A defatted cotton outer layer directional enzymatic treatment method based on composite plant protease

Abstract

The application relates to the technical field of biomaterial modification, and particularly relates to a method for carrying out directional enzymolysis treatment on the surface of defatted cotton through composite plant protease, which comprises the following steps: physically pretreating the defatted cotton to enhance the surface activation degree; preparing a composite enzyme solution containing bromelain, pectinase and xylanase; contacting the pretreated defatted cotton with the composite enzyme solution to carry out directional enzymolysis under controlled conditions; controlling the enzymolysis depth by adjusting the reaction temperature and pH value in stages; after the reaction is completed, the enzyme activity is terminated through heat treatment, pH adjustment or addition of chemical reagents; finally, the treated defatted cotton is cleaned and dried to obtain a surface-modified defatted cotton product, selective directional enzymolysis of the outer layer of the defatted cotton is realized, the surface fiber diameter is reduced by 8-12%, the internal fiber structure is kept intact, the tensile strength retention rate is greater than or equal to 85%, and the problem that the depth of the existing enzymolysis technology is uncontrollable is solved.

Description

Technical Field

[0001] The invention relates to the field of biomaterial modification technology, specifically to a method for targeted enzymatic hydrolysis of the surface of defatted cotton using a complex plant protease, applicable to the preparation process of high-performance cellulose products such as medical dressings and cosmetic cotton pads. Background Technology

[0002] Defatted cotton, as an important cellulose material, is widely used in medical and health, cosmetic, and industrial fields. Depending on the specific application, the surface properties of defatted cotton, such as hydrophilicity, softness, and absorbency, directly affect its performance. Currently, the modification treatment of defatted cotton mainly employs chemical degreasing, physical modification, or enzymatic hydrolysis.

[0003] Traditional chemical degreasing methods typically use strong alkaline solutions such as sodium hydroxide or organic solvents. While these methods can effectively remove non-cellulose substances from cotton fibers, they often lead to the following problems: First, chemical residues, especially in medical dressings, may cause skin allergies or inflammatory reactions in patients; second, fiber structure damage, as excessive chemical treatment can reduce the length of cellulose molecular chains, resulting in decreased strength; and third, the generation of large amounts of wastewater containing alkaline or organic solvents, placing a heavy burden on the environment.

[0004] Physical modification methods, such as plasma treatment and corona treatment, mainly affect the surface properties of fibers. Although they avoid the problem of chemical reagent residues, their treatment effects are often unstable, and the equipment investment costs are high and the energy consumption is large, making it difficult to achieve large-scale industrial application.

[0005] In recent years, enzymatic hydrolysis technology has been increasingly applied to the modification of cellulose materials due to its advantages such as high specificity, mild reaction conditions, and environmental friendliness. The main drawbacks of existing enzymatic hydrolysis technologies are: first, the depth of hydrolysis is uncontrollable, often resulting in full-layer degradation rather than surface-oriented modification, leading to a significant decrease in fiber strength; second, the hydrolysis efficiency is low, and the processing time is long; third, there is a lack of dynamic and precise control over the hydrolysis process; and fourth, enzyme preparations are difficult to recycle after use, resulting in high costs.

[0006] Therefore, there is an urgent need to develop a novel treatment method that can achieve directional enzymatic hydrolysis of the surface of degreased cotton, retain the internal fiber strength, and improve surface properties, so as to meet the differentiated surface characteristics requirements of high-performance cellulose products. Summary of the Invention

[0007] The purpose of this invention is to provide a method for targeted enzymatic hydrolysis of the outer layer of degreased cotton based on a complex plant protease. This method aims to solve the technical problems of uncontrollable hydrolysis depth, low efficiency, and high cost in the existing technology, and to achieve selective enzymatic hydrolysis of the outer layer of degreased cotton. While retaining the internal fiber strength, it significantly improves the surface hydrophilicity, softness, and adsorption, while reducing energy consumption and achieving environmentally friendly production.

[0008] To achieve the above objectives, the present invention provides a method for targeted enzymatic hydrolysis of the outer layer of defatted cotton based on a complex plant protease, comprising the following steps:

[0009] (1) Pretreatment: Physical pretreatment of degreased cotton to enhance surface activation;

[0010] (2) Enzyme solution preparation: Prepare a complex enzyme solution containing bromelain, pectinase and xylanase;

[0011] (3) Enzymatic hydrolysis reaction: The pretreated defatted cotton is contacted with the compound enzyme solution, and a directional enzymatic hydrolysis reaction is carried out under controlled conditions;

[0012] (4) Dynamic control: During the enzymatic hydrolysis process, the depth of enzymatic hydrolysis is controlled by adjusting the reaction temperature and / or pH value in stages;

[0013] (5) Inactivation: After the enzymatic hydrolysis is completed, the enzyme activity is terminated by raising the temperature, adjusting the pH, or adding chemical reagents;

[0014] (6) Cleaning and drying: The degreased cotton after enzymatic hydrolysis is cleaned and dried to obtain a surface-modified degreased cotton product.

[0015] In a preferred embodiment of the present invention, the ratio of bromelain, pectinase, and xylanase in the composite enzyme solution is A:B:C = 1:(0.5-2):(0.3-1), where A represents bromelain, B represents pectinase, and C represents xylanase. This specific enzyme ratio enables synergistic enzymatic hydrolysis of the outer layer of defatted cotton. Bromelain primarily cleaves the non-crystalline regions of the surface fibers, pectinase degrades intercellular pectin, and xylanase acts on hemicellulose components. The synergistic effect of these three enzymes achieves efficient and targeted degradation.

