A porphyra yeast fermentation filtrate, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation and cosmetic raw materials, and particularly relates to a perilla yeast fermentation filtrate, a preparation method and application thereof. The preparation method comprises the following steps: crushing dried perilla leaves, mixing with sterile water, and then performing ultrasonic activation treatment to obtain a fermentation substrate; inoculating the obtained fermentation substrate with a yeast seed liquid, and then performing aerobic fermentation; adding L-leucine guide to the fermentation material when the fermentation is performed for 24-48 h, and continuing the fermentation treatment for 24-48 h; terminating the fermentation by high-temperature inactivation of the fermentation material, then performing pressure filtration, collecting the filtrate, and obtaining the perilla yeast fermentation filtrate. The perilla yeast fermentation filtrate obtained by the present application is rich in natural linalool and specific active ingredients, has excellent aroma, soothing anti-sensitivity, antioxidant and skin barrier repair effects, and can be used for preparing high-end skin care products.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and cosmetic raw material technology, specifically relating to a perilla yeast fermentation filtrate, its preparation method, and its application. Background Technology

[0002] Perilla is a plant used both as food and medicine. Its leaves are rich in flavonoids (such as luteolin and apigenin), phenolic acids (such as rosmarinic acid), volatile oils, and other active ingredients. Traditionally, it has been believed to have effects such as relieving exterior syndromes, dispelling cold, regulating qi, and soothing the stomach. Modern research shows that perilla extract has good antioxidant, anti-inflammatory, anti-allergic, and antibacterial activities, and has broad application prospects in the cosmetics field.

[0003] Currently, the main methods for obtaining the active ingredients of perilla are solvent extraction (such as water extraction and alcohol extraction) or supercritical fluid extraction. However, these methods have the following drawbacks:

[0004] Limited extraction efficiency: The physical barrier of the cell wall makes it difficult for some active ingredients to be fully released.

[0005] Single composition: It mainly obtains free, small-molecule active substances, and has a low utilization rate of potential active ingredients in glycoside-bonded form (such as flavonoid glycosides).

[0006] Functional limitations: The functions of the obtained extracts are mainly based on their original components, and they lack the ability to generate new active substances through biotransformation.

[0007] Unpleasant aroma: Traditional perilla extract often has a distinct "grassy" or "medicinal" smell, which affects its application in high-end skincare products that emphasize user experience.

[0008] Fermentation technology is an effective means to solve the above problems. Existing technologies have reported the use of lactic acid bacteria or multiple microorganisms (such as a combination of yeast and lactic acid bacteria) to ferment perilla. However, lactic acid bacteria fermentation mainly produces a sour taste, which may mask the original fragrance of perilla; while dual-strain fermentation is complex, with many uncontrollable factors affecting metabolic products, resulting in poor product quality stability. Anaerobic fermentation is not conducive to yeast growth and the accumulation of certain aerobic metabolic products.

[0009] Therefore, developing a single yeast fermentation method that is simple, controllable, and can specifically enhance the effects of perilla while improving its sensory aroma has significant innovative value and practical significance. Summary of the Invention

[0010] In view of the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a method for preparing perilla yeast fermentation filtrate.

[0011] Another object of the present invention is to provide a perilla yeast fermentation filtrate prepared by the above method.

[0012] Another object of the present invention is to provide the application of the above-mentioned perilla yeast fermentation filtrate in skin care products.

[0013] The perilla yeast fermentation filtrate obtained by this invention is rich in natural linalool and specific active ingredients, and has excellent aroma, soothing and anti-allergic, antioxidant and skin barrier repair effects, and can be used to prepare high-end skin care products.

[0014] The objective of this invention is achieved through the following technical solution:

[0015] A method for preparing perilla yeast fermentation filtrate includes the following preparation steps:

[0016] (1) After the dried perilla leaves are crushed, they are mixed with sterile water and then subjected to ultrasonic activation treatment to obtain the fermentation substrate;

[0017] (2) Inoculate yeast seed liquid into the fermentation substrate of step (1), and then carry out aerobic fermentation. When the fermentation is carried out for 24~48h, add L-leucine guide to the fermentation material and continue fermentation treatment for 24~48h.

