Preparation method and application of tuberose fermentation liquid

Abstract

The invention discloses a preparation method and application of tuberose fermentation liquid, comprising: drying tuberose petals and then crushing them, passing through a 80-100 mesh sieve to obtain tuberose powder; sterilizing the fermentation medium obtained by adding the tuberose powder to a YPD liquid medium, inoculating brewer's yeast, wherein the activated yeast inoculation amount is 5% by mass relative to the tuberose fermentation medium, fermenting at 30°C for 48h, sterilizing at 121°C for 20min, and filtering to obtain the tuberose fermentation liquid. After deep liquid fermentation, the tuberose of the invention is enriched in active substances, the efficacy is enhanced, and it is safe and non-irritating, and belongs to a cosmetic raw material that is mild and friendly to the skin.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation, and particularly relates to a preparation method and application of tuberose fermentation liquid. Background Art

[0002] In modern society, the pursuit of beauty has gradually strengthened from "self-pleasing consumption" to "urgent consumption", and having healthy and firm skin is what everyone desires. Under normal physiological conditions, the body has a natural inhibitor of elastase degradation that is absolutely dominant in number, resulting in a dynamic balance between elastase and elastase inhibitors. However, with age, the dynamic balance between elastin and elastase is destroyed, elastin is degraded, causing skin function to degenerate, and the tissues composed of collagen and elastic fibers (elastin) in the body slowly lose elasticity, and the skin becomes loose and sagging. In addition, under the influence of ultraviolet rays, air pollution and other factors, the body will secrete excessive inflammatory factors such as interleukin (IL-6), necrotizing tumor factor α (TNF-α), and nitric oxide (NO), leading to skin inflammation such as redness, tingling, and red blood, thereby causing skin allergies and losing skin elasticity and luster.

[0003] Tuberose (Polianthus tuberososa) is a perennial herb of the Amaryllidaceae family and the genus Tuberose. It can clear the liver and improve eyesight, detoxify and regenerate skin. It is rich in active substances such as acidic polysaccharides and flavonoids, which have anti-inflammatory, soothing and skin tightening effects, making the skin full of elasticity.

[0004] At present, tuberose is rarely used in the cosmetics field, and most of them are compound products of tuberose solid powder or tuberose extracts; the content of active substances in directly used solid powder products is low and the efficacy is very limited; while the extracts have problems such as low extraction efficiency and unverified safety. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a tuberose fermentation broth, comprising:

[0008] The tuberose petals are dried and crushed, and passed through a 80-100 mesh sieve to obtain tuberose powder;

[0009] The fermentation medium prepared by adding tuberose powder to YPD liquid medium is sterilized, and then inoculated with brewer's yeast, and fermented at 30°C for 24 to 48 hours, and then sterilized and filtered to obtain tuberose fermentation liquid;

[0010] The brewer's yeast is Saccharomyces cerevisiae P2, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M 2021849.

[0011] As a preferred embodiment of the preparation method of the present invention, the percentage of tuberose powder in the fermentation medium to the total mass of the fermentation medium raw materials is 5-8%.

[0012] As a preferred embodiment of the preparation method of the present invention, the YPD liquid culture medium comprises: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract powder, a pH value of 6.5±0.2, and the solvent is water.

[0013] As a preferred embodiment of the preparation method of the present invention, the fermentation medium is sterilized under the following conditions: 121° C., 0.1 MPa, and sterilization for 30 min.

[0014] As a preferred embodiment of the preparation method of the present invention, wherein: the inoculated brewer's yeast, wherein the mass percentage of the seed liquid inoculation relative to the fermentation medium is 1% to 10%.

[0015] As a preferred embodiment of the preparation method of the present invention, wherein: the inoculated brewer's yeast, wherein the mass percentage of the seed liquid inoculation relative to the fermentation medium is 10%.

[0016] As a preferred embodiment of the preparation method of the present invention, the bacterial concentration of the seed solution is 10 7 ~10 8 CFU / mL.

[0017] As a preferred embodiment of the preparation method of the present invention, the filtration comprises removing solid insoluble matter through 200-300 mesh gauze, and filtering the clear liquid twice through a ceramic membrane with a filter diameter of 200 nm.

