CN117547566A - A fermented Rhodiola rosea extract with improved whitening and antioxidant capabilities, its preparation method and its application - Google Patents

Abstract

The invention provides a method for preparing Rhodiola rosea fermentation extract with improved whitening and antioxidant capabilities through a two-step fermentation method of yeast fermentation and lactobacillus fermentation. The present invention also relates to a Rhodiola rosea fermentation extract prepared by the two-step fermentation method and a pharmaceutical preparation or cosmetic composition containing the Rhodiola rosea fermentation extract. The present invention further relates to the use of the rhodiola rosea fermentation extract in preparing pharmaceutical or cosmetic compositions.

Description

A fermented Rhodiola rosea extract with improved whitening and antioxidant capabilities, and Preparation methods and applications

Technical field

The invention relates to a preparation method of Rhodiola rosea fermentation extract. The present invention also relates to a Rhodiola rosea fermentation extract with improved whitening and antioxidant capabilities prepared by the preparation method, a pharmaceutical preparation or a cosmetic composition containing the fermentation extract. The present invention further relates to the use of the rhodiola rosea fermentation extract in preparing pharmaceutical or cosmetic compositions.

Background technique

Rhodiola rosea is a perennial grass or subshrub of the Crassulaceae family. It is a rare wild plant growing in pollution-free areas at high altitudes. Rhodiola rosea is a traditional Chinese medicinal plant. It is rich in active compounds, including salidroside, tyrosol, flavonoids, coumarins, organic acids, etc. It has antioxidant, whitening, anti-aging, anti-inflammatory, Anti-fatigue and other effects. In the field of cosmetics, rhodiola rosea extract has whitening and antioxidant capabilities.

Chinese herbal medicine fermentation is a process that uses microorganisms to ferment Chinese herbal medicine as a substrate. After fermentation, the content of active substances in Chinese herbal medicines increases, the toxic and side effects are reduced, and it is easier to separate, purify and apply. The above changes benefit from the rich enzyme system and powerful biotransformation ability of microorganisms. In addition, the synergistic effect between metabolites of microbial fermentation and pharmaceutical ingredients may also enhance drug efficacy. The mild conditions, high quality and low price of fermentation also lay the foundation for its widespread development and application.

Existing technologies for research on Rhodiola rosea fermentation include:

CN110772460A discloses "a preparation method and application of Rhodiola rosea fermentation extract", which is characterized by first using cellulase and pectinase to enzymatically hydrolyze Rhodiola rosea root powder, and then adding nitrogen source, carbon source, inorganic Salt and water, the yeast seed liquid is inoculated into this medium for fermentation. This invention only uses a single strain of yeast for fermentation, and does not use other strains that may significantly improve the antioxidant capacity of Rhodiola rosea.

CN112472634A discloses "a rhodiola rosea extract, its preparation method and application", which is characterized in that the fermentation liquid obtained by mixing and fermenting Lactobacillus delbrueckii, rhodiola rosea root powder and culture medium is sterilized and then mixed with ethanol. , methanol, acetone, butylene glycol or pentanediol extraction. Since Rhodiola rosea has a strong inhibitory effect on the growth of bacteria, if Lactobacillus is directly added to a culture medium with a high Rhodiola rosea content, the growth will be inhibited and the effect of Lactobacillus cannot be fully exerted.

CN103131730A discloses "Method for manufacturing Rhodiola rosea fermentation product using adsorbent", which solves the problem that it is difficult to ferment Rhodiola rosea alone with lactic acid bacteria. The invention uses one or more adsorbents selected from acidic clay, bentonite, diatomite, activated carbon and weakly basic anion exchange resin to process Rhodiola rosea extract, and in the process of treating the filtrate that is not adsorbed on the adsorbent The step of inoculating lactic acid bacteria for fermentation. The adsorbent treatment of Rhodiola rosea extract can relieve the growth inhibition effect on lactic acid bacteria, but the adsorbent causes the loss of Rhodiola rosea active substances.