[0018] (3) The fermentation of the fermentation material in step (2) is inactivated by high temperature to terminate the fermentation, and then the material is filtered by pressure to collect the filtrate and obtain the perilla yeast fermentation filtrate.

[0019] Further, the pulverization mentioned in step (1) refers to pulverizing to 40-80 mesh, and the mass ratio of the material liquid mixed with sterile water is 1:5 to 1:15.

[0020] Furthermore, the ultrasonic activation treatment in step (1) has a power of 200~500W, a temperature of 40~60℃, and a time of 10~30min.

[0021] The purpose of ultrasonic activation pretreatment is to break down plant cell walls, release contents, and activate endogenous enzymes to provide richer substrates for subsequent yeast fermentation.

[0022] Furthermore, the yeast strain mentioned in step (2) is preferably *Saccharomyces cerevisiae* CGMCC No. 64857. This strain is characterized by its strong β-glucosidase activity: the β-glucosidase activity of *Saccharomyces cerevisiae* CGMCC No. 64857 is 120 U / mL, while that of ordinary *Saccharomyces cerevisiae* strains is only 15 U / mL, representing an approximately 8-fold increase in activity. It can efficiently hydrolyze flavonoid glycosides (such as luteolin) in perilla, releasing a more active aglycone (luteolin): after fermentation using CGMCC No. 64857, the release of luteolin from perilla is 8.5 mg / g, while that using ordinary yeast strains is only 1.2 mg / g, representing an approximately 7-fold increase in release efficiency. Meanwhile, the secondary metabolites of this bacterium contribute to the formation of a fresh aroma: 12 characteristic aroma substances (such as ethyl acetate and phenylethanol) were detected in the fermentation broth of CGMCC No. 64857, while only 3 were detected in the fermentation broth of ordinary yeast, indicating a significant lack of aroma complexity and freshness.

[0023] Furthermore, the inoculation percentage of the yeast seed liquid in step (2) is 0.5-5%.

[0024] Furthermore, the aerobic fermentation conditions described in step (2) are: a substrate layer thickness of 2-5 cm, a temperature of 25-30°C, and a relative humidity of 70-85%, ensuring a sufficient oxygen supply during the fermentation process. These conditions are controlled to promote vigorous yeast growth and the accumulation of target metabolites.

[0025] Furthermore, the amount of L-leucine added in step (2) is 0.1~0.5% of the total mass of the fermentation material. L-leucine is an important precursor for the synthesis of higher alcohols (such as linalool) by microorganisms. This step aims to guide the metabolic flow of yeast to promote the synthesis of linalool, thereby forming a characteristic aroma in the product.

[0026] Furthermore, the high-temperature inactivation mentioned in step (3) refers to heating at 85~95℃ for 10~15 minutes.

[0027] Furthermore, the filtrate in step (3) is further sterilized by filtration through a 0.22 μm microporous membrane.

[0028] A perilla yeast fermentation filtrate was prepared by the above method.

[0029] The above-mentioned application of perilla yeast fermentation filtrate in skin care products.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] The method of this invention uses a single yeast strain and achieves the following objectives through innovative pretreatment and fermentation process control:

[0032] (1) The cell structure was destroyed by ultrasonic pretreatment, which improved the release rate of components; the active ingredients in perilla were effectively transformed by brewer's yeast. The two worked synergistically to significantly enhance its soothing, anti-allergic and antioxidant effects.

[0033] (2) During the fermentation process, by selecting specific brewing yeast and combining aerobic fermentation with the addition of L-leucine precursor, linalool with a fresh floral and woody aroma was successfully introduced into the fermentation filtrate, completely masking the original grassy smell of perilla and giving the fermentation filtrate a natural and pleasant aroma. This overcomes the unpleasant odor of traditional extracts and allows it to be directly applied to products with high aroma requirements without the need for additional fragrance.

[0034] (3) Using a single yeast strain for aerobic fermentation makes the process conditions easy to control, the fermentation process stable, and the batch-to-batch differences small, which is conducive to industrial production and product quality standardization.

[0035] (4) The fermented filtrate is natural and contains no organic solvent residue. It is gentle on the skin and has been proven by skin patch test to be low in irritation and suitable for sensitive skin. Attached Figure Description

[0036] Figure 1 This is a product appearance diagram of the perilla yeast fermentation filtrate obtained in Example 1. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] Example 1

[0039] A method for preparing perilla yeast fermentation filtrate includes the following preparation steps:

[0040] (1) 100g of dried perilla leaves were crushed and passed through a 60-mesh sieve, mixed with 1000mL of sterile water, and then ultrasonically treated at 45℃ and 300W for 20min to obtain the fermentation substrate.

[0041] (2) Cool the fermentation substrate from step (1) to 28°C, inoculate with 30 ml of activated Saccharomyces cerevisiae (CGMCC No. 64857) seed culture and stir well. Spread the material evenly on a sterile tray with a layer thickness of about 3 cm, and place it in a constant temperature and humidity incubator at 28°C and 80% humidity for fermentation. Ensure sufficient oxygen supply during the fermentation process. After 36 hours of fermentation, evenly sprinkle in 3 g of L-leucine powder and continue fermentation until the total fermentation time is 72 hours.

[0042] (3) Heat the fermentation product from step (2) at 90°C for 10 min, then filter it using a filter press, collect the filtrate, and aseptically filter it through a 0.22 μm filter membrane to obtain a clear amber-colored perilla yeast fermentation filtrate (its appearance is shown in the figure below). Figure 1 As shown in the image, it has a refreshing floral and woody scent. The human skin patch test result is grade 0, indicating low irritation.

[0043] Example 2

[0044] A method for preparing perilla yeast fermentation filtrate includes the following preparation steps:

[0045] (1) 100g of dried perilla leaves were crushed and passed through a 40-mesh sieve, mixed with 1500mL of sterile water, and then ultrasonically treated at 60℃ and 500W for 10min to obtain the fermentation substrate.

[0046] (2) Cool the fermentation substrate from step (1) to 25°C, inoculate with 30 ml of activated Saccharomyces cerevisiae (CGMCC No. 64857) seed culture and stir well. Spread the material evenly on a sterile tray with a layer thickness of about 5 cm, and place it in a constant temperature and humidity incubator at 25°C and 70% humidity for fermentation. Ensure sufficient oxygen supply during the fermentation process. After 48 hours of fermentation, evenly sprinkle in 5 g of L-leucine powder and continue fermentation until the total fermentation time is 96 hours.

[0047] (3) The fermented material from step (2) was heated at 85°C for 15 minutes, then filtered using a filter press. The filtrate was collected and aseptically filtered through a 0.22 μm filter membrane to obtain a clear amber-colored perilla yeast fermentation filtrate with a fresh floral and woody aroma. The human skin patch test showed a test level of 0, indicating low irritation.

[0048] Example 3

[0049] A method for preparing perilla yeast fermentation filtrate includes the following preparation steps:

[0050] (1) 100g of dried perilla leaves were crushed and passed through an 80-mesh sieve, mixed with 500mL of sterile water, and then ultrasonically treated at 40℃ and 200W power for 30min to obtain the fermentation substrate.

[0051] (2) Cool the fermentation substrate from step (1) to 30°C, inoculate with 25 ml of activated Saccharomyces cerevisiae (CGMCC No. 64857) seed culture and stir well. Spread the material evenly on a sterile tray with a layer thickness of about 2 cm, and place it in a constant temperature and humidity incubator at 30°C and 85% humidity for fermentation. Ensure sufficient oxygen supply during the fermentation process. After 24 hours of fermentation, evenly sprinkle in 2.5 g of L-leucine powder and continue fermentation until the total fermentation time is 48 hours.

[0052] (3) The fermented material from step (2) was heated at 95°C for 10 minutes, then filtered using a filter press. The filtrate was collected and aseptically filtered through a 0.22 μm filter membrane to obtain a clear amber-colored perilla yeast fermentation filtrate with a fresh floral and woody aroma. The human skin patch test showed a test level of 0, indicating low irritation.

[0053] Comparative Example 1

[0054] The method for preparing perilla extract filtrate using traditional water extraction includes the following steps:

[0055] Take 100g of perilla leaf powder from the same source as in Example 1, add 1000mL of deionized water, reflux at 80℃ for 1 hour, filter, and concentrate the filtrate to the same volume as the fermentation filtrate in Example 1 for later use.

[0056] Comparative Example 2

[0057] A method for preparing perilla yeast fermentation filtrate, which is the same as in Example 1 except that ultrasonic treatment is not performed in step (1).

[0058] Comparative Example 3

[0059] A method for preparing perilla yeast fermentation filtrate, compared with Example 1, except that L-leucine powder was not added in step (2), and the rest are the same.

[0060] Effect verification experiment:

[0061] (1) The soothing efficacy of the filtrate products of Examples 1-3 and Comparative Examples 1-3 was tested (in vitro antihistamine test, determined by fluorescence method). The test results are shown in Table 1 below.

[0062] Table 1. Results of product soothing efficacy test

[0063] test sample histamine inhibition rate Example 1 88% Example 2 83% Example 3 87% Comparative Example 1 35% Comparative Example 2 65% Comparative Example 3 72%

[0064] As can be seen from the results in Table 1, the fermentation treatment, ultrasonic pretreatment, and the addition of L-leucine guide in this invention have a significant synergistic effect on the soothing efficacy of the product.

[0065] (2) Aroma component analysis: The relative content of linalool in the test sample was analyzed by GC-MS, and the results are shown in Table 2 below.

[0066] Table 2. Results of relative content analysis of linalool in the product

[0067] test sample relative content of linalool Example 1 18.5% Example 2 18.9% Example 3 17.8% Comparative Example 1 Not detected Comparative Example 2 9.8% Comparative Example 3 7.2%

[0068] Table 2 shows that the relative content of linalool in the filtrates of Examples 1-3 was 17.8%-18.9%, with a pleasant aroma; linalool was almost undetectable in Comparative Example 1, and the main components were grassy aroma components such as leaf alcohol; the linalool content in Comparative Example 2 was 9.8%, and the aroma was significantly weaker than that of Example 1. The linalool content in Comparative Example 3 was 7.2%, and the aroma was significantly weaker than that of Example 1. These results demonstrate the key role of the addition of L-leucine precursor in the targeted generation of the target aroma components, and the enhancing effect of ultrasonic pretreatment on the generation of the target aroma components.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing perilla yeast fermentation filtrate, characterized in that... The preparation steps include the following: (1) The dried perilla leaves were crushed and mixed with sterile water, and then ultrasonically activated to obtain the fermentation substrate; (2) Inoculate yeast seed liquid into the fermentation substrate of step (1), and then carry out aerobic fermentation. When the fermentation is carried out for 24~48h, add L-leucine guide to the fermentation material and continue fermentation treatment for 24~48h. (3) The fermentation of the fermentation material in step (2) is inactivated by high temperature to terminate the fermentation, and then the material is filtered by pressure to collect the filtrate and obtain the perilla yeast fermentation filtrate.

2. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The pulverization mentioned in step (1) refers to pulverizing to 40-80 mesh, and the mass ratio of the material liquid mixed with sterile water is 1:5 to 1:

15.

3. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The ultrasonic activation treatment in step (1) has a power of 200~500W, a temperature of 40~60℃, and a time of 10~30min.

4. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The yeast mentioned in step (2) is Saccharomyces cerevisiae CGMCC No. 64857.

5. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The inoculation percentage of the yeast seed liquid in step (2) is 0.5-5%.

6. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The aerobic fermentation conditions described in step (2) are: a material layer thickness of 2-5 cm, a temperature of 25-30°C, a relative humidity of 70-85%, and a sufficient oxygen supply during the fermentation process.

7. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The amount of L-leucine added in step (2) is 0.1~0.5% of the total mass of the fermentation material.

8. The method for preparing perilla yeast fermentation filtrate according to claim 1, characterized in that: The high-temperature inactivation mentioned in step (3) refers to heating at 85~95℃ for 10~15min; the filtrate is further filtered through a 0.22μm microporous membrane for sterilization.

9. A perilla yeast fermentation filtrate, characterized in that: It is prepared by the method described in any one of claims 1 to 8.

10. The application of the perilla yeast fermentation filtrate as described in claim 9 in skin care products.

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