[0018] Another object of the present invention is to supplement and improve the deficiencies of the prior art and provide a method for preparing a tuberose fermentation liquid to obtain a fermentation liquid.

[0019] Another object of the present invention is to supplement and improve the deficiencies of the prior art and provide an application of tuberose fermentation liquid in the preparation of cosmetics with soothing, anti-inflammatory and firming effects.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention provides a method for preparing a tuberose fermentation liquid having anti-inflammatory, soothing and firming effects. After the tuberose is deep fermented in liquid, the active substances are enriched and the efficacy is significantly enhanced. At the same time, the fermentation process of the present invention does not involve organic solvents, has low environmental pollution, is safe and non-irritating, and is a green cosmetic raw material that is mild to the skin.

[0022] (2) The present invention provides a tuberose fermentation liquid, which uses tuberose as a plant base and is inoculated with brewer's yeast for fermentation, so as to achieve the purpose of anti-inflammatory, soothing and skin tightening. The tuberose fermentation liquid inhibits elastin degradation and clears nitric oxide (NO) and interleukin (IL-6), two inflammatory factors, better than unfermented tuberose products and tuberose extracts; at the same time, the fermentation liquid has a higher Ca content than the fermented liquid. 2+ Intracellular Ca influx efficacy assay 2+ The content is also lower than that of unfermented tuberose products and tuberose extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 This is a comparison chart of the effects of the tuberose fermentation broth obtained by inoculating 7 different yeasts in the strain screening process of the present invention on the LPS-induced inflammatory factor NO; wherein, from left to right, they are respectively a blank group, an LPS model group, a tuberose fermentation broth obtained by inoculating Saccharomyces cerevisiae P2, a tuberose fermentation broth obtained by inoculating Saccharomyces cerevisiae A1, a tuberose fermentation broth obtained by inoculating Saccharomyces cerevisiae A2, a tuberose fermentation broth obtained by inoculating Pichia tricora A3, a tuberose fermentation broth obtained by inoculating Hansenula cacti A4, a tuberose fermentation broth obtained by inoculating Hansenula vitis A5, and a tuberose fermentation broth obtained by inoculating Saccharomyces cerevisiae CICC 32883.

[0025] Figure 2 This is a comparison chart of the effects of the tuberose fermentation broth obtained by inoculating 7 different yeasts in the strain screening process of the present invention on the LPS-induced inflammatory factor IL-6.

[0026] Figure 3 This is a comparison chart of the effects of tuberose fermentation broth obtained under different fermentation conditions in the fermentation optimization process of the present invention on the LPS-induced inflammatory factor NO.

[0027] Figure 4This is a comparison chart of the effects of tuberose fermentation broth obtained under different fermentation conditions in the fermentation optimization process of the present invention on the LPS-induced inflammatory factor IL-6.

[0028] Figure 5 This is a comparison chart of the expression of elastin produced by UVA-induced cells by the unfermented tuberose, the tuberose extract, and the tuberose fermentation liquid of the present invention.

[0029] Figure 6 The present invention is a tuberose unfermented product, a tuberose extract, and a tuberose fermentation liquid. 2+ Comparison chart of fluorescence detection values ​​of expression levels.

[0030] Figure 7 This is a comparison chart of the effects of the unfermented tuberose, the tuberose extract, and the tuberose fermentation liquid of the present invention on the inflammatory factor NO induced by LPS.

[0031] Figure 8 This is a comparison chart of the effects of the unfermented tuberose, tuberose extract, and tuberose fermentation broth of the present invention on the inflammatory factor IL-6 induced by LPS. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] The tuberose raw material in the embodiment of the present invention is a common commercially available product;

[0036] The brewer's yeast in the embodiment of the present invention is Saccharomyces cerevisiae P2, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO.M 2021849, the same as the brewer's yeast CCTCC NO.M 2021849 of Chinese patent application number: 2022117362409.

[0037] The activation method of yeast in the embodiment of the present invention is as follows: take out the yeast glycerol cryopreservation tube from the -80°C refrigerator, streak it onto the YPD solid medium (1% yeast powder, 2% peptone, 2% glucose, 2% agar, sterilized at 121°C for 20 min) aseptically, place it in a 30°C incubator, and invert and culture for 24 hours to grow a single colony;

[0038] Aseptically inoculate the strain into YPD medium and culture at 37°C, 200 rpm for 18-24 h. 7 ~10 8 CFU / mL, the activated yeast was obtained.