CN103127202B discloses "a method for producing a fermented product of Rhodiola rosea using red ginseng and a composition containing the fermented product for improving fatigue and improving exercise ability." This invention is also intended to solve the problem of difficulty in fermenting Rhodiola rosea alone with lactic acid bacteria, and provides a method for manufacturing a Rhodiola rosea fermented product, which method mixes red ginseng extract with Rhodiola rosea extract or combines Rhodiola rosea and red ginseng. A mixed extract of rhodiola rosea and red ginseng was produced by mixing, and the mixed extract was inoculated with lactic acid bacteria and fermented. This method requires adding red ginseng as raw material, and the allowed amount of Rhodiola rosea is low.

CN104257545B discloses "a Rhodiola rosea fermented puree cosmetic and its preparation method", which is characterized in that the fermentation liquid obtained by fermenting the initial system obtained by mixing Rhodiola rosea root powder, water and fermentation bacteria is sterilized and centrifuged The resulting supernatant is Rhodiola rosea fermentation puree cosmetics. The fermentation process does not add nutrients required for yeast growth, which limits the growth of yeast and may consume the active ingredients of Rhodiola rosea itself.

CN106265846A discloses "a fermentation method for improving the antioxidant capacity of Rhodiola rosea", which is characterized by crushing and drying Rhodiola rosea and mixing it with distilled water, extracting it twice at 100°C, filtering and centrifuging to remove impurities, and then obtaining a supernatant liquid. Glucose was added to the obtained extract, and Aspergillus niger seed liquid was inoculated for fermentation. The antioxidant capacity of Rhodiola rosea is enhanced after fermentation. When Rhodiola rosea water extract is used for fermentation, the active ingredients in the medicinal residue are not fully released, resulting in waste.

CN112754960A discloses "a cosmetic containing yeast and rhodiola rosea fermentation product extract, preparation method and application", which is characterized in that the ultrasonic extraction method is used to obtain the rhodiola rosea extract, and then the yeast seed liquid is inoculated for fermentation and sterilization Finally, 30-50% solvent is added, and the yeast and Rhodiola rosea fermentation product extracts are obtained by filtration. This invention uses a single yeast, and the fermentation process does not add nutrients required for yeast growth. The yeast cannot fully grow and metabolize, and consumes the active ingredients of Rhodiola rosea itself.

The problems existing in the existing technology are:

(1) In the prior art, rhodiola rosea fermentation generally uses a single bacterial species, that is, yeast or lactobacilli commonly used in plant fermentation;

(2) The antioxidant capacity of Rhodiola rosea extract obtained by fermentation of a single strain of yeast is poor;

(3) Although Lactobacillus fermentation of Rhodiola rosea can significantly improve the antioxidant capacity of Rhodiola rosea fermentation extract, because Rhodiola rosea has a strong inhibitory effect on the growth of bacteria, in the culture medium with high Rhodiola rosea content If Lactobacillus is added directly, its growth will be inhibited and the effect of Lactobacillus cannot be fully exerted;

(4) Using adsorbent to treat Rhodiola rosea extract can eliminate its growth inhibitory effect on Lactobacillus, but the adsorbent causes the loss of Rhodiola rosea active substances.

Therefore, there is still a strong demand for Rhodiola rosea fermentation extract with improved whitening and antioxidant capabilities and its preparation method and application.

Contents of the invention

The present invention obtains a Rhodiola rosea fermentation extract with improved whitening and antioxidant capabilities by performing a two-step fermentation of Rhodiola rosea using yeast first and then Lactobacillus, thereby solving the problem of Rhodiola rosea fermentation extraction in the prior art. It solves the problems existing in the liquid preparation method and overcomes the shortcomings existing in the prior art.

The technical solution of the present application adopts a two-step fermentation method, in which yeast is first inoculated for fermentation, and then lactobacilli are inoculated for fermentation. Lactobacillus can grow and proliferate in the Rhodiola rosea fermentation liquid after yeast fermentation. The antibacterial effect of the Rhodiola rosea extract after fermentation by yeast is weakened on Lactobacillus, which overcomes the problem that the higher Rhodiola rosea content in the fermentation process of the prior art inhibits the growth of Lactobacillus and thereby limits the fermentation of Rhodiola rosea by Lactobacillus. question. The Rhodiola rosea fermentation broth after two-step fermentation has a greater improvement in the inhibitory ability of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and tyrosinase.

In a first aspect, the present invention relates to a method for preparing Rhodiola rosea fermentation extract through two-step fermentation, wherein the method includes the following steps:

(a) using yeast to ferment Rhodiola rosea raw materials for the first time to obtain the first fermentation extract;

(b) Use Lactobacillus to ferment the first fermentation liquid obtained in step (a) for a second time to obtain a second fermentation extract.

The second aspect of the present invention relates to a Rhodiola rosea fermentation extract prepared by the method according to the first aspect of the present invention.

A third aspect of the present invention relates to a cosmetic composition comprising a Rhodiola rosea fermentation extract according to the second aspect of the present invention.

A fourth aspect of the invention relates to a pharmaceutical preparation comprising a Rhodiola rosea fermentation extract according to the second aspect of the invention.

The fifth aspect of the present invention relates to the use of Rhodiola rosea fermentation extract according to the second aspect of the present invention in the preparation of pharmaceutical or cosmetic compositions with whitening and/or antioxidant capabilities.

Detailed description of the invention

definition

As used herein, the terms "s", "min" and "h" represent "seconds", "minutes" and "hours" respectively.

The term "comprises" as used herein, which is synonymous with "includes," "contains," or "characterized by," is inclusive or open-ended and does not exclude additional, undescribed elements or method steps. However, throughout the text, each reference to "comprises" is intended to cover alternative embodiments "consisting essentially of" and "consisting of," where "consisting of." ....." excludes any elements or steps not specified, and "consisting essentially of" allows the inclusion of undescribed elements that are necessary or essential and do not materially affect the composition or method in question. Characteristics and other elements or steps of novelty characterization.

The term "about" used herein refers to ±10% of the numerical value modified by the term, more preferably ±5%, and most preferably ±2%, so those of ordinary skill in the art can clearly determine the value based on the modified value. Numerical values determine the scope of the term "about."

Preparation method of Rhodiola rosea fermentation extract

In a first aspect, the present invention relates to a method for preparing Rhodiola rosea fermentation extract through two-step fermentation, wherein the method includes the following steps:

(a) using yeast to ferment Rhodiola rosea raw materials for the first time to obtain the first fermentation extract;

(b) Use Lactobacillus to ferment the first fermentation liquid obtained in step (a) for a second time to obtain a second fermentation extract.

In a second aspect of the present invention, it relates to a method for preparing Rhodiola rosea fermentation extract through two-step fermentation, wherein the method includes the following steps:

(a) using yeast to ferment Rhodiola rosea raw materials for the first time to obtain the first fermentation extract;

(a’) inactivate the first fermentation extract, preferably by raising the temperature, to obtain the inactivated first fermentation extract;

(b) Add nutrients required for lactobacilli fermentation, preferably glucose, to the inactivated first fermentation extract obtained in step (a'), and add lactobacilli for a second fermentation to obtain a second fermentation extract; and optionally,

(c) Centrifuge and sterile filter the second fermentation extract obtained in step (b).

In a third aspect of the present invention, it relates to a method for preparing Rhodiola rosea fermentation extract through two-step fermentation, wherein the method includes the following steps:

(a) Add Rhodiola rosea raw materials and yeast to the fermentation culture liquid for fermentation to obtain the first fermentation extract;

(a’) inactivate the first fermentation extract, preferably by raising the temperature, to obtain the inactivated first fermentation extract;

(b) Add nutrients required for lactobacilli fermentation, preferably glucose, to the inactivated first fermentation extract obtained in step (a'), and add lactobacilli for a second fermentation to obtain a second fermentation extract; and optionally,

(c) Centrifuge and sterile filter the second fermentation extract obtained in step (b).

In some embodiments, the Rhodiola rosea raw material is Rhodiola rosea roots, Rhodiola rosea stems or other parts of Rhodiola rosea. In some embodiments, the Rhodiola rosea raw material is Rhodiola rosea root. In some embodiments, the Rhodiola rosea raw material is Rhodiola rosea root powder. In some embodiments, the particle size of the Rhodiola rosea root powder is 1-10 mesh, 10-20 mesh, 20-30 mesh, 30-40 mesh, 40-50 mesh, 50-60 mesh, 60-70 mesh , 70~80 mesh, 80~90 mesh, 90~100 mesh, 100~150 mesh, 150~200 mesh, 200~250 mesh, 250~300 mesh, 300~350 mesh, 350~400 mesh, 400~450 mesh , 450～500 mesh, 500～550 mesh, 550～600 mesh, 600～650 mesh, 650～700 mesh, 700～750 mesh, 750～800 mesh, 800～850 mesh, 850～900 mesh, 900～950 mesh Or 950~1000 mesh. In some embodiments, the particle size of the Rhodiola rosea root powder is 40-60 mesh.

In some embodiments, the content of the Rhodiola rosea raw material in the fermentation culture liquid is 1 to 10 g/L, 10 to 20 g/L, 20 to 30 g/L, 30 to 40 g/L, 40 to 50 g/L. L, 50～60g/L, 60～70g/L, 70～80g/L, 80～90g/L, 90～100g/L, 100～150g/L, 150～200g/L, 200～250g/L, 250～300g/L, 300～350g/L, 350～400g/L, 400～450g/L or 450～500g/L. In some embodiments, the content of the Rhodiola rosea raw material in the fermentation culture broth is 10-50 g/L.

In some embodiments, the fermentation culture broth is a commonly used fermentation culture broth for yeast fermentation known to those skilled in the art. In some embodiments, the fermentation broth includes a nitrogen source, a carbon source, and growth factors. In some embodiments, the fermentation broth includes a nitrogen source, a carbon source, growth factors, inorganic salts, and water. In some embodiments, the fermentation broth includes yeast extract, wherein the yeast extract provides a nitrogen source and growth factors to the fermentation broth. In some embodiments, the nitrogen source is a common nitrogen source for microbial culture, including inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, nitrate, etc.; and organic nitrogen sources such as peptone, yeast powder, fish meal, bacteria Silk body etc. In some embodiments, the carbon source is a carbon source commonly used for microbial culture, such as, but not limited to, sucrose, glucose, fructose, lactose, mannitol, glycerol, galactose or maltitol. In some embodiments, the inorganic salt is a commonly used inorganic salt for microbial culture, such as, but not limited to, K ₂ HPO ₄ , KH ₂ PO ₄ , MgSO ₄ , MnSO ₄ , CaCl ₂ , etc. In some embodiments, the fermentation broth includes tryptone, yeast extract, and glucose. In some embodiments, the fermentation broth includes tryptone, yeast extract, glucose, _MgSO4 · _7H2O , _{KH2PO4 ,} and water.

In some embodiments, the fermentation culture broth contains tryptone, wherein the content of tryptone is 5-10 g/L, 10-15 g/L, 15-20 g/L, 20-25 g/L, 25-30 g /L, 30~35g/L, 35~40g/L, 40~45g/L or 45~50g/L. In some embodiments, the fermentation culture broth contains tryptone, wherein the content of the tryptone is 15 to 20 g/L.

In some embodiments, the fermentation culture broth includes yeast extract, wherein the content of the yeast extract is 1 to 4g/L, 4 to 8g/L, 8 to 12g/L, 12 to 16g/L, 16 ~20g/L, 20~24g/L, 24~28g/L, 28~32g/L or 32~36g/L. In some embodiments, the fermentation culture broth includes yeast extract, wherein the content of the yeast extract is 8 to 12 g/L.

In some embodiments, the fermentation culture broth includes glucose, wherein the content of glucose is 5-10g/L, 10-15g/L, 15-20g/L, 20-25g/L, 25-30g/L , 30～35g/L, 35～40g/L, 40～45g/L, 45～50g/L, 50～60g/L, 60～70g/L, 70～80g/L, 80～90g/L or 90 ~100g/L. In some embodiments, the fermentation culture broth includes glucose, wherein the content of glucose is 20 to 30 g/L.

In some embodiments, the fermentation culture broth contains MgSO ₄ ·7H ₂ O, wherein the content of MgSO ₄ ·7H ₂ O is 0.1～0.3g/L, 0.3～0.6g/L, 0.6～0.8g/ L, 0.8～1.0g/L, 1.0～1.3g/L, 1.3～1.5g/L, 1.5～1.8g/L, 1.8～2.0g/L, 2.0～2.5g/L, 2.5～3.0g/L , 3.0～3.5g/L, 3.5～4.0g/L, 4.0～4.5g/L or 4.5～5.0g/L. In some embodiments, the fermentation culture broth includes MgSO ₄ ·7H ₂ O, wherein the content of MgSO ₄ ·7H ₂ O is 0.3 to 0.8 g/L.

In some embodiments, the fermentation culture broth contains KH ₂ PO ₄ , wherein the content of KH ₂ PO ₄ is 0.1 to 0.5 g/L, 0.5 to 1 g/L, 1 to 2 g/L, 2 to 3 g/L. L, 3～4g/L, 4～5g/L, 5～6g/L, 6～7g/L, 7～8g/L, 8～9g/L, 9～10g/L, 10～15g/L, 15～20g/L, 20～25g/L or 25～30g/L. In some embodiments, the fermentation culture broth includes KH ₂ PO ₄ , wherein the content of KH ₂ PO ₄ is 1 to 3 g/L.

In some embodiments, the yeast is selected from the group consisting of Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Candida, Hanseniaspora opuntiae, Pichia Fermentans ), Kluyveromyces and combinations thereof. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces cerevisiae, and combinations thereof.

In some embodiments, the amount of yeast added is 1.0×10 ⁴ to 1.0×10 ⁷ CFU/ml, preferably 1.0×10 ⁵ to 1.0×10 ⁶ CFU/ml, and most preferably 5.0×10 ⁵ ～ 9.0×10 ⁵ CFU/ml.

In some embodiments, the fermentation time with yeast fermentation is 1 to 12h, 12 to 24h, 24 to 36h, 36 to 48h, 48 to 60h, 60 to 72h, 72 to 84h, 84 to 96h, 96 to 108h or 108～120h. In some embodiments, the fermentation time with yeast is 24 to 48 hours.

In some embodiments, the fermentation temperature when fermenting with yeast is about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C ℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃. In some embodiments, the fermentation temperature when fermenting with yeast is 10°C to 50°C, preferably 25°C to 35°C, more preferably 25°C to 30°C, and most preferably about 28°C.

In some embodiments, the pH during fermentation with yeast is about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 , 5.8, 5.9, 6.0, 6.2, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In some embodiments, the pH during fermentation with yeast is from 4 to 7, preferably from 5 to 6, more preferably from 5.2 to 5.8, and most preferably about 5.5.

In some embodiments, after the end of the first fermentation and before the start of the second fermentation, the first fermentation extract is inactivated, preferably by increasing the temperature. In some embodiments, the temperature of inactivation is 50-60°C, 60-70°C, 70-80°C, 80-90°C, 90-100°C, 100-110°C, 110-120°C, 120-130°C , 130～140℃ or 140～150℃. In some embodiments, the temperature of inactivation is between 80°C and 100°C.

In some embodiments, the inactivation time for inactivating the first fermentation extract is 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 25 minutes, 25 minutes to 30 minutes , 30 minutes to 35 minutes, 35 minutes to 40 minutes, 40 minutes to 45 minutes, 45 minutes to 50 minutes, 50 minutes to 55 minutes or 55 minutes to 60 minutes. In some embodiments, the inactivation time for inactivating the first fermentation extract is 5 minutes to 1 hour, preferably 5 minutes to 40 minutes, more preferably 10 minutes to 30 minutes, most preferably about 20 minutes.

In some embodiments, when performing the second fermentation with Lactobacillus, nutrients required for Lactobacillus fermentation are additionally added to the fermentation broth. In some embodiments, the nutrient may be a carbon source. In some embodiments, the carbon source can be glucose, fructose, sucrose, arabinose, maltose. In some embodiments, when performing the second fermentation with lactobacilli, glucose is additionally added to the fermentation broth.

In some embodiments, when performing the second fermentation with Lactobacillus, the additional nutrients required for Lactobacillus fermentation added to the fermentation broth are pre-sterilized, preferably by high temperature sterilization. In some embodiments, the high temperature sterilization is performed at 80-90°C, 90-100°C, 100-110°C, 110-120°C, 120-130°C, 130-140°C, or 140-150°C , preferably at 110 to 120°C, and most preferably at 115°C. In some embodiments, the pre-sterilized sterilization time is 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 25 minutes, 25 minutes to 30 minutes, 30 minutes to 35 minutes minutes, 35 minutes to 40 minutes, preferably 10 minutes to 30 minutes, most preferably about 20 minutes.

In some embodiments, the amount of glucose added when performing the second fermentation with Lactobacillus is such that the final concentration of glucose in the yeast fermentation broth after addition is 1 to 5 g/L, 5 to 10 g/L, 10 ~15g/L, 15~20g/L, 20~25g/L, 25~30g/L, 30~35g/L, 35~40g/L, 40~45g/L, 45~50g/L, 50~60g /L, 60~70g/L, 70~80g/L, 80~90g/L or 90~100g/L. In some embodiments, the amount of glucose added when performing the second fermentation with Lactobacillus is such that the final concentration of glucose in the yeast fermentation broth after addition is 1 to 100 g/L, preferably 10 to 20 g/L.

In some embodiments, the Lactobacillus is selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus salivarius (Lactobacillus salivarius) and combinations thereof. In some embodiments, the Lactobacillus is selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, and combinations thereof.

In some embodiments, the amount of Lactobacillus added is 1.0×10 ⁴ to 1.0×10 ⁷ CFU/ml, preferably 1.0×10 ⁵ to 1.0×10 ⁶ CFU/ml, and most preferably 5.0×10 ⁵ to 9.0× 10 ⁵ CFU/ml.

In some embodiments, in the second fermentation with Lactobacillus, Lactobacillus is added in the form of Lactobacillus seed liquid, and the bacterial cell concentration of the Lactobacillus seed liquid is 1.0×10 ⁷ to 1.0×10 ⁸ CFU /ml, the inoculation amount of the Lactobacillus seed liquid is 0.5-2% of the Rhodiola rosea yeast fermentation liquid.

In some embodiments, the fermentation time of Lactobacillus fermentation is 1 to 12h, 12 to 18h, 18 to 24h, 24 to 26h, 26 to 28h, 28 to 30h, 30 to 32h, 32 to 36h, 36 to 42h, 42～48h, 48～60h or 60～120h. In some embodiments, the fermentation time with Lactobacillus fermentation is 18 to 32 hours.

In some embodiments, the fermentation temperature when fermenting with Lactobacillus is about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C ℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃. In some embodiments, the fermentation temperature when fermenting with Lactobacillus is 10°C to 50°C, preferably 25°C to 45°C, more preferably 30°C to 40°C, and most preferably about 35°C.

In some embodiments, the pH during fermentation with lactobacilli is about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 , 5.8, 5.9, 6.0, 6.2, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In some embodiments, the pH during fermentation with lactobacilli is 4-7, preferably 5.5-6.5, and most preferably about 6.

In some embodiments, the step of centrifuging and sterilizing the second fermentation extract is further included.

Rhodiola rosea fermentation extract

The fourth aspect of the present invention relates to a rhodiola rosea fermentation extract prepared by the method according to any one of the first to third aspects of the present invention.

In some embodiments, the Rhodiola rosea fermentation extract obtained through two-step fermentation has a stronger whitening ability than the Rhodiola rosea fermentation extract obtained only through yeast or lactobacillus fermentation under the same conditions.

In some embodiments, the Rhodiola rosea fermentation extract obtained through two-step fermentation has stronger antioxidant capacity than the Rhodiola rosea fermentation extract obtained only through yeast or lactobacillus fermentation under the same conditions.

Pharmaceutical preparations and cosmetic compositions

A fifth aspect of the present invention relates to a cosmetic composition comprising a Rhodiola rosea fermentation extract according to the fourth aspect of the present invention.

A sixth aspect of the present invention relates to a pharmaceutical preparation comprising a Rhodiola rosea fermentation extract according to the fourth aspect of the present invention.

In some embodiments, the pharmaceutical formulation or cosmetic composition is used for skin care. In some embodiments, the pharmaceutical preparation or cosmetic composition is used for whitening, antioxidant or anti-aging skin. In some embodiments, the pharmaceutical preparation or cosmetic composition includes Rhodiola rosea fermentation extract as the active ingredient and one or more pharmaceutically or cosmetically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical preparation or cosmetic composition is a cream, lotion, paste, ointment, mask, gel, lotion or serum. In some embodiments, the pharmaceutical preparation or cosmetic composition of the present invention may also contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, flavors and fragrances, pigments, preservatives, antioxidants, etc. Agents, moisturizers, UV absorbers, astringents, penetration aids, pH adjusters, etc. Those skilled in the art can make selections based on their common sense and specific needs.

Use of rhodiola rosea fermentation extract for preparing pharmaceutical or cosmetic compositions

The seventh aspect of the present invention relates to the use of Rhodiola rosea fermentation extract according to the fourth aspect of the present invention in the preparation of pharmaceutical or cosmetic compositions with whitening and/or antioxidant capabilities.

Detailed ways

The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in this field and can be replaced by instruments and equipment that meet corresponding standards.

The Chinese herbal raw materials used in the embodiments of the present invention are all commercially available unless otherwise specified. The reagents used in the embodiments of the present invention are all commercially available analytically pure chemical reagents unless otherwise specified.

The raw materials and equipment mentioned in the present invention are all commonly used raw materials and equipment in this field. They are only examples and are not intended to limit the scope of the present invention. Those skilled in the art can select equivalent raw materials and related equipment based on the disclosure of the present invention.

Experimental materials, reagents, equipment and statistical analysis methods

Sources of strains in the examples: Capsulata-coated yeast was purchased from the China General Microbial Culture Collection and Management Center (Address: Wuhan University), with the preservation number CGMCC 2.5608; Lactobacillus rhamnosus was a strain isolated by our laboratory, and this strain It has been deposited in the China Type Culture Collection Center (CCTCC). The relevant deposit information is as follows:

Rhodiola rosea raw materials: Rhodiola rosea, purchased from Shanghai Baiheyuan Pharmacy.

Experimental equipment: (1) 5L glass fermentation tank, Shanghai Baoxing BIOTECH-5JGY; (2) Constant temperature oscillator, Shanghai Zhicheng ZWY-2102C; (3) Clean workbench, Sujing Antai HVS-1300-U; (4 ) High-speed pulverizer, Zhejiang Hongjingtian Industry and Trade Co., Ltd. DE-300g.

Experimental reagents: Yeast extract and tryptone were purchased from OXOID in the UK, 500g/bottle; beef extract powder was purchased from Qingdao Haibo Biotechnology Co., Ltd., 250g/bottle; the remaining reagents were purchased from the General-reagent brand of Shanghai Titan Technology Co., Ltd. , packaging specification: 500g/bottle.

Statistical methods: The experimental results are the mean ± standard deviation of three parallel experimental data, and a paired t-test is used to test whether the differences between groups are statistically significant. The statistical software is SigmaPlot 14.0.

Example 1: Two-step fermentation of Rhodiola rosea using yeast and Lactobacillus rhamnosus

(1) