A sophorolipase hydrolysis product with acaricidal efficacy, and a preparation method and application thereof

Abstract

The present application relates to a sophorolipid enzymatic hydrolysate with de-acaricidal effect and a preparation method and application thereof, and belongs to the technical field of daily chemicals. The present application provides a preparation method of a sophorolipid enzymatic hydrolysate, comprising the following steps: S1: sophorolipid is enzymatically hydrolyzed by a composite enzyme to obtain a primary enzymatic hydrolysate; the composite enzyme comprises lipase and acetyl xylan esterase; S2: the primary enzymatic hydrolysate of step S1 is enzymatically hydrolyzed by beta-glucosidase to obtain a secondary enzymatic hydrolysate; S3: the secondary enzymatic hydrolysate of step S2 is purified to obtain the sophorolipid enzymatic hydrolysate. The substrate of the enzymatic hydrolysis reaction of the present application is mainly in the form of lactone sophorolipid, and the sophorolipid is enzymatically hydrolyzed by using a mild enzymatic hydrolysis method, the active substances of the obtained enzymatic hydrolysate synergistically act, and the final enzymatic hydrolysate has good de-acaricidal and repairing effects.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products technology, and in particular to a sophoroliposide enzymatic hydrolysis product with mite-removing effects, its preparation method, and its application. Background Technology

[0002] Dust mites have become a major carrier of indoor allergens, breeding in a wide range of environments including mattresses (where 1,000 to 5,000 mites can be found per gram of mattress dust), sofa fabrics, infant clothing, air conditioner filters, and plush toys. On one hand, the allergenic proteins such as Der p1 and Der f1 contained in dust mite excrement and molted skin can trigger allergic rhinitis and atopic dermatitis through inhalation or skin contact, posing a significant threat to sensitive populations such as infants and asthma sufferers. On the other hand, dust mite bites and the bacteria (such as Staphylococcus aureus) carried on their skin can also cause secondary skin infections, leading to conditions like impetigo, affecting the health of human skin, especially facial skin. Therefore, developing efficient and safe dust mite removal products has become an important research direction in the daily chemical, home textile, and health industries.

[0003] Currently, the mainstream methods for mite removal in the market include physical and chemical methods. Physical methods eliminate mites through high-temperature drying, ultraviolet irradiation, and vacuum cleaners. However, these methods have certain drawbacks. For example, while temperatures above 60℃ can kill adult mites, they damage natural fibers such as wool and silk, and cannot remove mite eggs deep within the fabric. Ultraviolet irradiation requires continuous exposure for more than 30 minutes to achieve a 50% mite removal rate, and its effectiveness is limited by fabric shading, with removal rates in shaded areas falling below 10%, making it difficult to meet comprehensive mite removal needs. Chemical methods primarily achieve their effect through acaricides such as pyrethroids and organophosphate compounds. While these products can achieve a certain mite-removal effect in the short term, their safety is insufficient: mite-removal agents are prone to leave residues in clothing, bedding, or air, which can irritate the skin, mucous membranes, and respiratory systems of infants, pregnant women, and sensitive individuals. Long-term exposure may also lead to chronic health risks. Secondly, they have poor environmental compatibility, with some components being difficult to degrade naturally, easily causing water and soil pollution. Thirdly, mites are prone to developing drug resistance, and long-term use will lead to a continuous decline in the mite-removal effect, requiring the concentration of the agent to be continuously increased, further exacerbating safety hazards.

[0004] To overcome the limitations of chemical acaricides, the acaricide industry has begun to shift its focus to the research and development of biological acaricides, with microbial metabolites becoming a research hotspot due to their "safety and easy degradation" characteristics. Sophorolipids, as a typical biosurfactant, are produced by Candida albicans (…). Candida bombicolaProduced through fermentation using sugars and vegetable oils as carbon sources, sophorolipids possess excellent surface activity, biocompatibility, and environmental friendliness, and have already found applications in the daily chemical and detergent industries. As an environmentally friendly biosurfactant, sophorolipids are non-toxic, biodegradable, heat- and salt-resistant, and have low surface tension, making them widely used in petroleum extraction (to reduce crude oil viscosity), agriculture (as a pesticide synergist), and daily chemicals (as a mild detergent). However, in existing technologies, the anti-mite activity of natural sophorolipids is relatively weak, limiting its promotion and application in the field of mite control. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sophoroliposide enzymatic hydrolysate with mite-removing effects, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing sophorolipase hydrolysis products, comprising the following steps: S1: Sophorolipase is hydrolyzed using a complex enzyme to obtain a primary hydrolysate; the complex enzyme includes lipase and acetylxylan esterase. S2: The primary hydrolysate from step S1 is hydrolyzed with β-glucosidase to obtain the secondary hydrolysate; S3: Purify the secondary enzymatic hydrolysate from step S2 to obtain the sophorolipid hydrolysate.