[0039] Filtration in the embodiment of the present invention: solid insoluble matter is removed by 200-300 mesh gauze, and the filtrate is filtered twice using a ceramic membrane with a pore size of 200 nm.

[0040] The test method of the effect of the samples on the inflammatory factors NO and IL-6 in the embodiments of the present invention:

[0041] RAW 264.7 cells were cultured at 2×10 4 Each well was inoculated into a 6-well plate, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator;

[0042] After 18 h, the culture medium was aspirated and the residual culture medium was washed with PBS. 2 mL of DMEM was added to the blank group, 1 μg / mL LPS was added to the LPS group, and 1 μg / mL LPS prepared in DMEM and 1% (v / v) DMEM were added to the experimental group and cultured together for 24 h.

[0043] Take the upper culture medium, centrifuge at 13000rpm for 5min, and aspirate the supernatant;

[0044] The NO / IL-6 detection kit was used according to the instructions to determine the effect of tuberose on the levels of LPS-induced inflammatory cytokines.

[0045] Example 1

[0046] This embodiment provides a strain screening process for yeast fermentation of tuberose, the main steps of which are:

[0047] (1) drying tuberose petals and crushing them, and passing them through a 80-100 mesh sieve to obtain tuberose powder;

[0048] (2) After the fermentation medium prepared by adding tuberose powder to YPD liquid medium is sterilized, 7 kinds of yeast are inoculated respectively, and the mass percentage of activated yeast inoculation relative to the tuberose fermentation medium is 10%. After fermentation at 30°C for 24 hours, the mixture is sterilized at 121°C for 20 minutes and then filtered to obtain the tuberose fermentation liquid.

[0049] (3) The obtained tuberose fermentation broth was taken to determine its effect on LPS-induced inflammatory factors NO and IL-6. The results are shown in Figures 1-2 .

[0050] Among them, 6 yeasts out of the 7 types of bacteria are from the self-built strain library of Jiangsu Ruiting Biotechnology Co., Ltd., namely, Saccharomyces cerevisiae P2, A1, and A2; Pichia tricora A3; Hansenula cactus A4; Hansenula vitis spores A5; the last one is a common commercially available brewer's yeast, which comes from the China Industrial Microbiology Culture Collection Center, with the strain number CICC 32883.

[0051] The screening results are summarized in Table 1.

[0052] Table 1

[0053]

[0054] By comprehensively comparing the contents of nitric oxide and interleukin in the seven fermentation broths in the chart, it can be found that the tuberose fermentation broth inoculated with brewer's yeast P2 has the lowest content of inflammatory factor IL-6 and a lower content of inflammatory factor NO. Brewer's yeast P2 is preferred. P2 is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO.M 2021849.

[0055] Example 2

[0056] This embodiment provides a fermentation optimization process of tuberose fermented by Saccharomyces cerevisiae P2, the main steps of which are:

[0057] The tuberose petals are dried and crushed, and passed through a 80-100 mesh sieve to obtain tuberose powder;

[0058] After the fermentation medium prepared by adding tuberose powder to YPD liquid medium is sterilized, the brewer's yeast P2 screened in Example 1 is inoculated, and the mass percentage of the activated yeast inoculation relative to the tuberose fermentation medium is 1% to 10%. After fermentation at 30°C for 24 to 48 hours, the mixture is sterilized at 121°C for 20 minutes and then filtered to obtain tuberose fermentation broths 1 to 9.

[0059] The obtained tuberose fermentation broth was taken to determine its effect on LPS-induced inflammatory factors NO and IL-6. The results are shown in Figures 3-4 .

[0060] The screening results are summarized in Table 2.

[0061] Table 2

[0062] NO content / (μM) IL-6 content / (pg / ml) Fermentation broth 1 2.96 338.15 Fermentation broth 2 2.43 245.79 Fermentation broth 3 2.77 243.42 Fermentation broth 4 2.65 254.81 Fermentation broth 5 2.03 206.55 Fermentation broth 6 1.96 183.29 Fermentation broth 7 2.37 239.54 Fermentation broth 8 2.25 254.61 Fermentation broth 9 2.89 266.93

[0063] Among them, fermentation liquid 1 is tuberose fermentation liquid obtained by fermenting for 24 hours with an inoculation amount of 1% of brewer's yeast P2; fermentation liquid 2 is tuberose fermentation liquid obtained by fermenting for 36 hours with an inoculation amount of 1%; fermentation liquid 3 is tuberose fermentation liquid obtained by fermenting for 48 hours with an inoculation amount of 1%; fermentation liquid 4 is tuberose fermentation liquid obtained by fermenting for 24 hours with an inoculation amount of 5%; fermentation liquid 5 is tuberose fermentation liquid obtained by fermenting for 36 hours with an inoculation amount of 5%; fermentation liquid 6 is tuberose fermentation liquid obtained by fermenting for 48 hours with an inoculation amount of 5%; fermentation liquid 7 is tuberose fermentation liquid obtained by fermenting for 24 hours with an inoculation amount of 10%; fermentation liquid 8 is tuberose fermentation liquid obtained by fermenting for 36 hours with an inoculation amount of 10%; fermentation liquid 9 is tuberose fermentation liquid obtained by fermenting for 48 hours with an inoculation amount of 10%.

[0064] By comparing the NO and IL-6 contents of the 9 fermentation broths in Table 2, it can be found that the activated yeast inoculation amount relative to the mass percentage of the tuberose fermentation medium is 5%, and the content of the two inflammatory factors in the tuberose fermentation broth with a fermentation time of 48h is low. This fermentation condition is preferred as the method for preparing the tuberose fermentation broth.

[0065] Example 3

[0066] This example provides a comparison of the firming and soothing effects of unfermented tuberose, tuberose extract, and tuberose fermented liquid:

[0067] Preparation method of unfermented tuberose:

[0068] Weigh 50.0g of tuberose powder, 20.0g of peptone, 20.0g of glucose, and 10.0g of yeast powder, disperse them in 1.0L of pure water, adjust the pH value to 6.5±0.2, and sterilize at 121℃ for 20min. The product is filtered through 200-mesh gauze and a ceramic membrane with a pore size of 200nm in turn, and the filtrate is the unfermented tuberose.

[0069] Preparation method of tuberose extract:

[0070] Weigh 50.0g of tuberose powder and 200.0g of butanediol, disperse in 1.0L of pure water, and extract at 55℃ for 2h. Centrifuge the product at 10000rpm for 10min, and then filter through 200-mesh gauze and a ceramic membrane with a filter diameter of 200nm. The filtrate is the tuberose extract.

[0071] Preparation method of tuberose fermentation liquid:

[0072] Weigh 50.0 g of tuberose powder, 20.0 g of peptone, 20.0 g of glucose, and 10.0 g of yeast powder, disperse them in 1.0 L of pure water, adjust the pH value to 6.5±0.2, sterilize at 121° C. for 20 min, inoculate with Saccharomyces cerevisiae P2, the mass percentage of the activated yeast inoculation amount relative to the tuberose fermentation medium is 5%, ferment at 30° C. for 48 h, and sterilize at 121° C.; filter the product through 200-mesh gauze and a ceramic membrane with a pore size of 200 nm in turn, and the filtrate is the tuberose fermentation liquid.

[0073] Comparative test of elastin expression in three samples:

[0074] HFF-1 cells were cultured at a rate of 1.5×10 5 Each well was inoculated with 100 μL of PBS and incubated at 37°C in a 5% CO2 incubator. After 18 h, the cells were washed with PBS three times and 100 μL of PBS was added at 15 J / cm 2 After UVA irradiation, remove PBS, add 100 μL DMEM or 100 μL 1% (v / v) DMEM, and incubate in an incubator for 24 h. Take the upper culture medium, centrifuge at 13000 rpm for 5 min, and aspirate the supernatant. According to the instructions, use the human elastin detection kit to determine the expression of elastin in the cells induced by UVA. The comparison results of elastin expression are shown in Figure 5 shown.

[0075] Effects of three samples on intracellular Ca 2+ Comparative test of expression level:

[0076] HaCaT cells were cultured at 2×10 4 Cells were inoculated in 6-well plates, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator. After 18 hours, the culture medium was discarded and the residual culture medium was washed with PBS. 2 mL of DMEM was added to the blank group, and 1 μg / mL CAP was prepared in DMEM to the CAP group. The sample with a final concentration of 1% and 1 μg / ml capsaicin (prepared in DMEM) were added to the experimental group and cultured together for 20 minutes. Fluo-4AM probe was then added for staining, protected from light, and incubated at 37°C for 30 minutes. After staining, the cells were digested and transferred to a black ELISA plate for fluorescence detection at 490nm / 525nm. The fluorescence value comparison results are shown as follows. Figure 6 shown.

[0077] The three samples were tested using NO detection kit and IL-6 detection kit respectively to compare the effects of the samples on LPS-induced inflammatory factors NO and IL-6. The results are shown in Figures 7-8 .

[0078] Table 3 Comparative summary of the efficacy of unfermented tuberose, tuberose extract and fermented tuberose

[0079]

[0080]

[0081] As shown in Table 3, in the elastin expression experiment, the amount of elastin produced by the tuberose fermentation liquid group on UVA damaged cells was higher than that of the tuberose unfermented material and tuberose extract groups; in the calcium influx and inflammatory factor expression experiments, the Ca2+ in the cells treated with the tuberose fermentation liquid was higher than that of the cells treated with the tuberose fermentation liquid. 2+ The content, NO and IL-6 expression levels were lower than those of the unfermented substance and the extract, indicating that the tuberose fermentation liquid has certain anti-inflammatory and soothing effects.

[0082] The invention provides a tuberose fermentation liquid, which uses tuberose as a plant base and is inoculated with brewer's yeast for fermentation, so as to achieve the purpose of anti-inflammatory, soothing and skin tightening. The invention provides a tuberose fermentation liquid, preferably brewer's yeast Rayting-P2 (Saccharomyces cerevisiae Rayting-P2) is inoculated for fermentation, preferably the activated yeast inoculation amount is 5% relative to the mass percentage of the tuberose fermentation medium, and the fermentation time is 48h. The tuberose fermentation liquid obtained by this fermentation method has the best skin tightening, anti-inflammatory and soothing effects.

[0083] The tuberose fermented liquid provided by the invention has excellent efficacy, is safe and pollution-free, is a green cosmetic raw material, has a light yellow color, and is suitable for the cosmetic field.

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing tuberose fermentation liquid, characterized in that: include, The tuberose petals are dried and crushed, and passed through a 80-100 mesh sieve to obtain tuberose powder; The fermentation medium prepared by adding tuberose powder to YPD liquid medium is sterilized, and then inoculated with brewer's yeast, and fermented at 30°C for 24 to 48 hours, and then sterilized and filtered to obtain tuberose fermentation liquid; The brewer's yeast is Saccharomyces cerevisiae P2, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M 2021849.

2. The preparation method according to claim 1, characterized in that: The percentage of tuberose powder in the fermentation medium to the total mass of the raw materials of the fermentation medium is 5-8%.

3. The preparation method according to claim 1 or 2, characterized in that: The YPD liquid culture medium comprises: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract powder, a pH value of 6.5±0.2, and a solvent of water.

4. The preparation method according to claim 3, characterized in that: The fermentation medium is sterilized under the following conditions: 121° C., 0.1 MPa, and sterilization for 30 min.

5. The preparation method according to claim 4, characterized in that: The inoculated brewer's yeast has a seed liquid inoculation amount of 1% to 10% by mass relative to the fermentation medium.

6. The preparation method according to claim 5, characterized in that: The inoculated brewer's yeast has a seed liquid inoculation amount of 10% by mass relative to the fermentation medium.

7. The preparation method according to claim 5 or 6, characterized in that: The bacterial concentration of the seed solution is 10 7 ~10 8 CFU / mL.

8. The preparation method according to claim 1, characterized in that: The filtration comprises removing solid insolubles through 200-300 mesh gauze, and filtering the clear liquid twice through a ceramic membrane with a filter diameter of 200 nm.

9. The tuberose fermentation liquid obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the tuberose fermented liquid according to claim 9 in preparing cosmetics with soothing, anti-inflammatory and firming effects.

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