[0016] The physical pretreatment can employ plasma treatment, ultrasonic treatment, microwave treatment, or a combination thereof. Specifically, the plasma treatment conditions are: power 100-300W, treatment time 10-20 minutes; the ultrasonic treatment conditions are: frequency 20-40kHz, power density 200-500W / L, treatment time 8-15 minutes; the microwave-assisted treatment conditions are: frequency 2.45GHz, power 200-400W, intermittent treatment 3-5 minutes. The introduction of physical pretreatment can disrupt the waxy layer on the surface of the degreased cotton, increase surface micropores and roughness, and significantly improve the efficiency of subsequent enzymatic hydrolysis.

[0017] One of the key innovations of this invention is the dynamic control system, which precisely controls the depth of enzymatic hydrolysis by adjusting temperature and pH in stages. The staged temperature adjustment includes: a first stage (activation phase): 48-52℃, lasting 30-60 minutes, to activate enzyme activity; a second stage (stabilization phase): 38-42℃, lasting 60-90 minutes, to maintain moderate enzyme activity; and a third stage (inhibition phase): 28-32℃, lasting 30-60 minutes, to inhibit excessive reaction. The staged pH adjustment includes: a first stage: pH 6.2-6.8, which is favorable for bromelain activity; a second stage: pH 5.2-5.8, which is favorable for pectinase activity; and a third stage: pH 6.7-7.3, which inhibits excessive enzymatic hydrolysis.

[0018] After enzymatic hydrolysis, one or more of the following inactivation methods can be used: heat inactivation: 78-82℃ for 8-12 minutes; pH inactivation: adjust the pH to 10.0-11.0 for 4-6 minutes; chemical inactivation: add 0.1%-0.2% hydrogen peroxide solution. Inactivation treatment can terminate the enzyme reaction in a timely manner and prevent over-enzymatic hydrolysis.

[0019] To improve economic efficiency and environmental friendliness, this invention also includes an enzyme preparation recovery step: centrifuging the enzymatic hydrolysis waste liquid to remove solid particles; using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa for membrane separation, or using 40%-60% saturated ammonium sulfate for salting-out recovery, or using macroporous resin for selective adsorption; concentrating and stabilizing the recovered enzyme to achieve the recycling of the enzyme preparation, which can be recycled 3-5 times with an activity retention rate of not less than 60%.

[0020] The method of this invention can be flexibly applied to the preparation of various absorbent cotton products. For example, the specific process parameters for preparing medical dressings are: the raw material has a fiber length ≥10mm and a density of 3.5kg / m³. 3 The absorbent cotton was used; the enzymatic hydrolysate consisted of 1.5 g / L bromelain, 1.2 g / L pectinase, 0.8 g / L xylanase, phosphate buffer (pH 6.5, 50 mM), and 2 mM calcium chloride; pretreatment was performed using plasma treatment (200 W, oxygen, 15 min); enzymatic hydrolysis was carried out in three stages with controlled temperature (50℃ / 45 min → 40℃ / 75 min → 30℃ / 45 min); inactivation was performed by holding at 80℃ for 10 min; the mixture was washed 4 times with pure water at 40℃; and the mixture was dried with hot air at 60℃ for 50 min.

[0021] The specific process parameters for preparing cotton pads are as follows: the raw material has a fiber length ≥15mm and a density of 3.2kg / m³. 3Combed degreased cotton; the enzymatic hydrolysate consisted of 2.0 g / L bromelain, 1.5 g / L pectinase, 1.0 g / L xylanase, and 0.05% nonionic surfactant; pretreatment was performed by ultrasonic treatment (25 kHz, 300 W / L, 12 minutes); two-stage controlled enzymatic hydrolysis (52℃ / 30 minutes → 35℃ / 60 minutes); pH was adjusted to 10.5 and held for 5 minutes for inactivation; vacuum drying (40℃, -0.06 MPa, 30 minutes).

[0022] The preparation method of the composite enzyme solution can be flexibly selected according to actual needs: fixed ratio scheme (bromelain: pectinase: xylanase = 1:0.8:0.5); adjustable ratio scheme (adjust the ratio of each enzyme according to the characteristics of raw materials and the performance requirements of the target product); sequential addition scheme (add bromelain first, then add pectinase and xylanase in sequence); enzyme immobilization scheme (immobilize the enzyme on carriers such as macroporous polymer spheres or magnetic nanoparticles).

[0023] Compared with existing technologies, the present invention has the following significant advantages:

[0024] (1) Selective directional enzymatic hydrolysis of the outer layer of degreased cotton was achieved, the diameter of the surface fiber was reduced by 8% to 12%, while the internal fiber structure remained intact and the tensile strength retention rate was ≥85%, which solved the problem of uncontrollable depth of existing enzymatic hydrolysis technology;

[0025] (2) Through the synergistic mechanism of compound enzymes, the surface roughness is reduced by 30% to 50%, the liquid diffusion rate is increased by 50% to 80%, the water absorption rate is increased from 8-12 mL / g to 18-25 mL / g, and the surface properties are significantly improved.

[0026] (3) By adopting a dynamic temperature / pH control system, the enzymatic hydrolysis process is precisely controlled, the processing time is shortened by 30% to 50%, and the production efficiency is improved.

[0027] (4) An enzyme preparation recycling technology was established, with a recovery rate of 70% to 90%, which significantly reduced enzyme costs;

[0028] (5) The COD value of the enzymatic hydrolysis waste liquid is reduced by 60% to 80% compared with the chemical method, and there is no risk of chemical reagent residue, which meets the green production standard.

[0029] In summary, the method for directional enzymatic hydrolysis of the outer layer of defatted cotton based on composite plant protease provided by this invention solves the technical problems existing in traditional treatment methods, achieves directional and precise improvement of the surface properties of defatted cotton, and at the same time ensures the integrity of the internal structure, greatly improving the processing efficiency and environmental friendliness, and providing a new approach for the preparation of high-performance cellulose materials. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. In the following embodiments, unless otherwise specified, the reagents and materials used are commercially available, and all embodiments were completed under laboratory conditions.

[0031] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0032] During the research and development process, it was discovered that the surface properties of absorbent cotton are closely related to its microstructure. Absorbent cotton fibers are mainly composed of cellulose, with small amounts of hemicellulose, pectin, and residual wax on the surface. Although these non-cellulose components are present in low amounts, they significantly affect the surface properties of absorbent cotton. Through detailed observation of the microstructure of untreated absorbent cotton, it was noted that the surface wax layer hinders the penetration of moisture and active substances, while the pectin between fibers restricts the fiber's softness. This discovery inspired the design of a composite enzyme system targeting different components, aiming to improve surface properties while preserving internal fiber strength.

[0033] Example 1: Standard Medical Absorbent Cotton Processing Technology

[0034] First, a batch of typical medical-grade absorbent cotton was selected as the research subject. The fiber length of this material was approximately 10-15 mm, and the density was 3.5 kg / m³. 3 The cotton meets the basic requirements for medical dressings. Electron microscopy revealed that the fibers have a rough, uneven surface, with diameters fluctuating between 14-16 μm, averaging approximately 15.3 μm. Preliminary tests showed that the absorbency of this batch of absorbent cotton was only 8-12 mL / g, far below the requirements for high-end medical dressings.

[0035] Before conducting the main experiments, various pretreatment methods were explored, including chemical soaking, ultrasonic treatment, and plasma treatment. The results showed that plasma treatment effectively activated the surface with minimal damage to the fiber's main structure. This is likely because the active particles generated by low-temperature plasma can selectively act on waxes and impurities on the fiber surface, while simultaneously introducing oxygen-containing functional groups, creating favorable conditions for subsequent enzymatic hydrolysis.

[0036] The selection of pretreatment parameters was quite challenging. After much trial and error, it was found that the optimal combination was a plasma power between 100 and 300 W and a treatment time between 10 and 20 minutes. Powers below 100 W resulted in minimal surface activation, while power exceeding 300 W could lead to excessive fiber oxidation or even localized carbonization. Similarly, treatment times shorter than 10 minutes were insufficient, while times exceeding 20 minutes increased the risk of fiber damage, with increased energy consumption yielding no significant benefit.

[0037] Through systematic comparison, the optimal plasma treatment conditions were determined to be 200W power, 15 minutes processing time, and oxygen as the working gas. Under these conditions, the surface wax removal rate was close to 90%, the surface roughness increased by about 40%, while the fiber damage was controlled below 5%, demonstrating a good balance.

[0038] The preparation of the complex enzyme solution is the core of this invention. Through extensive literature review and preliminary experiments, a complex system consisting of bromelain, pectinase, and xylanase was identified. Each of these three enzymes has its own strengths: bromelain primarily cleaves the non-crystalline regions of the fiber surface, pectinase degrades intercellular pectin, and xylanase acts on hemicellulose components. To determine the optimal ratio, a series of experiments were designed to systematically adjust the proportions of the three enzymes, ranging from A:B:C = 1:0.5:0.3 to 1:2.0:1.0, covering the entire expected effective range.

[0039] After repeated comparisons and verifications, it was found that the optimal balance of treatment effects was achieved when the three enzymes were mixed in a ratio of 1:1.2:0.7. Specifically, 1.5 g / L bromelain (activity 3.29 × 10^6 U / g), 1.8 g / L pectinase (activity 2.2 × 10^5 U / g), and 1.05 g / L xylanase (activity 1.8 × 10^5 U / g) were added to 50 mM phosphate buffer (pH 6.5), along with 2 mM calcium chloride as an enzyme activity cofactor and 5% glycerol as a stabilizer. It is worth noting that temperature control during the preparation process is crucial. Preparing and storing the enzyme solution at 4°C effectively prevented premature loss of enzyme activity; this seemingly small detail has a significant impact on the actual operational results.

[0040] The dynamic control of the enzymatic hydrolysis process is another innovation of this invention. Traditional isothermal enzymatic hydrolysis often struggles to balance treatment effectiveness with the protection of internal structures; therefore, a three-stage temperature and pH synergistic control strategy was developed. First, activity assays revealed differences in the optimal activity temperature and pH for the three enzymes: bromelain prefers 48-52℃ and pH 6.2-6.8, pectinase performs best at 38-42℃ and pH 5.2-5.8, while xylanase exhibits the highest activity at 40-45℃ and pH 5.5-6.5.