[0007] Sophorolipid molecules are composed of a hydrophilic sophorose group and a hydrophobic long-chain hydroxy fatty acid. In lactone-type sophorolipids, the carboxyl group (-COOH) at the end of the hydrophobic hydroxy fatty acid chain undergoes esterification with a hydroxyl group (-OH) on the hydrophilic sophorose group, forming a cyclic lactone structure. Due to its cyclic structure, lactone-type sophorolipids exhibit stronger antibacterial and antitumor bioactivity, higher emulsification stability, and greater temperature and salt tolerance. Furthermore, the excellent surface tension-reducing ability of lactone-type sophorolipids allows them to exert high surface activity at lower concentrations. In acidic sophorolipids, the carboxyl group at the end of the fatty acid chain remains free (-COOH) and does not form a cyclic structure with the sugar group, thus the molecule is open-ring. Due to the presence of its hydrophilic carboxyl group, acidic sophorolipids have superior foaming ability and solubility, making them suitable for applications requiring rich foam or high water solubility. This invention selects sophorolipids with a high content of lactones as the enzymatic hydrolysis substrate. On the one hand, lactone-type sophorolipids have stronger bactericidal properties, and the subsequent enzymatic hydrolysis products also have better mite-inhibiting effects. On the other hand, lactone-type and acid-type sophorolipids have different properties due to their structural differences, forming a complementary relationship in application. The product obtained by enzymatic hydrolysis of sophorolipids using this mixture has excellent mite-killing efficacy, as well as better repair effects and higher safety (lower irritation), making it more suitable for application in daily chemical products.

[0008] Further, in step S1, the sophorolipids include lactone-type sophorolipids and acid-type sophorolipids. Based on the mass of the sophorolipids, the mass percentage of lactone-type sophorolipids is preferably 70% to 90%, for example, including but not limited to any point value or any range of two points such as 70%, 75%, 80%, 85% and 90%.

[0009] Further, in step S1, the mass ratio of the lipase to the acetylxylan esterase is preferably lipase:acetylxylan esterase = (2~4):1, for example, including but not limited to any point value or any range value composed of any two points such as 2:1, 2.5:1, 3:1, 3.5:1 and 4:1.

[0010] Further, in step S1, the mass ratio of sophorolipid to the complex enzyme is preferably sophorolipid:complex enzyme = 100:(0.2~0.8), for example, including but not limited to any point value or any range of two points from 100:0.2, 100:0.25, 100:0.3, 100:0.35, 100:0.4, 100:0.45, 100:0.5, 100:0.55, 100:0.6, 100:0.65, 100:0.7, 100:0.75 and 100:0.8.

[0011] Further, in step S1, the sophorolipid mixture is enzymatically hydrolyzed with the composite enzyme. The sophorolipid mixture is a mixture of sophorolipid and water. The mass fraction of sophorolipid in the sophorolipid mixture is preferably 4% to 8%, for example, including but not limited to any point value or any range of any two points from 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, and 8%.

[0012] Further, in step S1, the preparation method of the sophorolipid mixture is as follows: the sophorolipid mixture is prepared by mixing sophorolipid and water uniformly through ultrasonic treatment, with the mixing temperature preferably being 30~55℃, for example including but not limited to any point value or any two points of 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, 45℃, 48℃, 50℃, 52℃ and 55℃; the ultrasonic treatment power is preferably 300~500W, for example including but not limited to any point value or any two points of 300W, 320W, 350W, 380W, 400W, 420W, 450W, 480W and 500W.

[0013] Further, in step S1, the enzymatic hydrolysis temperature is preferably 40~55℃, for example, including but not limited to any point value or any range of two points among 40℃, 42℃, 45℃, 48℃, 50℃, 52℃ and 55℃; the enzymatic hydrolysis time is preferably 150~300min, for example, including but not limited to any point value or any range of two points among 150min, 180min, 200min, 220min, 240min, 260min, 280min and 300min. In step S2, the enzymatic hydrolysis temperature is preferably 40~50℃, for example, including but not limited to any point value or any two points of 40℃, 42℃, 45℃, 48℃ and 50℃; the enzymatic hydrolysis time is preferably 120~240min, for example, including but not limited to any point value or any two points of 120min, 150min, 180min, 200min, 220min and 240min.

[0014] Further, in step S2, the pH of the primary enzymatic hydrolysate from step S1 is adjusted before enzymatic hydrolysis with β-glucosidase. The pH is preferably 5 to 6, for example, including but not limited to any point value or any range of two points from 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.

[0015] Further, in step S2, the pH of the primary enzymatic hydrolysate from step S1 is adjusted using a pH adjuster. The pH adjuster includes at least one of HCl solution, HNO3 solution, H2SO4 solution, NaH2PO4 solution, and KH2PO4 solution. The concentration of the pH adjuster is preferably 0.5~5 mol / L, for example, including but not limited to any point value or any range of any two points from 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, and 5 mol / L.

[0016] Further, in step S2, the mass ratio of sophorolipid to β-glucosidase is preferably 100:(0.1~0.4), for example, including but not limited to any point value or any range of two points among 100:0.1, 100:0.15, 100:0.2, 100:0.25, 100:0.3, 100:0.35 and 100:0.4.

[0017] Further, in step S1 or S2, after the enzymatic hydrolysis is completed, inactivation is performed. The inactivation temperature is preferably 70~90℃, for example, including but not limited to any point value or any two points of 70℃, 72℃, 75℃, 78℃, 80℃, 82℃, 85℃, 88℃ and 90℃; the holding time is preferably 15~30min, for example, including but not limited to any point value or any two points of 15min, 18min, 20min, 22min, 25min, 28min and 30min.

[0018] Further, in step S3, the secondary enzymatic hydrolysate from step S2 is purified by filtration through a membrane with a molecular weight cutoff of 1000-2000 Da, and then dried to obtain the sophoroliposide enzymatic hydrolysate. The molecular weight cutoff is preferably 1000-2000 Da, and may include, but is not limited to, any value from 1000 Da, 1200 Da, 1500 Da, 1800 Da, and 2000 Da, or a range consisting of any two of these values. Further, in step S3, the drying method includes at least one of heat drying, spray drying, and freeze drying; the temperature for heat drying is preferably 80~120℃, for example, including but not limited to any point value or any range of two points from 80℃, 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃, and 120℃; the inlet air temperature for spray drying is preferably 100~140℃, for example, including but not limited to any point value or any range of two points from 100℃, 105℃, 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, and 140℃; the outlet air temperature for spray drying is preferably 70~90℃. The preferred freezing temperature is -20 to 0°C, including but not limited to any point value or any range of two points among 70°C, 72°C, 75°C, 78°C, 80°C, 82°C, 85°C, 88°C, and 90°C; the preferred freeze-drying temperature is -20 to 0°C, including but not limited to any point value or any range of two points among -20°C, -18°C, -15°C, -12°C, -10°C, -8°C, -5°C, -2°C, and 0°C; the preferred freeze-drying vacuum degree is 50 to 120 Pa, including but not limited to any point value or any range of two points among 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, 100 Pa, 110 Pa, and 120 Pa.

[0019] Secondly, the present invention provides a sophoroliposide enzymatic hydrolysis product, which is prepared by the aforementioned preparation method.

[0020] Thirdly, the present invention provides the application of the sophoroliposide hydrolysis product in the preparation of acaricides.

[0021] Fourthly, the present invention provides an acaricide containing the sophoroliposide enzymatic hydrolysis product.

[0022] Furthermore, the species of mites include at least one of dust mites, flour mites, and demodex mites.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a two-stage enzymatic hydrolysis process for sophorolipids. The first enzymatic hydrolysis reaction involves deacetylation using a complex enzyme, yielding partially or fully deacetylated sophorolipids. The second enzymatic hydrolysis reaction uses β-glucosidase to partially hydrolyze the sophorolipids into sophorose and fatty acids. Compared to acetylated sophorolipids, deacetylated sophorolipids have their acetyl groups replaced by hydrophilic hydroxyl groups, resulting in better water solubility and easier penetration into mites during formulation. Fatty acids, as emulsifiers, can regulate the stability and skin feel of cosmetic formulations, and also promote the transdermal absorption of active ingredients, enhancing their skincare efficacy. The sophorolipid hydrolysis product of this invention uses a gentle enzymatic hydrolysis method to hydrolyze sophorolipids, resulting in a hydrolysate that retains the original sophorolipids while also yielding deacetylated sophorolipids, sophorose, and fatty acids. These active ingredients work synergistically, giving the final hydrolysate excellent mite-killing and repairing effects.

[0024] The substrate of the enzymatic hydrolysis reaction of the present invention is mainly lactone-type sophorolipid, which has excellent ability to reduce surface tension, antibacterial properties and emulsification stability. A large portion of lactone-type sophorolipid is also retained in the final enzymatic hydrolysis product, thus exhibiting strong mite-removing properties.

[0025] The enzymatic hydrolysis process of this invention is mild, which not only preserves the bioactivity of sophorolipids, but also better meets the requirements of green chemistry for strong alkaline hydrolysis. The resulting product has low skin irritation and higher safety performance. Attached Figure Description

[0026] Figure 1 This is a sample image of the sophorolipase hydrolysate with mite-removing effect from Example 1 of the present invention. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] The components and reagents involved in the embodiments and comparative examples of this invention are described below: The sophorolipids used in this invention are a mixture of lactone-type sophorolipids and acid-type sophorolipids, and are derived from Guangzhou Yingxuan Biotechnology Co., Ltd.

[0029] The CAS number of the acetylxylan esterase used in this invention is 9000-82-2.

[0030] The lipase used in this invention has the CAS number 9001-62-1.

[0031] The CAS number of the β-glucosidase used in this invention is 9001-22-3.

[0032] All commercially available enzymes corresponding to the above CAS numbers can be used in this invention.

[0033] Unless otherwise specified, all other components are commercially available.

[0034] Unless otherwise specified, the concentrations mentioned in this invention are mass concentrations.

[0035] Unless otherwise specified, the water mentioned in this invention is deionized water.

[0036] Example 1 This embodiment provides a sophoroliposide enzymatic hydrolysate with mite-removing effects and its preparation method, the steps of which are as follows: S1: Sophorolipids (composed of 80%±2% lactone-type sophorolipids and 20%±2% acid-type sophorolipids by mass) were added to water and mixed evenly under ultrasonic treatment at 40℃ and 400W to obtain a sophorolipid mixture with a sophorolipid mass fraction of 6%. Add 0.4% (by mass fraction) of a complex enzyme (the mass ratio of sophorolipid to complex enzyme is 100:0.4; the complex enzyme is lipase and acetylxylan esterase, with a mass ratio of 3:1) to the sophorolipid mixture, and perform one enzymatic hydrolysis at 45℃ for 240 min. After the enzymatic hydrolysis is completed, incubate at 80℃ for 20 min to inactivate the enzyme and obtain the primary hydrolysate. S2: Add 2 mol / L HCl solution to the primary enzymatic hydrolysate to adjust the pH to 5.5, add 0.2% β-glucosidase (mass ratio of sophorolipid to β-glucosidase is 100:0.2), and perform secondary enzymatic hydrolysis at 45℃ for 180 min. After the enzymatic hydrolysis is completed, incubate at 80℃ for 20 min to inactivate the enzyme, and obtain the secondary enzymatic hydrolysate. S3: The secondary enzymatic hydrolysate is purified by filtration through a ceramic membrane with a molecular weight cutoff of 1200 Da, and then spray-dried (conditions: inlet air temperature 120℃, outlet air temperature 80℃) to obtain a sophoroliposide enzymatic hydrolysate with mite-removing effects.

[0037] Example 2 This embodiment provides a sophoroliposide enzymatic hydrolysate with mite-removing effects and its preparation method, the steps of which are as follows: S1: Sophorolipids (composed of 70%±2% lactone-type sophorolipids and 30%±2% acid-type sophorolipids by mass) were added to water and mixed evenly under ultrasonic treatment at 30℃ and 500W to obtain a sophorolipid mixture with a sophorolipid mass fraction of 8%. Add 0.8% (by mass fraction of sophorolipid) of a complex enzyme (the mass ratio of sophorolipid to complex enzyme is 100:0.8; the complex enzyme is lipase and acetylxylan esterase, with a mass ratio of 4:1) to the sophorolipid mixture, and perform one enzymatic hydrolysis at 55℃ for 150 min. After the enzymatic hydrolysis is completed, incubate at 70℃ for 30 min to inactivate the enzyme and obtain the primary hydrolysate. S2: Add 0.5 mol / L H2SO4 solution to the primary enzymatic hydrolysate to adjust the pH to 6.0, add 0.4% β-glucosidase (mass ratio of sophorolipid to β-glucosidase is 100:0.4), and perform secondary enzymatic hydrolysis at 50℃ for 150 min. After the enzymatic hydrolysis is completed, incubate at 70℃ for 30 min to inactivate the enzyme, and obtain the secondary enzymatic hydrolysate. S3: The secondary enzymatic hydrolysate was purified by filtration through a ceramic membrane with a molecular weight cutoff of 2000 Da, and then freeze-dried (conditions: temperature -10℃, vacuum degree 80Pa) to obtain a sophoroliposide enzymatic hydrolysate with mite-removing effects (e.g., Figure 1 (As shown).

[0038] Example 3 This embodiment provides a sophoroliposide enzymatic hydrolysate with mite-removing effects and its preparation method, the steps of which are as follows: S1: Sophorolipids (composed of 90%±2% lactone-type sophorolipids and 10%±2% acid-type sophorolipids by mass) were added to water and mixed evenly under ultrasonic treatment at 55℃ and 300W to obtain a sophorolipid mixture with a sophorolipid mass fraction of 4%. Add 0.2% (by mass fraction) of a complex enzyme (the mass ratio of sophorolipid to complex enzyme is 100:0.2; the complex enzyme is lipase and acetylxylan esterase, with a mass ratio of lipase to acetylxylan esterase of 2:1) to the sophorolipid mixture, and perform one enzymatic hydrolysis at 40℃ for 300 min. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme and obtain the primary hydrolysate. S2: Add 5 mol / L HNO3 solution to the primary enzymatic hydrolysate to adjust the pH to 5.0, add 0.1% β-glucosidase (mass ratio of sophorolipid to β-glucosidase is 100:0.1), and perform secondary enzymatic hydrolysis at 40℃ for 240 min. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme, and obtain the secondary enzymatic hydrolysate. S3: The secondary enzymatic hydrolysate is purified by filtration through a ceramic membrane with a molecular weight cutoff of 1000 Da, and then heated and dried (condition: temperature 100℃) to obtain a sophoroliposide enzymatic hydrolysate with mite-removing effects.

[0039] Examples 4-13 Examples 4-13 provide a sophoroliposide enzymatic hydrolysate with mite-removing effects and its preparation method. The preparation methods of the sophoroliposide enzymatic hydrolysate in Examples 4-13 differ from those in Example 1 only in one step S1-S3; the rest are the same as in Example 1. The differences between Examples 4-13 and Example 1 are shown in Table 1 below.

[0040] Table 1. Differences between Examples 4-13 and Example 1 Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the sophorolipid composed of 80%±2% lactone-type sophorolipid and 20%±2% acid-type sophorolipid in step S1 of Example 1 is replaced with a sophorolipid composed of 50±2% lactone-type sophorolipid and 50±2% acid-type sophorolipid. The remaining steps and parameters are the same as in Example 1.

[0041] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that in Comparative Example 2, the sophorolipid composed of 80%±2% lactone-type sophorolipid and 20%±2% acid-type sophorolipid in step S1 of Example 1 is replaced with a sophorolipid composed of 96%±2% lactone-type sophorolipid and 4%±2% acid-type sophorolipid. The remaining steps and parameters are the same as in Example 1.

[0042] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the complex enzyme in step S1 of Example 1 is replaced with lipase in Comparative Example 3, and the mass ratio of sophorolipid to lipase is 100:0.4. The remaining steps and parameters are the same as in Example 1.

[0043] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the complex enzyme in step S1 of Example 1 is replaced with acetylxylan esterase in Comparative Example 4, and the mass ratio of sophorolipid to acetylxylan esterase is 100:0.4. The remaining steps and parameters are the same as in Example 1.

[0044] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the mass ratio of lipase to acetylxylan esterase in the complex enzyme in step S1 of Example 1 is replaced by a mass ratio of 1:1 instead of 3:1. The remaining steps and parameters are the same as in Example 1.

[0045] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that the lipase in step S1 of Example 1 is replaced with carboxylesterase in Comparative Example 6, while the remaining steps and parameters are the same as in Example 1.

[0046] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that in Comparative Example 7, the acetylxylan esterase in step S1 of Example 1 is replaced with carboxyl esterase, while the remaining steps and parameters are the same as in Example 1.

[0047] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that the enzymatic hydrolysis time in step S1 of Example 1 was changed from 240 min to 400 min in Comparative Example 8, while the remaining steps and parameters were the same as in Example 1.

[0048] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that in Comparative Example 9, β-glucosidase in step S2 of Example 1 is replaced with endoglucanase, while the remaining steps and parameters are the same as in Example 1.

[0049] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that in Comparative Example 10, β-glucosidase in step S2 of Example 1 is replaced with α-glucosidase, while the remaining steps and parameters are the same as in Example 1.

[0050] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that the enzymatic hydrolysis time in step S2 of Example 11 was changed from 180 min to 300 min, while the remaining steps and parameters were the same as in Example 1.

[0051] Comparative Example 12 The difference between Comparative Example 12 and Example 1 is that in Comparative Example 12, the sophorolipid composed of 80%±2% lactone-type sophorolipid and 20%±2% acid-type sophorolipid in step S1 of Example 12 is replaced with a sophorolipid composed of 20%±2% lactone-type sophorolipid and 80%±2% acid-type sophorolipid. The remaining steps and parameters are the same as in Example 1.

[0052] Comparative Example 13 Comparative Example 13 provides a sophoroliposide enzymatic hydrolysate with mite-removing effects and its preparation method, the steps of which are as follows: S1: Sophorolipids (composed of 80%±2% lactone-type sophorolipids and 20%±2% acid-type sophorolipids by mass) were added to water and mixed evenly under ultrasonic treatment at 40℃ and 400W to obtain a sophorolipid mixture with a sophorolipid mass fraction of 6%. The pH of the sophorolipid mixture was adjusted to 5.5 by adding 2 mol / L HCl solution. β-glucosidase (mass ratio of sophorolipid to β-glucosidase of 0.2% by mass of sophorolipid) was added and enzymatic hydrolysis was performed once at 45℃ for 180 min. After the enzymatic hydrolysis was completed, the mixture was incubated at 80℃ for 20 min to inactivate the enzyme and obtain the primary hydrolysate. S2: Add 0.4% (by mass fraction) of a complex enzyme (the mass ratio of sophorolipid to complex enzyme is 100:0.4; the complex enzyme is lipase and acetylxylan esterase, and the mass ratio of lipase to acetylxylan esterase is 3:1) to the primary enzymatic hydrolysate, and perform a second enzymatic hydrolysis at 45℃ for 240 min. After the enzymatic hydrolysis is completed, incubate at 80℃ for 20 min to inactivate the enzyme, and obtain the secondary enzymatic hydrolysate. S3: The secondary enzymatic hydrolysate is purified by filtration through a ceramic membrane with a molecular weight cutoff of 1200 Da, and then spray-dried (conditions: inlet air temperature 120℃, outlet air temperature 80℃) to obtain a sophoroliposide enzymatic hydrolysate with mite-removing effects.

[0053] Example of effect I. Experimental Methods 1. Eye irritation test The sophorolipolysis products of Examples 1-13 and Comparative Examples 1-13 of this invention were subjected to eye irritation tests according to the standard SN / T2329-2009 "Eye Irritation and Corrosiveness Test of Chicken Embryo Villi Allantoic Membrane in Cosmetics". The results were evaluated using the irritation scoring method. The test results are shown in the table below.

[0054] Irritation classification: IS < 1, non-irritating; 1 ≤ IS < 5, mildly irritating; 5 ≤ IS < 9, moderately irritating; IS ≥ 9, strongly irritating / corrosive.

[0055] The chicken embryos used were BWEL-SPF chickens from Xinxing Dahua Agricultural Poultry and Egg Co., Ltd., 0 days old, and qualified according to GB / T17999.1-2008.

[0056] Test samples: The enzymatic hydrolysis products of sophorolipids from Examples 1-13 and Comparative Examples 1-13 were prepared into a 15% solution with deionized water; a sophorolipid with a mass fraction of 80% ± 3% lactone was prepared into a 15% solution with DMSO as a control.

[0057] 2. Mite removal test Test samples: Sophoroliposide hydrolysate of Examples 1-13 and Comparative Examples 1-13, prepared into a 0.5% solution with sterile water, and sterile physiological saline as the blank control group.

[0058] Test procedure: The filter paper was uniformly sprayed using a Potter standard spray tower (the filter paper diameter was 9 cm, matching the petri dish). The spray volume for the test sample or blank control group was 1 mL. The spray pressure and spray distance were kept consistent during the spraying process. The sprayed filter paper was placed in a sterile environment at room temperature to air dry for 30 min. After drying, 30 adult house dust mites and 10 mg of mite feed were added to the filter paper. The petri dish was then covered and cultured in a constant temperature and humidity incubator (temperature 25±1℃, humidity 75%±5%) for 24 h. After the culture was completed, the status of the mites was observed under a 400x microscope. The mites were considered dead if their chelicerae or claws did not move. The mite removal rate was calculated based on the number of dead house dust mites. The method for calculating the house dust mite removal rate is shown in the following formula (1).

[0059] House dust mite removal rate (%) = House dust mite corrected mortality rate (%) = [(number of surviving house dust mites in blank control group - number of surviving house dust mites in sample group) / number of surviving house dust mites in blank control group] × 100% Equation (1).

[0060] The test was conducted in three parallel trials, and the average value of the results was taken.

[0061] 3. Repair test (IVL gene expression level test) Test Principle: The skin's barrier function is primarily located in the stratum corneum (SC), the outermost layer of the skin. Keratin within stratum corneum cells, along with extracellular membrane proteins such as filaggrin (FLG), involucrin (IVL), and loricrin (LOR), as well as the extracellular matrix and tight junctions (TJs), interconnect to form the complete skin barrier. IVL is a key structural protein synthesized in the early stages of terminal differentiation of human keratinocytes (HaCaT). It plays a crucial role in the formation of the keratin capsule. When the skin is stimulated by external factors, the normal differentiation process of the epidermis is disrupted, leading to a significant decrease in IVL gene and protein expression, and a loosening and fragility of the keratin capsule structure. When the test sample can promote increased IVL gene or protein expression, it indicates that the component can promote healthy terminal differentiation of keratinocytes, promote keratin capsule maturation, strengthen the skin barrier, and exhibit a repairing effect.

[0062] Test samples: Sophoroliposide enzymatic hydrolysates from Examples 1-13 and Comparative Examples 1-13 were prepared into a 0.5% (w / w) test solution using H-DMEM medium containing 10% (v / v) FBS and 1.5 mM CaCl2.

[0063] Test procedure: HaCaT cells in the logarithmic growth phase were placed in 6-well culture plates, and 2 × 10⁻⁶ cells were added to each well. 5 Cells were cultured in H-DMEM medium containing 10% FBS for 24 h at 37°C, 5% CO2, and saturated humidity. The culture medium was then discarded. 1 mL of 200 ng / mL LPS (lipopolysaccharide) solution was added to the model control group and sample group, while 1 mL of culture medium was added to the negative control group. Cells were cultured at 37°C, 5% CO2, and saturated humidity for 6 h. After culture, the culture medium was discarded. 1 mL of the corresponding test solution was added to each sample group, while 1 mL of H-DMEM medium containing 10% FBS and 1.5 mM CaCl2 was added to the negative control group. Cells were cultured for another 24 h. After culture, the cells were lysed to collect RNA, which was reverse transcribed into cDNA and stored at -80°C. Finally, quantitative real-time PCR was performed to determine the expression level of the IVL gene. Three routine experiments were performed for each group, and the average results were taken.

[0064] 4. Repair Test (Zebrafish Tail Fin Repair Test) Testing Principle: When injured, the skin must regenerate rapidly to repair the skin barrier. In the embryonic stage, wound healing is very rapid and leaves no scar; however, after the embryonic stage, wound healing involves steps such as coagulation, inflammation, skin regeneration, angiogenesis, and the formation of granulation tissue, ultimately resulting in a scar. In zebrafish, the wound repair process is identical to that in humans, except for the absence of coagulation. Zebrafish wounds heal very quickly, followed by the migration of inflammatory cells to the wound to form granulation tissue composed of macrophages, fibroblasts, blood vessels, and collagen. Therefore, the main steps and principles of wound healing in zebrafish and humans are very similar, making zebrafish a suitable model for screening and testing the efficacy of human skin repair.

[0065] Test samples: The sophorolipolysis products of Examples 1-13 and Comparative Examples 1-13 were prepared into test sample solutions with a mass fraction of 0.5% using fish embryo culture medium.

[0066] Test Procedure: Damaged zebrafish embryos were transferred to 96-well plates, with one embryo and 0.2 mL of test sample solution per well (the blank control group consisted of 0.2 mL of embryo culture medium). Both the blank control group and the test sample group were incubated at 28°C for 3 hours. After incubation, the zebrafish embryos were anesthetized with tricaine, and the caudal fin length of each embryo was counted under a stereomicroscope. The caudal fin repair promotion rate of the zebrafish embryos was calculated using the following formula. Ten parallel experiments were conducted for each group, and the average result was taken.

[0067] Promotion rate = (TC) / C × 100% Equation (2); In equation (2), T represents the average length of the caudal fin of zebrafish embryos in the test sample group; C represents the average length of the caudal fin of zebrafish embryos in the blank control group.

[0068] II. Experimental Results As shown in Table 2, under high concentration conditions, the sophoroliposide enzymatic hydrolysates of Examples 1-13 of this invention are not irritating, and are therefore very safe. In Comparative Example 6, lipase was replaced with carboxylesterase; in Comparative Example 7, acetylxylan esterase was replaced with carboxylesterase; and in Comparative Example 12, the proportion of lactone-type sophoroliposide in the sophoroliposide mixture was altered. These changes resulted in variations in the proportion of active substances in the final enzymatic hydrolysates, leading to differences in the irritant properties of the hydrolysates.

[0069] Table 2. Results of eye irritation test on sophorolipolysis products Table 3. Results of mite removal and repair tests on sophorolipase hydrolysate. As shown in Table 3, the sophoroliposide hydrolysates of Examples 1-13 of this invention exhibit excellent mite-killing effects against house dust mites, with a mite-killing rate exceeding 90%, achieving highly efficient mite-killing. The sophoroliposide hydrolysates of Examples 1-13 can also promote the increase of IVL gene expression levels in damaged HaCaT cells and promote the repair of zebrafish tail fins, indicating that the sophoroliposide hydrolysates of Examples 1-13 possess excellent skin barrier enhancement and damage repair effects. Among these, the mite-killing effect (mite-killing rate) and repair effect (relative expression level of IVL mRNA, tail fin promotion rate) of Examples 1-13 are better than commercially available sophoroliposides, which suffer from inherent defects such as high acetylation degree, poor water solubility, and a single active ingredient. This invention employs a two-stage, stepwise, targeted enzymatic hydrolysis process to hydrolyze sophorolipids. The first step replaces the acetyl groups on the sophorolipids with hydrophilic hydroxyl groups, enhancing the transdermal permeability of the final product. The second enzymatic hydrolysis step involves the targeted hydrolysis of some glycosidic bonds of the sophorolipids by β-glucosidase, generating sophorose and fatty acids with synergistic effects of promoting penetration, repairing, and emulsifying. The synergistic effect among multiple components, including lactone-type sophorolipids, acidic sophorolipids, deacetylated sophorolipids, free sophorose, and fatty acids, results in a final product exhibiting excellent mite-killing and repairing efficacy.

[0070] The mite-killing and repair effects of Examples 1-13 are better than those of Comparative Examples 1-2 and 12, indicating that the types of sophorolipids (lactone-type and acidic sophorolipids) have a significant impact on the efficacy of the enzymatic hydrolysis products. Lactone-type sophorolipids exhibit better antibacterial activity, emulsification stability, and temperature and salt resistance, while acidic sophorolipids have superior foaming ability and solubility. The different functional groups they contain result in significant differences in the types of active substances in their enzymatic hydrolysis products. Lactone-type sophorolipids are the core component ensuring mite-killing activity and emulsification stability. If the proportion is too low, the amount of lactone-type sophorolipids participating in the enzymatic hydrolysis reaction decreases, leading to a reduction in the amount of lactone-type sophorolipids retained in the final product. This weakens the final product's ability to reduce surface tension and reduces its antibacterial effect. A high proportion of lactone-type sophorolipids leads to a low content of acidic sophorolipids, resulting in a reduced final content and fewer enzymatic hydrolysis products. This results in insufficient foaming ability and solubility of the final product, affecting its synergistic effect and spreadability on the target material, thus weakening its mite-killing and repairing effects. In this invention, using sophorolipids with a lactone-type content of 70%–90% as the enzymatic hydrolysis raw material yields enzymatic hydrolysis products with better mite-killing and repairing effects. The mite-killing and repairing effects of Examples 1–13 are better than those of Comparative Examples 4–7 and 9–10, indicating that the type of enzyme used in the enzymatic hydrolysis has a significant impact on the final enzymatic hydrolysis products. The type of enzyme affects the degree of enzymatic hydrolysis and the type of final product, influencing the synergistic effect of various active ingredients and significantly impacting the mite-killing and repairing effects of the final product. The mite-killing and repairing effects of Examples 1-13 are better than those of Comparative Examples 8 and 11. Excessive enzymatic hydrolysis time (Comparative Examples 8 and 11) leads to over-hydrolysis of sophorolipids, affecting the synergistic effect of various active ingredients in the hydrolysate products and consequently impacting the efficacy of the final product. The better mite-killing and repairing effects of Examples 1-13 compared to Comparative Example 13 indicate that the enzymatic hydrolysis step has a significant impact on the final product. This invention preferably uses a composite enzyme of lipase and acetylxylan esterase to hydrolyze sophorolipids, followed by β-glucosidase hydrolysis, and has determined the optimal hydrolysis time.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing sophorolipase hydrolysate, characterized in that, Includes the following steps: S1: Sophorolipase is hydrolyzed using a complex enzyme to obtain a primary hydrolysate; the complex enzyme includes lipase and acetylxylan esterase. S2: The primary hydrolysate from step S1 is hydrolyzed with β-glucosidase to obtain the secondary hydrolysate; S3: Purify the secondary enzymatic hydrolysate from step S2 to obtain the sophorolipid hydrolysate.

2. The preparation method according to claim 1, characterized in that, In step S1, the sophorolipids include lactone-type sophorolipids and acid-type sophorolipids. Based on the mass of the sophorolipids, the mass percentage of lactone-type sophorolipids is 70% to 90%.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of the lipase to the acetylxylan esterase is: lipase:acetylxylan esterase = (2~4):

1.

4. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of sophorolipid to the complex enzyme is: sophorolipid: complex enzyme = 100: (0.2~0.8).

5. The preparation method according to claim 1, characterized in that, In step S1, the sophorolipid mixture is hydrolyzed with the compound enzyme. The sophorolipid mixture is a mixture of sophorolipid and water, and the mass fraction of sophorolipid in the sophorolipid mixture is 4% to 8%.

6. The preparation method according to claim 1, characterized in that, In step S1, the enzymatic hydrolysis temperature is 40~55℃, and the enzymatic hydrolysis time is 150~300min; In step S2, the enzymatic hydrolysis temperature is 40~50℃, and the enzymatic hydrolysis time is 120~240min.

7. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of sophorolipid to β-glucosidase is: sophorolipid:β-glucosidase = 100:(0.1~0.4).

8. A sophorolipase hydrolysis product, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the sophorolipase hydrolysis product according to claim 8 in the preparation of acaricides.

10. A mite remover, characterized in that, The acaricide contains the sophorolipase hydrolysis product as described in claim 8.

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