; anti-interference ability is defined as "resistance to heavy metal ions (such as Cu) within 10 ppm"). 2+ Fe 3+ "In a 5% high-salt environment, the activity retention rate of the additive is ≥90%"; the anti-degradation ability is defined as "after 30 days of constant temperature storage at 50℃, the sulfide content decay rate of the additive is ≤5%"; and the application effect is limited under complex working conditions (such as high temperature, multi-component system) due to structural adjustment requirements.

[0005] Insufficient compatibility: Poor compatibility with bio-based raw materials or green polymer materials, which can easily lead to system stratification and performance fluctuations, thus restricting the development and promotion of biodegradable and environmentally friendly products.

[0006] In the synthesis of polymer materials, workability-enhancing biopolymers serve as important auxiliary additives, improving the flowability and stability of the polymerization system and promoting the synthesis and performance optimization of the target product. Traditional workability-enhancing biopolymer mother liquors often require the addition of chain transfer agents during preparation to control the polymerization rate and molecular weight distribution; however, the use of chain transfer agents can easily lead to an irritating odor in the mother liquor, affecting the production environment and the health of operators.

[0007] In addition, the core functional components of traditional additive mother liquors mostly rely on chemical synthesis processes, which have problems such as complex preparation processes, high energy consumption, and poor environmental friendliness. Moreover, the functions of the synthesized substances are limited, making it difficult to meet the demand for high-performance, low-pollution additives in the field of polymer materials.

[0008] To address the aforementioned issues, the industry urgently needs to develop a new generation of green, environmentally friendly, multifunctional, and highly compatible polymerization auxiliaries. While existing bio-based auxiliaries have made breakthroughs in environmental friendliness, they generally suffer from low activity, complex preparation processes, and high costs. Furthermore, they lack targeted design for the "six-carbon structure," which, due to its high molecular stability and good compatibility with most organic / inorganic systems, is an ideal molecular framework for improving the overall performance of auxiliaries. Therefore, developing a biopolymerization auxiliary mother liquor based on a six-carbon structure, possessing high activity and environmentally friendly properties, has become a critical technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0009] To address the shortcomings of the existing technologies, a six-carbon easy-to-use biopolymerization auxiliary mother liquor, its preparation method, and its application are provided. The aim is to obtain a polymerization auxiliary mother liquor with high activity, high irritation, no irritating odor, and green environmental protection characteristics through bio-metabolic engineering modification and targeted synthesis process, while simplifying the preparation process, reducing production costs, and meeting the application needs of multiple fields.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is a mother liquor of a six-carbon-based workable biopolymerization auxiliary, comprising a bottom material and a dropper. The bottom material comprises, by weight, 340 parts water, 320 parts monomer, and 1.5 parts caustic soda flakes. The dropper comprises, by weight, 45 parts water, 30 parts six-carbon-based workable biopolymerization auxiliary, 1.5 parts caustic soda flakes, 6 parts acrylic acid, 3 parts hydrogen peroxide with a weight concentration of 27.5%, and 1.5 parts ferrous sulfate solution with a weight concentration of 1%.

[0011] The aforementioned six-carbon-compatible biopolymerization aid mother liquor is produced by selecting microorganisms, subjecting them to high activation, high stimulation, and nitrogen balance treatment to optimize the metabolic environment, and using microbial metabolic engineering technology to introduce key enzyme genes of the six-carbon synthesis pathway into the microorganisms to construct recombinant microbial strains for the directed synthesis of six-carbon structural intermediates; the fermentation products of the recombinant microorganisms are separated and purified at high temperature to obtain biolipids; the biolipids are then matured and subjected to sulfonation to obtain the finished six-carbon-compatible biopolymerization aid liquid.

[0012] The aforementioned mother liquor for a six-carbon and efficacious biopolymerization aid uses microorganisms selected from *Saccharomyces cerevisiae* or *Lactobacillus delbrueckii*. The microbial screening criteria are: "After inoculation into LB liquid medium (yeast) or MRS liquid medium (lactic acid bacteria), and incubation at 35°C for 48 hours, the accumulation of a six-carbon precursor (such as glucose-6-phosphate) is ≥5 g / L." The key enzyme genes in the six-carbon synthesis pathway include hexokinase genes and phosphofructokinase genes. The sulfonation reaction uses concentrated sulfuric acid as the sulfonating agent. Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate, and phosphofructokinase further catalyzes its conversion to fructose-1,6-bisphosphate. The two work synergistically to construct an efficient six-carbon synthesis pathway, ultimately leading to the targeted synthesis of six-carbon intermediates.

[0013] The gene transfer process for the aforementioned six-carbon and easy-to-polymerize biopolymerization aids is as follows:

[0014] The pET-28a(+) plasmid was used as the vector, containing an ampicillin resistance gene (selective marker), a T7 promoter (regulating target gene expression), and a T7 terminator. The vector and target genes (hexokinase gene and phosphofructokinase gene) were double-digested with restriction endonucleases EcoRI and XhoI. The digestion system consisted of 50 μL of the vector / gene (1 μg), EcoRI (1 μL), XhoI (1 μL), and 10×Buffer (5 μL), incubated at 37°C for 2 h. T4 DNA ligase was used for... The ligation system consisted of "20 μL of system containing 3 μL of digested vector, 5 μL of digested target gene, 1 μL of T4 ligase, and 2 μL of 10×Buffer, ligated overnight at 16℃". After the ligation product was introduced into competent cells, it was inoculated into LB solid medium containing ampicillin (50 μg / mL) and cultured at 37℃ for 48 h. Single colonies were picked for PCR verification. The PCR products were detected by 1.5% agarose gel electrophoresis. The presence of "1200 bp band of hexokinase gene and 1500 bp band of phosphofructokinase gene" indicated a positive recombinant strain.

[0015] The preparation method of the above-mentioned six-carbon easy-to-polymerize biopolymer mother liquor includes the following steps:

[0016] (1) Add the bottom material to the reactor. The bottom material includes 340 parts water, 320 parts monomer, and 1.5 parts caustic soda by weight. After stirring evenly, heat to 60-65℃ and keep warm for 10-15 minutes. This step allows the acrylic acid monomer to be initially neutralized with the caustic soda, adjusting the pH of the system to slightly alkaline (pH 7.0-7.5, measured by a precision pH meter with an accuracy of ±0.01), creating a stable environment for the subsequent dropwise addition reaction. The monomer is acrylic acid monomer.

[0017] (2) Add the feed material to the reactor at a constant rate and stir. The feed material includes 45 parts water, 30 parts carbon six-component biopolymerization auxiliaries, 1.5 parts caustic soda flakes, 6 parts acrylic acid, and 3 parts 27.5% hydrogen peroxide by mass. The addition time is 40-45 min. During the addition, the stirring rate is maintained at 200-300 r / min, and the reaction temperature is kept stable at 60-65℃ by the reactor temperature control system. The addition acceleration rate is adjusted according to the effective volume of the reactor, specifically "3 mL / (min·L reactor volume)", for example, the addition acceleration rate of a 500L reactor is 1.5 kg / min (corresponding to 40 min) or 1.2 kg / min (corresponding to 45 min). This stirring rate can ensure uniform mixing of materials and avoid side reactions caused by excessive local concentration. The constant temperature can ensure the stability of the oxidizing activity of hydrogen peroxide.

[0018] (3) After the addition is complete, keep it at 60-65℃ for 60 min to ensure that the polymerization reaction is fully carried out; then add 1.5 parts of 1% ferrous sulfate solution and stir for 10-15 min; terminate the activity of hydrogen peroxide through redox reaction to avoid over-reaction; the ferrous sulfate solution needs to be quantitatively detected by the "o-phenanthroline spectrophotometric method" for Fe²+ concentration: take 1 mL of ferrous sulfate solution, add 2 mL of 0.1% o-phenanthroline solution and 5 mL of acetate-sodium acetate buffer solution with pH=4.5, make up to 25 mL, react at room temperature in the dark for 15 min (avoid strong light irradiation to avoid Fe²+ oxidation), measure the absorbance at a wavelength of 510 nm, and calculate the concentration by referring to the standard curve (Fe²+ concentration 0-10 μg / mL). The Fe²+ concentration is required to be ≥0.95% (mass concentration), and the solution is light green without yellow precipitate.

[0019] (4) Add 216 parts of water to neutralize and dilute, and cool to room temperature (25±2℃) to obtain a 40% hexacarbon and easy-to-use biopolymerization auxiliary mother liquor.

[0020] The above-mentioned method for preparing a mother liquor of a six-carbon easy-to-polymerize biopolymer auxiliary, in step (2), the core of the method for preparing the six-carbon easy-to-polymerize biopolymer auxiliary lies in combining microbial metabolic engineering and directed synthesis technology to ensure the accurate construction of the six-carbon structure and the integration of the auxiliary function, including the following steps:

[0021] (1) Select microorganisms with the potential for six-carbon skeleton synthesis, and optimize the microbial metabolic environment through high activation treatment, high stimulation induction and nitrogen balance regulation;

[0022] (2) Using microbial metabolic engineering technology, the key enzyme genes of the six-carbon synthesis pathway were introduced into the above-mentioned microorganisms to construct recombinant microbial strains, regulate their metabolic pathways, and directionally synthesize six-carbon intermediates;

[0023] (3) The fermentation products of recombinant microorganisms were placed in a high-temperature separation device to remove impurities and moisture, and the bio-lipids were purified. Fermentation process: The initial pH of the GYM fermentation substrate (20 g / L glucose, 10 g / L yeast extract, 5 g / L peptone) was 6.8-7.0. During the fermentation process, the pH was measured every 12 hours. If the pH was lower than 6.5, 1 mol / L NaOH solution was added to adjust it to 6.8-7.0. At the same time, the concentration of the six-carbon intermediate was detected (the detection method is described below). Fermentation was terminated when the concentration was ≥10 g / L.

[0024] (4) The bio-lipids are matured and then sulfonated to obtain a six-carbon and easy-to-use biopolymerization auxiliary liquid product. This product has the function of synthesizing multiple substances such as poly-sulfides, sulfonated sulfur, and sodium, and has a biaxial reduction mechanism. The sulfonation reaction drop acceleration rate is adjusted according to the bio-lipid mass, specifically "1mL / (min·100g bio-lipids)", for example, 300g bio-lipids corresponds to a concentrated sulfuric acid drop acceleration rate of 3mL / min.

[0025] The above-mentioned method for preparing a mother liquor of a six-carbon and easy-to-polymerize biopolymer auxiliary includes the following steps: In step (1), the high activation treatment is performed at a temperature of 35-40℃ and a pH of 6.5-7.2; the high activation induction uses 0.1-0.3% glucose as an inducer; the metabolic pathway is directed towards six-carbon synthesis; and the nitrogen source concentration is 0.5-0.8 mol / L in the nitrogen balance regulation. This provides sufficient nitrogen nutrition for microbial growth and gene expression.

[0026] In the above-mentioned method for preparing a mother liquor of a six-carbon easy-to-polymerize biopolymer, in step (3), the temperature of the high-temperature separation device is 80-90℃ and the pressure is 0.12-0.15 MPa. This removes bacterial residues, unmetabolized small molecule impurities, and water from the fermentation broth, purifying it to obtain biolipids. These temperature and pressure parameters ensure efficient separation while maintaining the activity of the biolipids, preventing component degradation.

[0027] The above-mentioned method for preparing a mother liquor of a six-carbon easy-to-use biopolymerization auxiliary includes a maturation temperature of 50-55℃ and a maturation time of 2-3h in step (4) to improve the uniformity of the subsequent sulfonation reaction. In the sulfonation reaction, the amount of sulfonating agent is 5-8% of the biolipid content, the sulfonation temperature is 45-50℃, and the sulfonation time is 1.5-2h. The sulfonation reaction can introduce sulfonic acid groups onto the biolipid molecules, significantly improving the water solubility and dispersibility of the auxiliary, and finally obtaining a six-carbon easy-to-use biopolymerization auxiliary liquid with the functions of synthesizing multiple substances such as polysulfides, sulfonated sulfur, and sodium, and with a biaxial reduction mechanism. The biaxial reduction mechanism can simultaneously achieve polymerization promotion and system impurity reduction, breaking through the limitation of the single function of traditional auxiliaries.

[0028] Detection method for six-carbon structure intermediates:

[0029] High-performance liquid chromatography (HPLC, Agilent 1260) was used with a Zorbax SB-C18 column (4.6 × 250 mm, 5 μm), a mobile phase of methanol-water (30:70 v / v), a flow rate of 1.0 mL / min, a detection wavelength of 210 nm, and a column temperature of 30 °C. For standard preparation: 0.2 g, 5 g, 8 g, 10 g, and 12 g of D-fructose-1,6-diphosphate disodium standard were accurately weighed, dissolved in the mobile phase, and diluted to 100 mL. The solutions were then degassed by sonication for 10 min (300 W) to prepare standard solutions of 2, 5, 8, 10, and 12 g / L. 1 mL of fermentation broth was centrifuged at 12000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic phase filter membrane, and 20 μL was injected. The intermediate concentration was calculated based on the peak area against the standard curve.

[0030] Methods for detecting hyperactivity:

[0031] 1. Anti-interference ability test: Take 0.5g of hexacarbon-based easy-to-polymerize biopolymerization aid, add 10ppm Cu 2 Add 50 mL of an aqueous solution of CuSO4·5H2O, stir at 25°C for 30 min, and determine the sulfonic acid group content in the additive using potentiometric titration (refer to GB / T11276-2021). Calculate the sulfonic acid group retention rate (retention rate = sulfonic acid group content after treatment / sulfonic acid group content before treatment × 100%), requiring a retention rate ≥ 90%.

[0032] 2. Degradation resistance test: Take 10g of hexacarbon-based easy-to-polymerize biopolymer, seal it, and store it at 50℃ for 30 days. Use the "gravimetric method" to test the sulfide content (refer to GB / T17041-2012), and calculate the sulfide decay rate (decay rate = (initial sulfide content - sulfide content after storage) / initial sulfide content × 100%). The decay rate should be ≤5%.

[0033] Biaxial reduction mechanism chemical reaction equation

[0034] 1. Reduction of Fe by hydroxyl groups (-OH) 3 +:

[0035] 2R-OH+2Fe 3 +→2R-O·+2Fe 2 ++2H+

[0036] (R represents the six-carbon easy biopolymerization auxiliary molecular skeleton, and the product RO· is further stable in the system without toxic byproducts.)

[0037] 2. Sulfonate groups (-SO3Na) reduce hydrogen peroxide:

[0038] R-SO3Na+H2O2+H+→R-SO4H+Na++H2O

[0039] (The R-SO4H generated in the reaction still has dispersibility, which can help improve the stability of the system.)

[0040] The application of a C6 workability biopolymerization admixture mother liquor in the construction materials field is as follows: In the construction materials field, the mother liquor is used as a concrete admixture to improve the workability and compressive strength of concrete. Concrete workability testing is conducted according to GB / T50080-2016 "Standard for Test Methods of Performance of Ordinary Concrete Mixtures," testing the initial slump and 1-hour slump loss rate, requiring an initial slump ≥200mm and a 1-hour slump loss rate ≤15%. Compressive strength testing is conducted according to GB / T50081-2019 "Standard for Test Methods of Physical and Mechanical Properties of Concrete," testing the 28-day compressive strength, requiring ≥45MPa. In the coatings and chemical industry, the mother liquor is used as a coating dispersant and stabilizer. Coating dispersibility testing is conducted according to GB / T6753.1-2007 "Determination of Grinding Fineness of Paints, Varnishes and Printing Inks," requiring a fineness ≤30μm. m; Storage stability testing refers to GB / T6753.3-1986 "Test Method for Storage Stability of Coatings", which requires storage at 50℃ for 30 days, with no stratification or clumping; In the field of polymer polymerization, the mother liquor is used as a polymerization reaction aid to improve monomer polymerization conversion rate and control polymer molecular weight distribution; Polymerization conversion rate is tested using the "gravimetric method": After the reaction, methanol is added to precipitate the polymer, which is then filtered and dried to constant weight, and the conversion rate is calculated (conversion rate = polymer mass / total monomer mass × 100%), requiring ≥95%; Molecular weight distribution is tested using gel permeation chromatography (GPC, model: Waters1515), with a Styragel HR4E column, tetrahydrofuran as the mobile phase, a flow rate of 1 mL / min, and a column temperature of 35℃, requiring a molecular weight distribution dispersion index (PDI) ≤1.8.

[0041] The beneficial effects of the six-carbon easy-to-use biopolymerization auxiliary mother liquor, its preparation method, and its application are that, based on the design of the six-carbon molecular skeleton, the mother liquor has excellent compatibility with bio-based raw materials, organic polymer materials, and inorganic fillers, which can effectively avoid system stratification and performance fluctuations, and is suitable for multi-field composite systems.

[0042] By combining "microbial metabolic engineering" (biosynthesis technology) with "introduction of key enzyme genes in the six-carbon synthesis pathway" (directed synthesis of six-carbon structures), an auxiliary agent with both "six-carbon structural stability" and "bio-based environmental friendliness" is prepared through a continuous process of "high activation, gene modification, high-temperature purification, and sulfonation".

[0043] The core additives are prepared through bio-metabolic engineering. The finished product has no irritating odor and the production process has no toxic or harmful byproduct emissions, which meets the requirements of green environmental protection and solves the environmental pain points of traditional chemical synthesis additives.

[0044] Through bio-activation, high-excitation, and nitrogen-balanced treatment, followed by microbial metabolic engineering to introduce key enzyme genes in the synthetic pathway, regulating the structure of large and small molecules, and high-temperature separation and purification, bio-lipids are obtained. Finally, through maturation and sulfonation, the finished liquid product is obtained. This finished liquid product has the synthetic function of multiple substances such as polysulfides and sodium sulfonate. It possesses high activity and high excitability, and has a biaxial reduction mechanism, which can simultaneously achieve polymerization promotion (the six-carbon structure enhances molecular reactivity), system stability (sulfonic acid groups enhance dispersibility), and impurity reduction (the biaxial reduction mechanism removes impurities from the system), breaking through the limitations of traditional additives with single performance.

[0045] The additives of this invention form "biaxial reduction sites" by the hydroxyl groups (-OH) on the bio-lipid molecules and the sulfonate groups (-SO3Na): the hydroxyl groups can reduce trace metal impurities (such as Fe) in the polymerization system. 3 +), to avoid side reactions; the sulfonate group can reduce hydrogen peroxide residue, precisely terminate the polymerization reaction, and achieve the integration of three functions: "polymerization promotion, impurity removal and reaction rate control". Traditional additives require 2-3 additives to achieve the same effect.

[0046] Existing bio-based additives suffer from complex preparation processes requiring multiple enzymatic catalysis steps and incurring high costs—2-3 times that of chemical additives—making large-scale application difficult. This invention, however, requires only four steps: microbial culture, gene delivery, high-temperature purification, and sulfonation. The preparation process eliminates the need for complex equipment, and key parameters (such as temperature and dropping time) are easily controlled. The raw materials are primarily water, monomers, and bio-lipids, eliminating the need for expensive enzyme catalysts. The low cost and readily available raw materials make it suitable for large-scale production, solving the problems of high cost and complex processes in existing bio-based additive preparations. This shortens the production cycle, making bio-based additives economically competitive with chemical additives and driving the industry's upgrade from "chemical synthesis" to "biosynthesis."

[0047] Based on a six-carbon molecular framework design, its molecular structure exhibits excellent compatibility with bio-based raw materials (such as polylactic acid), organic polymers (such as polyethylene), and inorganic fillers (such as cement particles), effectively avoiding system stratification and performance fluctuations. It is suitable for composite systems in multiple fields such as building materials, coatings, chemicals, and polymer polymerization. The mother liquor of this invention demonstrates excellent performance in the three major fields of polymer polymerization, coatings, and building materials.

[0048] In the construction and building materials industry, the core additives are prepared using a biological method, resulting in no toxic byproducts during production and no irritating odor in the finished product. This helps the construction and coating industries overcome the transformation bottleneck of "difficulty in balancing environmental protection and performance." For example, in the field of indoor construction, concrete using the mother liquor of this invention can directly meet the GB / T18883-2022 "Indoor Air Quality Standard" without additional odor treatment, reducing environmental costs for enterprises. As a concrete admixture, it can improve the workability and compressive strength of concrete. Through the adsorption of the six-carbon structure on the surface of cement particles, it reduces the frictional resistance between particles, increases the initial slump of concrete, and reduces the slump loss rate. At the same time, the polysulfides in the additive can react with cement hydration products to generate stable hydration products, improving the 28-day compressive strength of concrete.

[0049] In the field of coatings and chemicals, as a dispersant and stabilizer, it can improve the uniformity and storage stability of coating systems. The hydrophilic-hydrophobic balance of the six-carbon structure can improve the dispersion of pigments and fillers in coatings and prevent particle agglomeration. At the same time, the high efficacity of the additive can inhibit the stratification and deterioration of coatings during storage, extending the shelf life of coatings from the traditional 6 months to 10 months. It also has excellent compatibility with water-based coatings and bio-based coatings (such as polylactic acid-based coatings), and the mixed system does not exhibit stratification or precipitation.

[0050] In the field of polymer polymerization, as a polymerization reaction aid, it can improve monomer polymerization conversion rate and regulate polymer molecular weight distribution. In the preparation of general-purpose plastics such as polyethylene and polypropylene, as well as biodegradable plastics (such as polycaprolactone), the high activity of the aid can accelerate the monomer polymerization reaction and improve the polymerization conversion rate; at the same time, the biaxial reduction mechanism can precisely regulate the polymer chain growth process, reduce the molecular weight distribution dispersion index, and ensure the stability of the mechanical and processing properties of polymer products. In the preparation of biodegradable plastics (PLA), it can simultaneously improve the tensile strength and elongation at break of PLA, solving the problem of traditional aids "increasing strength inevitably reduces toughness", and providing a new solution for the performance optimization of biodegradable materials. Detailed Implementation

[0051] The present invention will now be described in detail with reference to specific embodiments.

[0052] Example 1

[0053] A method for preparing a six-carbon-based, workable biopolymerization auxiliary includes the following steps:

[0054] (1) Select Saccharomyces cerevisiae (strain number: ATCC26603), inoculate it into LB liquid medium, set the high activation temperature to 35℃ and pH 6.5, and culture for 24h; then add 0.1% glucose solution as an inducer and continue to culture for 18h to complete the high activation; finally add ammonium chloride to adjust the nitrogen source concentration to 0.5mol / L, culture for 12h to achieve nitrogen balance, and obtain the pretreated yeast.

[0055] (2) The hexokinase gene and phosphofructokinase gene were introduced into pretreated yeast using electroporation (the gene introduction vector was pET-28a(+)). The yeast with the target gene was inoculated into a selection medium containing ampicillin (concentration 50 μg / mL) and cultured at 37°C for 48 h. Positive recombinant strains were screened by PCR verification.

[0056] (3) The recombinant yeast was inoculated into a 50L fermenter and fermented for 72h at 32℃, dissolved oxygen 20% and stirring rate 200r / min using glucose, yeast extract and peptone (GYM) as fermentation substrate. During fermentation, samples were taken every 12h and the pH was adjusted to 6.8-7.0 with 1mol / L NaOH. At the same time, the concentration of the six-carbon intermediate was detected by HPLC. The concentration of the intermediate was 10.2g / L at 72h and fermentation was terminated. After fermentation, the fermentation liquid was transferred to a high-temperature separation device, and the temperature was set at 80℃ and the pressure at 0.12MPa. The liquid was distilled under reduced pressure for 4h to remove impurities and water to obtain crude biolipids. The crude biolipids were purified by recrystallization with anhydrous ethanol to obtain refined biolipids with a purity of 96.1%.

[0057] (4) The refined bio-lipids were placed in a constant temperature reactor, and the maturation temperature was set to 50℃ and the maturation time to 2h (lower limit of maturation parameters) to complete the maturation. 5% concentrated sulfuric acid (calculated based on the mass of bio-lipids, with a dropping rate of 1mL / (min·100g bio-lipids)) was added, and the sulfonation temperature was set to 45℃ and the sulfonation time to 1.5h. The reaction was stirred. After the reaction was completed, the residue was removed by washing with deionized water three times and then dried under vacuum at 60℃ and 0.08MPa for 2h to obtain a six-carbon easy-to-polymerize biopolymer (named B-870L additive). The additive was tested and found to have no irritating odor, a sulfide content of 11.8%, an activity retention rate of 94.8%, no precipitation, and a six-carbon structure intermediate conversion rate of 92.0%. High susceptibility testing showed that the sulfonic acid group retention rate was 91.2% after treatment with 10ppm Cu²+, and the sulfide decay rate was 4.8% after 30 days of storage at 50℃, which met the functional requirements.

[0058] Example 2

[0059] Preparation of 40% C6 and workability biopolymerization auxiliaries mother liquor:

[0060] Raw material preparation (by weight)

[0061] Bottom material: Water 340g, acrylic acid monomer 320g (purity ≥99.5%), caustic soda flakes 1.5g (purity ≥98%)

[0062] Additives: 45g water, 30g B-870L additive (prepared in Example 1, six-carbon intermediate conversion rate 92.0%), 1.5g caustic soda flakes, 6g acrylic acid, 3g 27.5% hydrogen peroxide

[0063] Other: 1.5g of 1% ferrous sulfate solution (Fe²⁺ concentration 0.96% as determined by o-phenanthroline spectrophotometry), 216g of neutralizing water.

[0064] Preparation process:

[0065] 1. Add the bottom material to a 500L stainless steel reactor, start stirring, set the stirring speed to 200r / min, and heat to 60℃; keep at this temperature for 15min to ensure that the acrylic acid monomer and caustic soda are fully neutralized, and take a sample to test the pH of the system to 7.0.

[0066] 2. The feed material is added to the reactor at a constant rate through a dropping funnel. Based on a reactor volume of 500L, the dropping rate is 1.5kg / min (3mL / (min·L×500L)), ensuring that the addition is completed within 40 minutes. During the addition process, the temperature is maintained at 60℃ by temperature control of the reactor jacket, and a stirring rate of 200r / min is used to ensure that the B-870L additive is evenly mixed with other materials to avoid side reactions caused by excessively high local concentrations.

[0067] 3. After the addition is complete, continue to keep warm at 60℃ for 60 minutes to ensure the polymerization reaction is complete; add 1.5 parts of 1% ferrous sulfate solution and stir for 15 minutes to completely stop the activity of hydrogen peroxide and avoid over-reaction.

[0068] 4. Finally, add 216 parts of neutral water and stir for 20 minutes to dilute evenly; turn on the cooling system to lower the temperature to 25°C to obtain a 40% hexacarbon and workable biopolymerization aid mother liquor.

[0069] Mother liquor performance testing:

[0070] The mother liquor was tested according to relevant national standards, and the results are as follows: solid content 39.7% (close to the design requirement of 40%), pH 7.2 (neutral range, suitable for multiple systems), viscosity (25℃): 860 mPa·s (good fluidity, easy to store and use), no irritating odor, acrylic acid polymerization conversion rate: 95.8% (gravimetric method); VOC content: 8.5 g / L. All indicators meet the standards. Although some indicators are slightly lower than the intermediate limit parameters of the product, there is no performance degradation, proving that this lower limit combination is feasible for production.

[0071] Example 3

[0072] A method for preparing a six-carbon-based, workable biopolymerization auxiliary includes the following steps:

[0073] (1) Saccharomyces cerevisiae was selected and inoculated into LB liquid medium. The temperature was set at 38℃ and the pH at 7.0, and high activation culture was carried out for 24h. Then, 0.2% glucose solution was added as an inducer, and the culture was continued for 18h to complete the high activation. Finally, ammonium chloride was added to adjust the nitrogen source concentration to 0.6mol / L, and the culture was carried out for 12h to achieve nitrogen balance. The activation temperature of 38℃ and the inducer concentration of 0.2% glucose were selected because when the temperature is below 35℃, the activity of the strain is only 1.0 and the yield of six-carbon intermediate is reduced by 18%; when the temperature is above 40℃, the strain's metabolism is disordered and the by-products increase by 12%; at the concentration of 0.2% glucose, the activity of the key enzyme in the six-carbon synthesis pathway is increased by 25% compared with the concentration of 0.1% glucose, and the by-products are reduced by 9% compared with the concentration of 0.3%, which proves that this parameter is the optimal innovative choice for the balance of "activity-by-products".

[0074] (2) The hexokinase gene and phosphofructokinase gene were introduced into pretreated yeast by electroporation (the vector and ligation method are the same as in Example 1). The yeast with the target gene was inoculated into a selection medium containing ampicillin (concentration 50 μg / mL) and cultured at 37°C for 48 h. Positive recombinant strains were screened by PCR verification.

[0075] Unlike traditional bio-adjuvants that rely on random synthesis through natural microbial metabolism, this invention constructs a directed six-carbon synthesis pathway of 'glucose → glucose-6-phosphate → fructose-1,6-bisphosphate' by introducing hexokinase and phosphofructokinase genes. This increases the conversion rate of six-carbon intermediates to over 92% (compared to only 60%-70% in traditional biological methods), solving the problems of "uncontrollable structure and low yield" in biosynthesis.

[0076] (3) The recombinant yeast was inoculated into a 50L fermenter and fermented for 72h at 32℃, dissolved oxygen 20% and stirring rate 200r / min using glucose, yeast extract and peptone (GYM) as fermentation substrate. Samples were taken at 48h of fermentation and the concentration of the six-carbon intermediate was 11.5g / L by HPLC. The concentration was 12.1g / L when fermentation continued for 72h and fermentation was terminated. After fermentation, the fermentation liquid was transferred to a high-temperature separation device and distilled under reduced pressure at 85℃ and 0.13MPa for 4h to remove impurities and water to obtain crude biolipids. The crude biolipids were purified by recrystallization with anhydrous ethanol to obtain refined biolipids with a purity ≥96%.

[0077] (4) The refined bio-lipid was placed in a constant temperature reactor and matured at 52℃ for 2.5h; then 7% concentrated sulfuric acid (dropping rate 1mL / (min·100g bio-lipid)) was added, and the mixture was stirred at 48℃ for 1.8h; after the reaction, the residue was removed by washing three times with deionized water, and then dried under vacuum at 60℃ and 0.08MPa for 2h to obtain a six-carbon easy-to-polymerize biopolymer auxiliary (Auxiliary B-870). The auxiliary agent was found to have no irritating odor, a sulfide content of 12.5%, an activity retention rate of 95.3%, no precipitation, and a six-carbon intermediate conversion rate of 92.8%; high susceptibility testing showed that after treatment with 10ppm Cu²+, the sulfonic acid group retention rate was 93.5%, and the sulfide decay rate after 30 days of storage at 50℃ was 3.2%, meeting the functional requirements.

[0078] By employing low-temperature curing at 50-55℃ (traditional curing temperatures of 60-70℃ can easily lead to biolipid degradation) and precise sulfonation at 45-50℃, the uniformity of the reaction between the sulfonating agent (concentrated sulfuric acid) and the biolipid is improved. The content of polysulfides in the final additive is stable, and the content of sodium sulfide is ≥8%, avoiding the problem of "local over-sulfonation leading to decreased activity" in traditional processes.

[0079] Example 4

[0080] Preparation of 40% C6 and workability biopolymerization auxiliaries mother liquor:

[0081] Raw material preparation (by weight):

[0082] Bottom material of the reactor (water 340, acrylic acid monomer 320, caustic soda flakes 1.5)

[0083] Additives (water 45, B-870 additive 30, caustic soda 1.5, acrylic acid 6, 27.5% hydrogen peroxide 3)

[0084] 1% ferrous sulfate solution 1.5 (Fe²+ concentration 0.98%), neutralizing water 216; all raw materials are industrial grade, of which acrylic acid monomer purity ≥99.5% and caustic soda flakes purity ≥98%.

[0085] Preparation process:

[0086] 1. Add the bottom material to a 500L stainless steel reactor, start stirring (speed 250r / min), heat to 62℃, hold for 12min, and take a sample to test the pH of the system. The pH is 7.1, which meets the initial requirements of the reaction.

[0087] 2. The feed material is added dropwise to the reactor at a constant rate using a dropping funnel. Based on a reactor volume of 500L, the dropping rate is 1.2 kg / min (3 mL / (min·L×500L)), ensuring the addition is completed within 45 minutes. During the dropping process, the temperature is maintained at 62℃ using the reactor jacket temperature control to prevent hydrogen peroxide decomposition due to temperature fluctuations. If the dropping time is less than 40 minutes, the local hydrogen peroxide concentration is too high, leading to explosive polymerization of acrylic acid monomers and a mother liquor viscosity fluctuation of ±20%. If the dropping time is longer than 50 minutes, the reaction is incomplete, resulting in an 8% reduction in the content of active components. At a stirring rate of 250 r / min, the mother liquor uniformity (particle size distribution variation coefficient) is 0.12, lower than 0.25 at 200 r / min and 0.18 at 300 r / min, proving that this process can achieve the optimal effect of "complete reaction - uniform system".

[0088] 3. After the addition is complete, continue to keep the temperature at 62℃ for 60 minutes to ensure the polymerization reaction is complete; then add 1.5 parts of 1% ferrous sulfate solution and stir for 12 minutes to terminate the reaction;

[0089] 4. Finally, add 216 parts of neutral water, stir for 20 minutes to dilute evenly, turn on the cooling system to lower the temperature to 28℃, and obtain a 40% hexacarbon and workable biopolymerization aid mother liquor.

[0090] Mother liquor performance testing:

[0091] The mother liquor was tested in accordance with relevant national standards, and the results are as follows: solid content 40.2% (meets the 40% design requirement), pH 7.3 (neutral range, suitable for multiple systems), viscosity (25℃): 800mPa·s (good fluidity, easy to store and use), no irritating odor, acrylic acid polymerization conversion rate 97.2% (gravimetric method), and all indicators meet the standards.

[0092] Application scenarios:

[0093] The 40% hexacarbon-based easy-to-polymerize biopolymerization auxiliary mother liquor described in this invention can be widely used in the following fields:

[0094] In the construction materials field: the appropriate cement type is PO42.5 ordinary Portland cement (refer to GB175-2020 "General Portland Cement"). The concrete aggregate is continuously graded crushed stone (particle size 5-20mm) and medium sand (fineness modulus 2.3-3.0), with a water-cement ratio of 0.45. Add mother liquor at 0.4% of the cement mass. The workability of the concrete is tested according to GB / T50080-2016 "Standard for Test Methods of Performance of Ordinary Concrete Mixtures", testing the initial slump and 1-hour slump loss rate, requiring an initial slump ≥200mm and a 1-hour slump loss rate ≤15%. The compressive strength is tested according to GB / T50081-2019 "Standard for Test Methods of Physical and Mechanical Properties of Concrete", testing the 28-day compressive strength, requiring ≥45MPa.

[0095] As a concrete admixture, it can improve the workability of concrete, reduce slump loss, and enhance the compressive strength of concrete. It has no irritating odor and meets indoor building environmental protection standards. "The admixture can simultaneously synthesize polysulfides (enhancing polymerization activity) and sodium sulfonate (enhancing dispersibility). In the concrete system, polysulfides can react with cement hydration products (Ca(OH)2) to form stable sulfoaluminate, increasing compressive strength; sodium sulfonate can reduce the surface tension of cement particles, improving workability and resolving the contradiction of traditional admixtures where 'single function makes it difficult to balance workability and strength.'" Application testing: Adding 0.4% mother liquor to C35 concrete resulted in an initial slump of 230mm, a 1-hour slump loss of 12%, and a 28-day compressive strength of 49.8MPa, meeting GB / T50080 and GB / T50081 standards.

[0096] In the coatings and chemical industry: Suitable for water-based latex paints (PVC 50%, film-forming substance is styrene-acrylic emulsion), add the mother liquor at 0.8% of the total mass of the paint; the paint dispersibility test refers to GB / T6753.1-2007 "Determination of grinding fineness of paints, varnishes and printing inks", requiring a fineness ≤30μm; the storage stability test refers to GB / T6753.3-1986 "Test method for storage stability of coatings", store at 50℃ for 30 days, requiring no stratification or clumping.

[0097] As a dispersant and stabilizer in coatings, it can improve the uniformity of coating systems and extend the shelf life of coatings from 6 months to 10 months. It also exhibits excellent compatibility with water-based and bio-based coatings, showing no stratification. Application testing: Adding 0.8% of the mother liquor to water-based latex paint, with a fineness of 28μm, resulted in no stratification after 30 days of storage at 50℃, meeting GB / T6753.1 and GB / T6753.3 standards.

[0098] In the field of polymer polymerization, when applied to the preparation of polycaprolactone (PCL), the polymerization reaction temperature is 140℃, the pressure is 0.1MPa, the catalyst is stannous octoate (added at 0.1%, based on the mass of caprolactone monomer), the reaction time is 4h, and the mother liquor is added 30min after the start of the polymerization reaction; when applied to acrylamide polymerization, the mother liquor is added at 1.5% of the monomer mass; the polymerization conversion rate is detected by the "gravimetric method": after the reaction is completed, methanol is added to precipitate the polymer, which is then filtered and dried to constant weight (dried at 105℃ until the mass change is ≤0.1%), and the conversion rate is calculated (conversion rate = polymer mass / total monomer mass × 100%), requiring ≥95%; the molecular weight distribution is detected by gel permeation chromatography (GPC, model: Waters1515), the chromatographic column is Styragel HR4E, the mobile phase is tetrahydrofuran, the flow rate is 1mL / min, the column temperature is 35℃, and the molecular weight distribution dispersion index (PDI) is required to be ≤1.8.

[0099] As a polymerization accelerator, it can improve monomer polymerization conversion and regulate polymer molecular weight distribution. In the preparation of general-purpose plastics such as polyethylene and polypropylene, as well as biodegradable plastics (such as polycaprolactone), the high activity of the accelerator can accelerate the monomer polymerization reaction and improve the polymerization conversion rate. At the same time, the biaxial reduction mechanism can precisely regulate the polymer chain growth process, reduce the molecular weight distribution dispersion index, and ensure the stability of the mechanical and processing properties of polymer products. In the preparation of biodegradable plastics (PLA), it can simultaneously improve the tensile strength and elongation at break of PLA, solving the problem of traditional accelerators "increasing strength inevitably reduces toughness", and providing a new solution for the performance optimization of biodegradable materials. Application testing: Adding 1.5% mother liquor to acrylamide polymerization resulted in a conversion rate of 98.5%, a PAM molecular weight of 12 million, and a PDI of 1.7, meeting the performance requirements of polymer materials.

[0100] Example 5

[0101] Verification of the application effects of mother liquor in multiple fields

[0102] To verify the practical application value of the mother liquor of this invention, application tests were conducted in three core fields: building materials, coatings and chemicals, and polymer polymerization. The results were compared with those of traditional chemical synthesis auxiliary agent mother liquor (control group). The results are as follows:

[0103] Application of Concrete Admixtures in the Construction and Building Materials Sector

[0104] Test procedure: In the preparation of C35 concrete, the mother liquor of this invention was added at 0.4% of the cement mass (experimental group), and the control group was added with the same amount of traditional polycarboxylate admixture mother liquor; the initial slump, 28-day compressive strength and odor characteristics of the concrete were tested (refer to GB / T18883-2022 "Indoor Air Quality Standard").

[0105] Test results: The initial slump of the concrete in the experimental group was 230 mm (compared to 190 mm in the control group, an increase of 21%), and the 28-day compressive strength was 49.8 MPa (compared to 42.1 MPa in the control group, an increase of 18.3%). It also had no irritating odor (TVOC content 0.3 mg / m³). 3 The control group concrete met the limits specified in GB / T18883-2022; the control group concrete had a slightly pungent odor (TVOC content 0.8 mg / m³). 3 This aligns with the environmental and performance advantages of this invention.

[0106] Application of dispersants in coatings and chemicals: water-based latex paints

[0107] Test procedure: In the preparation of white interior wall water-based latex paint (PVC 50%), the mother liquor of this invention was added at 0.8% of the total mass of the paint (experimental group), and the control group was added with the same amount of traditional sodium pyrophosphate dispersant; the fineness of the paint (GB / T6753.1), storage stability (50℃ constant temperature storage for 30 days, GB / T6753.3) and compatibility were tested.

[0108] Test results: The experimental group coating had a fineness of 28 μm (compared to 55 μm in the control group, a reduction of 49.1%), and showed no stratification or clumping after 30 days of storage (compared to slight stratification in the control group after 20 days of storage), demonstrating complete compatibility with the latex paint system; this verifies the dispersion stability and compatibility advantages of the mother liquor of this invention.

[0109] Applications of Polyacrylamide (PAM) as a Polymerization Aid in Polymerization

[0110] Test procedure: During the polymerization of acrylamide monomers to prepare PAM, the mother liquor of this invention (experimental group) was added at 1.5% of the monomer mass, and the control group was added with the same amount of traditional sodium bisulfite auxiliary agent; the monomer polymerization conversion rate (gravimetric method), PAM molecular weight and dissolution time were detected (GB / T13940-2008 "Polyacrylamide").

[0111] Test results: The monomer polymerization conversion rate of the experimental group was 98.5% (compared to 85.2% in the control group, an increase of 15.6%), the molecular weight of PAM was 12 million (compared to 8 million in the control group, an increase of 50%), and the dissolution time was 30 min (compared to 60 min in the control group, a decrease of 50%); fully demonstrating the high activity and molecular weight regulation capability of the mother liquor of this invention.

[0112] Therefore, based on the high stability of the six-carbon molecular skeleton and the multifunctional integrated properties of the additives, this mother liquor can be widely used in the following fields, and its application effect is significantly better than that of traditional additives:

[0113] In the construction materials industry: As a concrete admixture, it can improve the workability and compressive strength of concrete. Through the adsorption of the six-carbon structure on the surface of cement particles, it reduces the frictional resistance between particles, increasing the initial slump of concrete by 20-30% and reducing the slump loss rate by 15-20%. At the same time, the polysulfides in the admixture can react with cement hydration products to generate stable hydration products, increasing the 28-day compressive strength of concrete by 10-12%. Moreover, the mother liquor has no irritating odor and meets indoor building environmental protection standards (such as GB / T18883-2022 "Indoor Air Quality Standard").

[0114] In the coatings and chemical industry: As a dispersant and stabilizer, it can improve the uniformity and storage stability of coating systems. The hydrophilic-hydrophobic balance of the six-carbon structure can increase the dispersion of pigments and fillers in coatings by 25-30%, preventing particle agglomeration. At the same time, the high efficacity of the additive can inhibit the stratification and deterioration of coatings during storage, extending the shelf life of coatings from the traditional 6 months to 10 months. It also has excellent compatibility with water-based coatings and bio-based coatings (such as polylactic acid-based coatings), and the mixed system does not exhibit stratification or precipitation.

[0115] In the field of polymer polymerization: as a polymerization reaction aid, it can improve monomer polymerization conversion rate and regulate polymer molecular weight distribution. In the preparation of general-purpose plastics such as polyethylene and polypropylene, as well as biodegradable plastics (such as polycaprolactone), the high activity of the aid can accelerate the monomer polymerization reaction, increasing the polymerization conversion rate from the traditional 85% to over 98%. At the same time, the biaxial reduction mechanism can precisely regulate the polymer chain growth process, reducing the molecular weight dispersion index from 2.5 to 1.8, ensuring the stability of the mechanical and processing properties of polymer products.

[0116] Example 6

[0117] A method for preparing a six-carbon-based, workable biopolymerization auxiliary includes the following steps:

[0118] (1) Select Saccharomyces cerevisiae, inoculate into LB liquid medium, set high activation temperature 40℃ and pH 7.2, and culture for 24h; add 0.3% glucose solution (high activation inducer concentration limit) as inducer, continue to culture for 18h to complete high activation; add ammonium chloride to adjust nitrogen source concentration to 0.8mol / L (nitrogen balance nitrogen source concentration limit), culture for 12h to achieve nitrogen balance, and obtain pretreated yeast.

[0119] (2) Same as in Example 1 (vector is pET-28a(+), enzyme digestion and ligation are consistent with PCR verification method), ensuring no difference in gene introduction scheme, only changing microbial pretreatment parameters.

[0120] (3) The fermentation process was the same as in Example 1. After 60 hours of fermentation, the concentration of the six-carbon intermediate was 12.8 g / L as detected by HPLC, and the fermentation was terminated. After the fermentation broth was transferred to a high-temperature separation device, the temperature was set to 90℃ and the pressure to 0.15 MPa, and the pressure was reduced for 4 hours to remove impurities and water, and crude biolipids were obtained. After recrystallization and purification with anhydrous ethanol, the purity of the refined biolipids reached 97.2%.

[0121] (4) The refined bio-lipids were placed in a constant temperature reactor, and the maturation temperature was set at 55℃ for 3 hours to complete the maturation. 8% concentrated sulfuric acid (dropping rate 1 mL / (min·100g bio-lipids)) was added, and the sulfonation temperature was set at 50℃ for 2 hours. The mixture was stirred and reacted. Subsequent washing and drying steps were the same as in Example 1, yielding a six-carbon easy-to-polymerize biopolymer auxiliary (B-870H auxiliary). Testing showed that the auxiliary had no irritating odor, a sulfide content of 13.2%, an activity retention rate of 95.5%, no precipitation, a six-carbon intermediate conversion rate of 93.5%, and a high-temperature stable dispersion time of 49 hours. High-temperature stability test: 10 ppm Cu 2 The sulfonic acid group retention rate after treatment is 95.1%, and the sulfide decay rate is 2.5% after 30 days of storage at 50℃, which meets the functional requirements.

[0122] Example 7

[0123] Preparation of 40% C6 and workability biopolymerization auxiliaries mother liquor:

[0124] Raw material preparation (by weight)

[0125] Bottom material: Water 340g, acrylic acid monomer 320g (purity ≥99.5%), caustic soda flakes 1.5g (purity ≥98%)

[0126] Additives: 45g water, 30g B-870L additive (prepared in Example 6, six-carbon intermediate conversion rate 93.5%), 1.5g caustic soda flakes, 6g acrylic acid, 3g 27.5% hydrogen peroxide

[0127] Other: 1% ferrous sulfate solution 1.5 (Fe 2 +concentration 0.99%), neutralized water 216

[0128] Preparation process:

[0129] 1. Add the bottom material to the 500L reactor, start stirring, set the stirring speed to 300r / min, heat to 65℃, and hold for 10min to prevent premature polymerization of acrylic acid monomers. Take a sample to test the pH of the system, which is 7.3.

[0130] 2. The additive is added to the reactor at a constant rate through a dropping funnel. Based on a reactor volume of 500L, the dropping rate is 1.2kg / min, ensuring that the addition is completed within 45 minutes. During the dropping process, the temperature is maintained at 65℃, and a high stirring rate of 300r / min is used to further improve the dispersion uniformity of the additive and enhance its synergistic effect with other materials.

[0131] 3. After the addition is complete, continue to keep warm at 65℃ for 60 minutes to ensure the polymerization reaction is complete; add 1.5 parts of 1% ferrous sulfate solution and stir for 10 minutes to precisely stop the hydrogen peroxide activity and avoid excessive reduction.

[0132] 4. Finally, add 216 parts of neutral water and stir for 20 minutes to dilute evenly; turn on the cooling system to lower the temperature to 30°C to obtain a 40% hexacarbon and workable biopolymerization aid mother liquor.

[0133] Mother liquor performance testing:

[0134] The mother liquor was tested according to relevant national standards, and the results are as follows: solid content 40.4% (meets the 40% design requirement), pH 7.1 (neutral range, suitable for multiple systems), viscosity (25℃): 770 mPa·s (good fluidity, easy to store and use), no irritating odor, acrylic acid polymerization conversion rate: 99.3% (gravimetric method); VOC content: 7.6 g / L. High-temperature stable dispersion time was 48.5 h, and all indicators met the standards. Based on the mother liquor prepared with the six-carbon workability biopolymerization aid in Example 6, the mother liquor polymerization conversion rate, fluidity, and stability are all superior to the lower limit combination, and there are no side reactions, proving that this higher limit combination can be safely used in production and can further improve product performance.

[0135] The gene transfer (electroconversion) operation parameters described in this invention are as follows:

[0136] Preparation of competent cells: Using 0.1 mol / L CaCl2 solution (pre-cooled to 4°C), the bacterial cells were incubated on ice for 30 min, centrifuged at 5000 r / min for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in sterile physiological saline to prepare competent cells (concentration 1×10⁻⁶). 8 -1×10 9 CFU / mL, concentration verified by a hemocytometer.

[0137] Electroporation conditions: The electroporator was a Bio-RadGenePulserXcell, with parameters set as follows: voltage 1.5-2.0kV, capacitance 25-50μF, resistance 200Ω, and electroporation time 2-5ms. The concentrations of the introduced hexokinase gene and phosphofructokinase gene were both 100ng / μL, and 10μL of gene solution was added to every 100μL of competent cells.

[0138] Resuscitation after electroporation: Immediately after electroporation, 1 mL of LB liquid medium (containing 50 μg / mL ampicillin) was added to competent cells, and the cells were cultured at 37°C and 200 r / min for 1 h with shaking to restore cell viability and expression of resistance genes, and then inoculated into selection medium.

[0139] Screening for positive recombinant strains: The concentration of ampicillin in the screening medium was precisely 50 μg / mL (prepared with sterile water and filtered for sterilization). After incubation at 37℃ for 48 h, PCR verification was performed. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles, and a final extension at 72℃ for 10 min. The amplified products were detected by 1.5% agarose gel electrophoresis. The presence of the expected bands (hexokinase gene band length 1200 bp, phosphofructokinase gene band length 1500 bp) indicated a positive strain.

[0140] Fermentation process control:

[0141] GYM fermentation substrate ratio (mass concentration): glucose 20g / L, yeast extract 10g / L, peptone 5g / L. Adjust the substrate pH to 6.8-7.0 with 1mol / L sodium hydroxide. Autoclave at 121℃ for 20min. The aeration rate of the 50L fermenter is 1-2L / min, the stirring rate is 200r / min, the dissolved oxygen level is monitored in real time using a dissolved oxygen electrode and maintained at 20±2%, the fermentation temperature is 32±1℃, and the concentration of the six-carbon intermediate is sampled every 12 hours during the 72h fermentation period. The concentration of the intermediate should be ≥10g / L.

[0142] Detection of six-carbon intermediates: High-performance liquid chromatography (HPLC, model: Agilent 1260) was used with a Zorbax SB-C18 column (4.6×250mm, 5μm), a mobile phase of methanol-water (volume ratio 30:70), a flow rate of 1.0 mL / min, a detection wavelength of 210 nm, and a column temperature of 30℃. The concentration of six-carbon intermediates in the fermentation broth was calculated by external standard method (the six-carbon intermediate standard was D-fructose-1,6-diphosphate disodium, purity ≥98%, concentration 10 g / L), and it should reach 8.0-12.0 g / L.

[0143] The high-temperature separation and purification parameters are as follows:

[0144] The high-temperature separation device was a SHZ-D(III) circulating water vacuum pump, with a temperature of 80-90℃ (error ±1℃) and a pressure of 0.12-0.15MPa (monitored with a precision pressure gauge). The reduced pressure distillation time was 4 hours, and the temperature range for collecting the distillate during distillation was 75-85℃. A 50L rotary evaporator was used, with a material feed rate of 5L / h (based on equipment volume optimization; too fast a rate may lead to incomplete separation, while too slow a rate reduces efficiency). The temperature was 80-90℃, and the pressure was 0.12-0.15MPa, with the pressure monitored in real-time by a precision pressure gauge (accuracy 0.001MPa). The crude bio-lipids were recrystallized with anhydrous ethanol (bio-lipids to anhydrous ethanol volume ratio 1:3), allowed to stand at 4℃ for 12 hours, and then filtered. The filter cake was dried under vacuum at 60℃ and 0.08MPa for 2 hours to obtain refined bio-lipids (purity ≥96%, detected by HPLC under the same conditions as the six-carbon intermediate).

[0145] Maturation process: The constant temperature reactor model is GR-50, the maturation temperature is 50-55℃ (error ±0.5℃), the stirring speed is 150r / min, the maturation time is 2-3h, and after maturation, the acid value of the bio-lipids is detected by titration (refer to GB / T5530-2022 "Determination of Acid Value and Acidity of Animal and Vegetable Oils"). The acid value needs to be reduced to ≤3.5mgKOH / g.

[0146] Sulfonation reaction: The sulfonating agent is 98% concentrated sulfuric acid (analytical grade), with a dosage of 5-8% of the biolipid content, added slowly dropwise (dropping rate 1 mL / (min·100g biolipid)), sulfonation temperature 45-50℃ (error ±0.5℃), stirring speed 200 r / min, sulfonation time 1.5-2 h; after the reaction, wash three times with deionized water (each time the water volume is twice the biolipid content), and the pH of the aqueous phase after washing is measured with a pH meter, which must be ≥6.0; finally, the sulfonic acid group content is determined by potentiometric titration (refer to GB / T11276-2021 "Determination of Critical Micelle Concentration of Surfactants"), and the sulfonic acid group content must be ≥7.5%.

[0147] Acrylic acid monomer: purity ≥99.5%, polymerization inhibitor (hydroquinone) content ≤50ppm (detected by high performance liquid chromatography, refer to GB / T17529.1-2017).

[0148] Sodium hydroxide flakes: purity ≥98%, sodium carbonate impurity content ≤1% (refer to GB / T209-2018 "Industrial Sodium Hydroxide");

[0149] 27.5% hydrogen peroxide: The stabilizer is phosphoric acid (content 0.01-0.03%), and there are no other heavy metal impurities (refer to GB / T1616-2021 "Industrial Hydrogen Peroxide").

[0150] Ferrous sulfate: 1% mass concentration solution, freshly prepared, Fe²⁺ concentration ≥ 0.95% is detected by "o-phenanthroline spectrophotometry" (solution is light green, no yellow precipitate).

[0151] Experimental verification data of the "biaxial reduction mechanism"

[0152] 1. Hydroxyl groups (-OH) reduce trace metallic impurities (Fe³+).

[0153] Experimental conditions: In a Fe-containing environment 3 Add 0.5% mother liquor to the polymerization system (concentration 10ppm) and stir at 25℃ for 30min;

[0154] Detection method: Fe³⁺ residue was detected using an atomic absorption spectrophotometer (AA-7000, Shimadzu).

[0155] Results: Fe³⁺ residue ≤ 0.5 ppm, reduction efficiency ≥ 95%.

[0156] 2. Sulfonate groups (-SO3Na) reduce hydrogen peroxide residue.

[0157] Experimental conditions: Add 0.3% stock solution to the system containing hydrogen peroxide (concentration 0.5%), and stir at 25℃ for 20 min;

[0158] Detection method: Iodometric titration to determine hydrogen peroxide residue;

[0159] Results: Hydrogen peroxide residue ≤0.05%, reduction efficiency ≥90%.

[0160] The specifications of the core raw materials and reagents of this invention are as follows:

[0161] Monomer: The monomer is industrial grade acrylic acid (purity ≥99.5%, polymerization inhibitor is hydroquinone, content ≤50ppm, refer to GB / T17529.1-2017 "Industrial Acrylic Acid"). Its molecular structure is stable and can be fully neutralized with caustic soda, ensuring the controllability of subsequent polymerization reactions.

[0162] Culture medium formulation:

[0163] LB liquid medium for yeast: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0 with 1 mol / L NaOH, autoclaved at 121℃ for 20 min, cooled and ready for use (for the cultivation of Saccharomyces cerevisiae ATCC26603).

[0164] MRS liquid culture medium for lactic acid bacteria: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 mL / L Tween-80, 0.2 g / L MgSO4·7H2O, 0.05 g / L MnSO4·4H2O. Adjust the pH to 6.2-6.4 with 1 mol / L NaOH, autoclave at 121℃ for 20 min, and cool before use (for the culture of Lactobacillus delbrueckii ATCC11842).

[0165] Auxiliary reagents:

[0166] Ferrous sulfate: Use industrial-grade ferrous sulfate heptahydrate (FeSO4·7H2O, purity ≥99%, heavy metal content ≤0.001%, refer to GB / T10531-2016 "Ferrous Sulfate for Water Treatment") to prepare a 1% ferrous sulfate solution. It must be dissolved in deionized water and stirred until completely clear. It should be used within 24 hours after fresh preparation.

[0167] Caustic soda flakes: Industrial grade sodium hydroxide (purity ≥98%, sodium carbonate impurity content ≤1%, refer to GB / T209-2018 "Industrial Sodium Hydroxide"), which needs to be crushed to a particle size ≤5mm for easy dissolution.

[0168] Hydrogen peroxide: 27.5% industrial grade hydrogen peroxide (stabilizer is phosphoric acid, content 0.01-0.03%, free of heavy metal impurities, refer to GB / T1616-2021 "Industrial Hydrogen Peroxide"), storage temperature ≤25℃, avoid direct sunlight.

[0169] Details of microbial pretreatment:

[0170] High activation treatment: temperature 35-40℃, pH 6.5-7.2, treatment time 24h (based on strain metabolic cycle optimization, less than 20h strain activity is insufficient, more than 28h easily produces by-products), during the culture process the stirring rate is maintained at 150r / min, and the dissolved oxygen content is controlled at 20±2%.

[0171] High-induction stimulation: Use 0.1-0.3% glucose solution (based on culture medium volume) as the inducer, add it to the culture medium all at once, and continue to culture for 18 hours after induction. Keep the culture temperature at 35℃ to avoid temperature fluctuations that may cause induction failure.

[0172] Nitrogen balance control: The nitrogen source is ammonium chloride (industrial grade, purity ≥99%). The concentration is adjusted to 0.5-0.8 mol / L. After adding, stir for 30 min to ensure uniform distribution of the nitrogen source. Incubate for 12 h. During this period, samples are taken every 6 h to test the nitrogen content of the culture medium to ensure stable nitrogen concentration.

[0173] Biaxial reduction mechanism verification scheme:

[0174] Experimental design: Two control groups and two experimental groups were set up, with each group undergoing three parallel experiments. The average reduction efficiency was calculated.

[0175] Control group 1: Polymerization system containing only 10 ppm Fe³+ (added in the form of FeCl3) (without mother liquor), stirred at 25°C for 30 min;

[0176] Control group 2: The system containing only 0.5% hydrogen peroxide (without adding the mother liquor) was stirred at 25°C for 20 minutes;

[0177] Experimental group 1: Polymerization system containing 10 ppm Fe³+ and 0.5% mother liquor, stirred at 25℃ for 30 min;

[0178] Experimental group 2: The system containing 0.5% hydrogen peroxide and 0.3% mother liquor was stirred at 25°C for 20 minutes.

[0179] Elimination of interfering factors: Before the experiment, the content of other reducing substances (such as sulfite and thiosulfate) in the polymerization system should be tested, and their concentration should be ≤0.01% (detected by ion chromatography, model: Dionex ICS-1100) to avoid interference with the reduction detection of Fe³⁺ and hydrogen peroxide.

[0180] Test results record: Fe³⁺ residue was detected using an atomic absorption spectrophotometer (AA-7000, Shimadzu), hydrogen peroxide residue was detected using the iodometric method, and the reduction efficiency of each group was recorded. The reduction efficiency of experimental group 1 was required to be ≥95%, and the reduction efficiency of experimental group 2 was required to be ≥90%.

[0181] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A mother liquor for a six-carbon easy-to-polymerize biopolymer, comprising bottom feed and drop feed, characterized in that: The bottom material comprises, by weight, 340 parts water, 320 parts monomer, and 1.5 parts caustic soda flakes; the dripping material comprises, by weight, 45 parts water, 30 parts C6 easy-to-polymerize biopolymerization auxiliaries, 1.5 parts caustic soda flakes, 6 parts acrylic acid, 3 parts hydrogen peroxide with a mass concentration of 27.5%, and 1.5 parts ferrous sulfate solution with a mass concentration of 1%.

2. The mother liquor of a six-carbon easy-to-polymerize biopolymerization aid according to claim 1, characterized in that, The six-carbon and easy-to-polymerize biopolymerization aid is produced by selecting microorganisms, subjecting them to high activation, high stimulation, and nitrogen balance treatment to optimize the metabolic environment, and using microbial metabolic engineering technology to introduce key enzyme genes of the six-carbon synthesis pathway into the microorganisms to construct recombinant microbial strains for the directed synthesis of six-carbon structural intermediates; the fermentation products of the recombinant microorganisms are separated and purified at high temperature to obtain biolipids; the biolipids are then matured and subjected to sulfonation to obtain the finished liquid six-carbon and easy-to-polymerize biopolymerization aid.

3. The mother liquor of a six-carbon easy-to-polymerize biopolymerization aid according to claim 2, characterized in that, The microorganisms are selected from Saccharomyces cerevisiae or Lactobacillus delbrueckii; the key enzyme genes of the six-carbon synthesis pathway include hexokinase gene and phosphofructokinase gene; the sulfonation reaction uses concentrated sulfuric acid as the sulfonating agent.

4. The mother liquor of a six-carbon easy-to-polymerize biopolymerization aid according to claim 3, characterized in that, The gene transfer process for six-carbon and workable biopolymerization aids is as follows: The pET-28a(+) plasmid was used as a vector containing the ampicillin resistance gene, the T7 promoter, and the T7 terminator. The vector and the target gene were double-digested with restriction endonucleases EcoRI and XhoI. The DNA was ligated using T4 DNA ligase. After the ligation product was introduced into competent cells, it was inoculated into LB solid medium containing ampicillin and cultured at 37°C for 48 h. Single colonies were picked for PCR verification. The PCR products were detected by 1.5% agarose gel electrophoresis. The presence of a 1200 bp band for the hexokinase gene and a 1500 bp band for the phosphofructokinase gene indicated a positive recombinant strain.

5. A method for preparing a mother liquor of a six-carbon easy-to-polymerize biopolymer according to any one of claims 1-4, characterized in that: Includes the following steps: (1) Add bottom material to the reactor, which includes 340 parts water, 320 parts monomer and 1.5 parts caustic soda by mass. After stirring evenly, heat to 60-65℃ and keep warm for 10-15 minutes. The monomer is acrylic acid monomer. (2) Add the feed material to the reactor at a constant rate and stir. The feed material includes 45 parts water, 30 parts C6 easy biopolymerization auxiliaries, 1.5 parts caustic soda flakes, 6 parts acrylic acid, and 3 parts 27.5% hydrogen peroxide by mass. The addition time is 40-45 minutes. During the addition process, the stirring rate is maintained at 200-300 r / min, and the reaction temperature is kept stable at 60-65℃ by the reactor temperature control system. The dropping rate is adjusted according to the effective volume of the reactor. (3) After the addition is complete, keep it at 60-65℃ for 60 minutes to ensure that the polymerization reaction is fully carried out; then add 1.5 parts of 1% ferrous sulfate solution and stir for 10-15 minutes; terminate the activity of hydrogen peroxide through redox reaction to avoid over-reaction; (4) Add 216 parts of water to neutralize and dilute, cool to room temperature, and obtain 40% hexacarbon and easy-to-use biopolymerization auxiliary mother liquor.

6. The method for preparing a six-carbon easy-to-polymerize biopolymer mother liquor according to claim 5, characterized in that, In step (2), the preparation method of the six-carbon easy-to-polymerize biopolymer is based on combining microbial metabolic engineering and directed synthesis technology to ensure the accurate construction of the six-carbon structure and the integration of the additive function, including the following steps: (1) Select microorganisms with the potential for six-carbon skeleton synthesis, and optimize the microbial metabolic environment through high activation treatment, high stimulation induction and nitrogen balance regulation; (2) Using microbial metabolic engineering technology, the key enzyme genes of the six-carbon synthesis pathway were introduced into the above-mentioned microorganisms to construct recombinant microbial strains, regulate their metabolic pathways, and directionally synthesize six-carbon intermediates; (3) The fermentation products of recombinant microorganisms were placed in a high-temperature separation device to remove impurities and water, and the bio-lipids were purified. Fermentation process: The initial pH of the GYM fermentation substrate was 6.8-7.

0. During the fermentation process, the pH was measured every 12 hours. If the pH was lower than 6.5, 1 mol / L NaOH solution was added to adjust it to 6.8-7.

0. At the same time, the concentration of the six-carbon intermediate was detected. When the concentration was ≥10 g / L, the fermentation was terminated. (4) The bio-lipids are matured and then sulfonated to obtain a liquid product of six-carbon and easy-to-use biopolymerization auxiliaries.

7. The method for preparing a six-carbon easy-to-polymerize biopolymer mother liquor according to claim 6, characterized in that, In step (1), the high activation treatment is performed at a temperature of 35-40℃ and a pH of 6.5-7.2; the high activation induction uses 0.1-0.3% glucose as an inducer; the metabolic pathway is directed towards six-carbon synthesis; and the nitrogen source concentration is 0.5-0.8 mol / L in the nitrogen balance regulation.

8. The method for preparing a six-carbon easy-to-polymerize biopolymer mother liquor according to claim 7, characterized in that, In step (3), the temperature of the high-temperature separation device is 80-90℃ and the pressure is 0.12-0.15Mpa.

9. The method for preparing a six-carbon easy-to-polymerize biopolymer mother liquor according to claim 8, characterized in that, In step (4), the aging temperature is 50-55℃ and the aging time is 2-3h; in the sulfonation reaction, the amount of sulfonating agent is 5-8% of the amount of biological lipids, the sulfonation temperature is 45-50℃ and the sulfonation time is 1.5-2h.

10. The application of a mother liquor of a six-carbon easy-to-polymerize biopolymer, characterized in that: In the field of building materials, the mother liquor is used as a concrete admixture to improve the workability and compressive strength of concrete; in the field of coatings and chemicals, the mother liquor is used as a coating dispersant and stabilizer; in the field of polymer polymerization, the mother liquor is used as a polymerization reaction aid.