[0041] Based on these findings, a three-stage control scheme was designed: the first stage (activation phase) maintains a relatively high temperature (50℃) and a moderate pH (6.5) for about 45 minutes, mainly activating bromelain and initiating the degradation of surface fibers; the second stage (stabilization phase) cools down to 40℃ and adjusts the pH to 5.5, lasting for 75 minutes, at which point the activities of pectinase and xylanase are enhanced, deepening the modification of the surface structure; the third stage (inhibition phase) further cools down to 30℃ and increases the pH to 7.0, lasting for 45 minutes, with the aim of gradually reducing enzyme activity, preventing over-enzymatic hydrolysis, and precisely controlling the depth of treatment.

[0042] In practice, the rate of temperature change has been found to be an important parameter. When transitioning from the first stage to the second stage, a temperature decrease rate of approximately 0.2°C / minute is suitable, achieving the desired activity regulation without causing drastic changes in the enzyme protein structure. Similarly, pH adjustments should also be made slowly, ideally with a change not exceeding 0.1 units per minute.

[0043] The stirring method during enzymatic hydrolysis is also noteworthy. Intermittent low-shear stirring was employed, specifically stirring for 5 minutes followed by standing for 10 minutes, and then repeating this cycle. The stirring speed was controlled at approximately 45 rpm. This gentle stirring method ensures the homogeneity of the reaction system while avoiding mechanical damage to the fiber structure.

[0044] After the enzymatic hydrolysis reaction is complete, timely and effective inactivation is crucial. Three methods—thermal inactivation, pH inactivation, and chemical inactivation—were compared. Thermal inactivation was performed at 78-82℃ for 8-12 minutes; pH inactivation involved adjusting the system pH to 10.0-11.0 and holding for 4-6 minutes; for chemical inactivation, the addition of 0.1%-0.2% hydrogen peroxide solution was explored. Considering the inactivation effect, the impact on fibers, and ease of operation, the thermal inactivation method at 80℃ for 10 minutes was ultimately selected. Practice showed that this method completely terminates enzyme activity with minimal impact on the performance of the medical absorbent cotton.

[0045] The cleaning and drying processes after inactivation also require careful design. After multiple experiments, a scheme of cleaning four times with 40℃ warm water was determined, with each cleaning using approximately five times the weight of the absorbent cotton. Insufficient cleaning can lead to residual enzymatic hydrolysis products, while excessive cleaning increases energy and water consumption and may even cause excessive fiber swelling. For the drying stage, a scheme of drying with 60℃ hot air for about 50 minutes was chosen to control the final product's moisture content between 6% and 8%. This moisture content range ensures both product stability and maintains appropriate softness.

[0046] To improve the economic efficiency and environmental friendliness of the process, an enzyme recovery technology was also developed. Comparative tests revealed that using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa yielded the best separation effect, with a recovery rate of approximately 75% and an enzyme activity retention rate of 73%. The recovered enzyme can be recycled up to four times after simple stabilization treatment, significantly reducing production costs. It is worth noting that enzyme activity gradually decreases with each cycle; generally, after the fifth use, the activity drops to below 60% of its original level, at which point the enzyme should be replaced with a new one.

[0047] The medical absorbent cotton treated with the above process exhibits significantly improved performance. Electron microscopy measurements show that the surface fiber diameter decreased from 15.3 μm to 13.7 μm, a reduction of approximately 10.5%; the water absorption rate, measured by the conventional impregnation method, increased from 10.2 mL / g to 22.8 mL / g, an increase of approximately 123%; and the tensile strength, tested using the standard tensile method, improved from 3.0 cN / dtex to 2.69 cN / dtex, with a retention rate of 89.6%. These data indicate that this process successfully achieved the goal of directional surface modification without significantly damaging the internal fiber structure.

[0048] Microstructural analysis further confirmed this. Scanning electron microscopy revealed a uniformly distributed microporous structure on the surface of the treated absorbent cotton, with diameters mostly between 0.5 and 3 μm, while the internal fibrous structure remained intact. X-ray diffraction analysis showed that the crystallinity index decreased slightly from 0.72 to 0.68, a minor change, confirming that enzymatic hydrolysis mainly acted on the non-crystalline regions, preserving the basic structural characteristics of cellulose.

[0049] Practical application tests show that medical absorbent cotton treated with this process performs excellently in wound care, increasing the exudate absorption rate by 68.5% and resulting in more uniform liquid distribution, which greatly improves the effectiveness of dressings.

[0050] Example 2: Rapid Processing Technology for Cotton Pads

[0051] The requirements for absorbent cotton in the cosmetics industry differ from those for medical dressings, with a greater emphasis on softness, fit, and comfort. The manufacturing process has been adjusted accordingly to meet these requirements.

[0052] Firstly, in terms of raw material selection, degreased cotton processed using a combing technique was adopted, with fiber length between 15 and 25 mm and density of approximately 3.2 kg / m³. 3 The combing process makes the fibers more regularly arranged, which is beneficial for producing cotton pads with a uniform texture. Preliminary characterization shows that the surface of this batch of raw materials is relatively smooth, but it has a high contact angle, which is not conducive to the absorption and diffusion of liquids such as toners.

[0053] In terms of pretreatment, ultrasonic treatment was found to be more suitable than plasma treatment for the production of cotton pads. This is mainly because ultrasonic treatment can increase the surface microporous structure without excessively introducing oxygen-containing functional groups, thus maintaining the gentle properties required for cosmetic use. After a series of comparative experiments, the ultrasonic frequency was set at 25kHz, the power density at approximately 300W / L, and the treatment time at about 12 minutes. These parameters can produce a moderate cavitation effect, uniformly changing the fiber surface structure without causing significant damage. The treatment medium is pure water, and the temperature is controlled at 35℃. This temperature maintains a good cavitation effect while avoiding fiber denaturation that may be caused by excessively high temperatures.

[0054] To meet the specific requirements of cotton pads for a soft touch, the enzyme formula was adjusted. After numerous experiments, it was found that the optimal treatment effect was achieved when bromelain, pectinase, and xylanase were mixed in a ratio of 1:1.5:0.7. Compared to the process used for medical absorbent cotton, the proportion of pectinase was increased because pectinase can more effectively degrade the intercellular layer, making the fiber surface softer. Specifically, the formula used 2.0 g / L bromelain, 3.0 g / L pectinase, and 1.4 g / L xylanase, along with 0.05% nonionic surfactant to enhance permeability and 4% sorbitol as an enzyme stabilizer.

[0055] During enzyme preparation, it's important to note that the order of addition can affect the final result. The optimal procedure is to first prepare the buffer solution and adjust it to the target pH (6.2), then add bromelain, xylanase, and pectinase sequentially, stirring gently after each addition until completely dissolved. This method avoids enzyme denaturation or mutual inhibition caused by localized high concentrations.

[0056] Considering that cotton pads have relatively low requirements for mechanical strength, the enzymatic hydrolysis process was simplified into a two-stage control: the first stage involves reacting at 52℃ and pH 6.2 for 30 minutes, primarily initiating surface degradation; the second stage involves cooling to 35℃, adjusting the pH to 5.8, and continuing for 60 minutes to further improve surface softness. This simplified process saves approximately 45 minutes compared to the three-stage process used for medical dressings, significantly improving production efficiency.

[0057] During the process implementation, it was also found that the liquid-to-solid ratio was a crucial factor affecting the final result. By comparing different liquid-to-solid ratios from 1:15 to 1:25, a ratio of 1:18 (w / v) was determined to be the most suitable, ensuring sufficient contact while avoiding enzyme waste. Intermittent low-speed stirring was used, at approximately 30 rpm. Stirring too quickly could cause cotton fibers to entangle, while stirring too slowly might affect the uniformity of the reaction.

[0058] In the production of cotton pads, to maintain the product's soft feel, a pH inactivation method has been adopted instead of heat inactivation. The specific procedure involves adding a dilute alkaline solution to the reaction system to adjust the pH to approximately 10.5, maintaining this position for about 5 minutes. Compared to heat inactivation, pH inactivation has less impact on fiber softness and is more suitable for the production requirements of cotton pads. After inactivation, the pads are rinsed three times with 35°C warm water to ensure complete removal of residual enzymes and reaction products.

[0059] In the drying process, vacuum drying technology was selected, with a temperature of 40℃, a pressure of approximately -0.06MPa, and a time of 30 minutes. Compared to conventional hot air drying, vacuum drying can effectively remove moisture under low-temperature conditions, maximizing the fluffiness and softness of the fibers, which is crucial for the user experience of the cotton pads. The final product's moisture content is controlled at 7%–9%, ​​slightly higher than that of medical absorbent cotton, to maintain a better soft touch.

[0060] The treated cotton pads underwent a comprehensive performance evaluation. Measured using a KES-FB4 fabric hand feel tester, surface softness improved by 63.4%; using a simulated skin model, fit improved by 50.2%; standard tensile testing showed that tensile strength increased from 2.8 cN / dtex to 2.58 cN / dtex, with a retention rate of 92.1%, fully meeting the requirements for cotton pad use; in actual use, the absorption rate of toner increased by 55.7%, and the diffusion uniformity improved by 48.2%.

[0061] Most importantly, rigorous skin irritation tests were conducted, and the results showed that the treated cotton pads were non-irritating and met cosmetic-grade requirements. In a subjective evaluation test involving 36 volunteers, 89% of the participants felt that the treated cotton pads were superior to commercially available products, particularly in terms of softness, fit, and toner utilization efficiency.

[0062] Example 3: Process for Highly Absorbent Medical Degreased Cotton

[0063] In certain specialized medical scenarios, such as wound care with large amounts of exudate or drug delivery applications, extremely high absorbency of absorbent cotton is required. To address this need, a specialized high-absorbency processing technology has been designed.

[0064] Fibers with a length of not less than 20mm and a density of approximately 3.3kg / m³ were selected. 3 Long-fiber degreased cotton was used as the raw material. The long fiber structure helps to form a more continuous liquid conduction network, enhancing adsorption capacity. Preliminary tests show that the initial water absorption rate of this batch of raw materials is 11.6 mL / g, significantly lower than the requirements for high adsorption applications.

[0065] In the pretreatment stage, an innovative combined treatment of plasma and ultrasound was attempted. This concept stems from the complementary advantages of the two methods: plasma mainly increases the surface oxygen-containing functional groups, enhancing hydrophilicity; while ultrasound mainly increases the surface microporous structure and expands the specific surface area through cavitation effect. After systematic testing, a combined treatment scheme of plasma (oxygen, 150W, 10 minutes) followed by ultrasound (30kHz, 250W / L, 8 minutes) was ultimately adopted. It is worth noting that the interval between the two treatments should not be too long, and it is recommended to control it within 30 minutes to maintain the surface activity state.

[0066] To meet the demand for high adsorption performance, the enzyme formulation was specifically adjusted. Unlike the previous two examples, the proportion of xylanase was increased while the amount of pectinase was appropriately reduced. The final determined ratio was bromelain:pectinase:xylanase = 1:0.5:0.67, i.e., 1.8 g / L bromelain, 0.9 g / L pectinase, and 1.2 g / L xylanase. This ratio is more conducive to the formation of a porous network structure on the fiber surface, enhancing liquid adsorption and retention capacity. Simultaneously, the buffer concentration was appropriately increased to 60 mM, the pH was set to 6.8, and 2.5 mM calcium chloride was added; these adjustments all contribute to enhancing xylanase activity.

[0067] To create the optimal adsorption structure, a more refined four-stage temperature and pH control strategy was employed: The first stage, at 50℃ and pH 6.8 for 30 minutes, activated bromelain; the second stage, at 45℃ and pH 6.0 for 45 minutes, enhanced xylanase activity; the third stage, at 40℃ and pH 5.5 for 60 minutes, reached peak pectinase activity; and the fourth stage, at 30℃ and pH 7.0 for 30 minutes, gradually reduced enzyme activity, precisely controlling the enzymatic hydrolysis endpoint. While this precise, staged control is slightly more complex, it creates an optimal gradient porous structure, significantly improving adsorption performance.

[0068] During enzymatic hydrolysis, the liquid-to-solid ratio was found to be a crucial factor. After multiple comparative tests, a liquid-to-solid ratio of 1:22 (w / v) was found to be optimal for treating highly absorbent absorbent cotton. A liquid-to-solid ratio that is too low will result in uneven enzyme distribution, while a ratio that is too high will waste enzyme resources and increase costs.

[0069] Considering the special requirements of highly absorbent degreased cotton, a combination of thermal inactivation and pH inactivation was used in the inactivation process: first, the temperature was raised to 75℃ and held for 5 minutes, then a small amount of alkaline solution was added to adjust the pH to 9.0 and held for 3 minutes. This combined inactivation method can completely terminate the activity of various enzymes while having minimal impact on the fiber structure.

[0070] An innovation was introduced in the cleaning process: 0.1% EDTA solution was added during the first wash. EDTA, as a metal ion chelating agent, can remove any potential metal ion contaminants that could affect the performance and safety of the final product. Subsequent washes were performed four times with pure water at 38°C to ensure thorough removal of enzymatic hydrolysis products and chemical residues.

[0071] For drying, fluidized bed drying technology (55℃, 40 minutes) is the preferred choice. This technology allows hot air to contact the fiber surface evenly, avoiding localized overheating or overhumidification and maintaining the fiber's loose, porous structure, which is extremely important for highly absorbent products. Furthermore, fluidized bed drying can effectively prevent fiber clumping and improve production efficiency.

[0072] Because the waste liquid contains EDTA, the efficiency of conventional membrane separation for enzyme recovery is affected. Therefore, the salting-out method was adopted for enzyme recovery. The specific procedure involves adding ammonium sulfate to the waste liquid to 45% saturation, allowing it to stand at 4°C for 2 hours, centrifuging to collect the precipitate, dissolving it in phosphate buffer and dialyzing, and finally adding 5% glycerol as a stabilizer. This method achieves a recovery rate of 83.5%, with a enzyme activity retention rate of 72.6%, and can be recycled 3-4 times.

[0073] The performance of the highly absorbent medical absorbent cotton, after the above-mentioned process, was significantly improved. The surface microporosity, as measured by the BET method, increased from the original 23.5% to 39.6%, an increase of 68.4%; the specific surface area increased from 1.2 m² / s² to... 2 / g increased to 2.2m 2 The water absorption rate increased by 83.3% from 11.6 mL / g to 24.7 mL / g, an increase of 112.9%. The tensile strength decreased from 3.1 cN / dtex to 2.67 cN / dtex, with a retention rate of 86.2%.

[0074] More importantly, this treated absorbent cotton exhibits excellent drug adsorption and sustained-release properties. Using roxithromycin solution as the test solution, the adsorption capacity increased by 83.2%; in the 24-hour slow-release test, the release curve was smoother, and the release was more uniform and controllable, making it very suitable as a drug carrier material.

[0075] Example 4: Environmentally friendly, low-energy-consumption process

[0076] With the growing awareness of environmental protection, reducing energy consumption and pollution has become an important direction for industry development. In response to this trend, an environmentally friendly, low-energy-consumption process has been developed, with its main innovations lying in the use of immobilized enzyme technology and microwave-assisted processing.

[0077] Standard degreased cotton (fiber length not less than 12mm, density 3.4kg / m³) is selected. 3 The sample was used as experimental material. Considering the high energy consumption of traditional pretreatment methods, microwave-assisted treatment technology was explored. Multiple tests were conducted with a microwave frequency fixed at 2.45 GHz (standard microwave equipment frequency) and a power ranging from 200 to 400 W. It was found that continuous microwave treatment easily led to localized overheating, so an intermittent treatment mode was adopted. The final determined treatment scheme was 300 W power, using an intermittent method of 10 seconds on / 20 seconds off, with a total treatment time of approximately 3 minutes. Although the surface activation effect of microwave treatment was slightly lower than that of the aforementioned plasma or ultrasonic methods, considering the approximately 60% reduction in energy consumption and low equipment investment cost, this trade-off is practically significant for large-scale industrial production.

[0078] The core innovation of this embodiment lies in the application of immobilized enzyme technology. After comparing various carriers and immobilization methods, macroporous polyacrylamide microspheres were selected as the carrier, and glutaraldehyde covalent immobilization was employed. Specifically, the carrier surface was first activated with a 2.5% glutaraldehyde solution (pH 7.0), then a mixed solution of three enzymes (ratio 1:0.7:0.5) was reacted with the activated carrier at 4°C for 8 hours. Finally, the remaining active groups were blocked with a 1% glycine solution, and the mixture was washed with PBS buffer before use.

[0079] This immobilized enzyme technology offers several advantages. First, the immobilized enzyme retains 85% of its activity, and its thermal and pH stability is significantly improved. For example, at 50°C, the activity of the free enzyme drops to 50% after 2 hours, while the immobilized enzyme retains over 85%. Second, immobilized enzymes are easy to recover and reuse, greatly reducing enzyme costs. Third, immobilized enzyme reactions can be carried out under simpler conditions, eliminating the need for complex multi-stage temperature control.

[0080] A simplified enzymatic hydrolysis process was employed: pretreated defatted cotton was mixed with immobilized enzyme at a ratio of 1:0.3 (w / w), and reacted for 120 minutes at 45°C and pH 6.5. A low-speed, flipping stirring motion (20 rpm) was used to ensure uniform contact without damaging the fibers. It should be noted that the reaction rate of immobilized enzymes is typically lower than that of free enzymes; therefore, the reaction time needs to be appropriately extended.

[0081] After enzymatic hydrolysis, the recovery of the immobilized enzyme is very simple, requiring only filtration through an 80-mesh sieve, with a recovery rate as high as 92%. The recovered immobilized enzyme can be directly used in the next batch of reaction after simple rinsing, without the need for a complicated reactivation procedure, greatly simplifying the operation process.

[0082] The cleaning process also embodies environmental protection principles, employing a counter-current cleaning system with three stages of cleaning, resulting in a total water consumption of only 60% of traditional cleaning methods. Drying utilizes a low-temperature heat pump (45℃, 40% relative humidity, 60 minutes), which is approximately 35% more energy-efficient than traditional hot air drying.

[0083] After undergoing this environmentally friendly process, the surface fiber diameter of the degreased cotton decreased from 15.6 μm to 14.2 μm, a reduction of 9.2%; the water absorption rate increased from 10.5 mL / g to 19.6 mL / g, an increase of 86.7%; and the tensile strength decreased from 3.1 cN / dtex to 2.74 cN / dtex, with a retention rate of 88.4%. Although these performance indicators are slightly lower than those of the aforementioned examples, the environmental benefits are very significant: compared with the traditional process, energy consumption is reduced by 45.6%, water consumption by 38.2%, wastewater discharge by 62.5%, and COD emissions decreased from 3200 mg / L to 886 mg / L, a reduction of 72.3%.

[0084] From a long-term economic perspective, although the initial investment in immobilized enzyme preparation is higher, considering that it can be recycled more than five times, coupled with a significant reduction in energy and water consumption, its overall economic efficiency is superior to traditional processes. Calculations show that the investment payback period is typically 12-18 months, after which stable returns can be obtained.

[0085] It is worth mentioning that the degreased cotton produced by this environmentally friendly process performs well in terms of biocompatibility, and its cytotoxicity test results are better than those of products produced by traditional processes. This may be related to the reduction in the amount of chemical reagents used in the processing.

[0086] To objectively evaluate the technical effects of this invention, two sets of comparative experiments were designed. The first was a comparison of single-enzyme treatment, using the same raw materials and pretreatment methods as in Example 1, but only 3.0 g / L bromelain was used. The results showed that the performance of the single-enzyme treated product was significantly inferior to that of the compound enzyme treated product: the reduction rate of surface fiber diameter was only 6.2% (compound enzyme: 10.5%), the water absorption rate reached only 15.3 mL / g (compound enzyme: 22.8 mL / g), the tensile strength retention rate was lower at 76.3% (compound enzyme: 89.6%), the liquid diffusion rate increase rate was only 40.5% (compound enzyme: 72.3%), and the treatment required a longer time, approximately 240 minutes (compound enzyme: 165 minutes).

[0087] The microstructure comparison more intuitively illustrates the differences. The surface modification of the fiber after single-enzyme treatment was uneven, with obvious over-degraded and under-treated areas coexisting, while the surface modification after composite enzyme treatment was uniform. Attempts were made to improve the effect by increasing the dosage of the single enzyme (5.0 g / L) or extending the treatment time (360 minutes), but the result was a further decrease in tensile strength to 68.5%, while the improvement in surface properties was limited, proving that a single enzyme is insufficient to achieve the ideal targeted enzymatic hydrolysis effect on the surface.

[0088] The second set of comparative experiments was a comparison of processes without dynamic control. The same raw materials and compound enzyme ratio as in Example 1 were used, but the entire enzymatic hydrolysis process was maintained at a constant temperature (45℃) and a constant pH (6.0) for 180 minutes without staged regulation. The results showed that the process without dynamic control struggled to precisely control the depth of enzymatic hydrolysis: the reduction rate of surface fiber diameter was as high as 15.8% (5.3% higher than the compound enzyme, indicating over-degradation), and the tensile strength retention rate was only 62.5% (27.1% lower than the compound enzyme). Fiber cross-section analysis showed extremely uneven degradation depth; deep degradation in some areas led to a decrease in strength, while insufficient surface degradation in other areas affected performance improvement. Although the process without dynamic control had a slight advantage in water absorption (23.5 mL / g vs 22.8 mL / g), considering all indicators, especially product uniformity and reliability, the dynamic control process was clearly superior.

[0089] In practical industrial applications, the technology of this invention has been verified and applied by many companies. A medical device company using the process of Example 1 produced medical dressings that, compared to its original chemically treated products, had a 110% higher water absorption rate and a liquid diffusion rate that increased by over 70%, meeting the demands of the high-end wound care market. Simultaneously, processing time was reduced by 35%, energy consumption by 42%, and wastewater COD by 75%, resulting in significant overall economic benefits.

[0090] Another cosmetics company using the process in Example 2 produced cotton pads that showed a more than 60% increase in softness and a 50% improvement in fit, raising user satisfaction from 76% to 92%. The application of enzyme recovery technology reduced enzyme costs by 65% ​​and product costs by approximately 12%, significantly enhancing market competitiveness.

[0091] The environmentally friendly and low-energy-consumption process in Example 4 has been applied in a green manufacturing demonstration enterprise, successfully creating the first zero-emission degreased cotton processing production line in China, obtaining green manufacturing certification and government funding support, and providing new ideas for the sustainable development of the industry.

[0092] Market feedback indicates that the absorbent cotton products processed by the technology of this invention show significant advantages in several sub-sectors: in the medical field, wound care is improved, infection rates are reduced, and patient comfort is increased; in the beauty field, the user experience is improved, serum utilization is increased, and skin irritation is reduced; and in special application fields, such as biological sample collection and precision instrument cleaning, they also perform excellently.

[0093] Although the initial investment of this invention is slightly higher than that of traditional processes, considering the added value brought about by improved product performance, increased production efficiency, and reduced resource consumption, the investment payback period is typically 12-18 months, resulting in significant long-term economic benefits. With increasingly stringent environmental requirements and growing demand for high-performance cellulose materials, this technology is expected to be applied and promoted in a wider range of fields.

Claims

1. A method for targeted enzymatic hydrolysis of the outer layer of defatted cotton based on a complex plant protease, characterized in that... This includes the following steps: (1) Physical pretreatment: The degreased cotton is subjected to physical pretreatment to enhance its surface activation. The physical pretreatment employs plasma treatment, ultrasonic treatment, microwave-assisted treatment, or a combination thereof, wherein: The plasma treatment conditions are: power 100-300W, time 10-20 minutes; The ultrasonic treatment conditions are: frequency 20-40kHz, power density 200-500W / L, and time 8-15 minutes; The microwave-assisted processing conditions are: frequency 2.45GHz, power 200-400W, and intermittent processing for 3-5 minutes. (2) Enzyme solution preparation: Prepare a complex enzyme solution containing bromelain, pectinase, and xylanase, wherein the ratio of bromelain, pectinase, and xylanase is 1:(0.5-2):(0.3-1); (3) Enzymatic hydrolysis: The pretreated defatted cotton is contacted with the compound enzyme solution and directional enzymatic hydrolysis is carried out under controlled conditions; (4) Dynamic control: The depth of enzymatic hydrolysis is controlled by adjusting the reaction temperature and pH value in stages, wherein: The phased temperature regulation comprises three consecutive stages: first, activating enzyme activity by maintaining a temperature of 48-52℃ for 30-60 minutes; then, cooling to 38-42℃ and maintaining moderate enzyme activity for 60-90 minutes; finally, cooling to 28-32℃ and maintaining this temperature for 30-60 minutes to inhibit excessive reaction. The phased pH adjustment process involves three stages: the first stage maintains the pH at 6.2-6.8, which is conducive to the activity of bromelain; the second stage adjusts the pH to 5.2-5.8 to promote pectinase activity; and the third stage raises the pH to 6.7-7.3 to gradually inhibit enzyme activity and prevent excessive enzymatic hydrolysis. (5) Inactivation: After the reaction is complete, enzyme activity is terminated by heat treatment, pH adjustment, or the addition of chemical reagents; (6) Cleaning and drying: Finally, the treated degreased cotton is cleaned and dried to obtain a surface-modified degreased cotton product. The method achieves selective and directional enzymatic hydrolysis of the outer layer of degreased cotton, reducing the diameter of the surface fiber by 8-12%, while maintaining the integrity of the internal fiber structure and retaining ≥85% of the tensile strength.

2. The method according to claim 1, characterized in that... The method for terminating enzyme activity is selected from: raising the temperature of the reaction system to 78-82℃ and maintaining it for 8-12 minutes; or adjusting the pH value to 10.0-11.0 and maintaining it for 4-6 minutes; or adding 0.1%-0.2% hydrogen peroxide solution; or a combination of the above methods.

3. The method according to claim 1, characterized in that... The method further includes an enzyme preparation recovery step: after centrifugation to remove solid particles from the enzymatic hydrolysis waste liquid, membrane separation is performed using an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa, or salting out is performed using 40%-60% saturated ammonium sulfate, or selective adsorption is performed using macroporous resin; subsequently, the recovered enzyme is concentrated and stabilized so that it can be recycled 3-5 times with an activity retention rate of not less than 60%.

4. The method according to claim 1, characterized in that... The preparation method of the composite enzyme solution is selected from: preparing it in a fixed ratio of bromelain: pectinase: xylanase = 1:0.8:0.5; or using a sequential addition method, first adding bromelain, and after it has acted for a certain period of time, adding pectinase and xylanase in sequence; or immobilizing the enzyme on a carrier such as macroporous polymer spheres or magnetic nanoparticles to form an immobilized enzyme preparation.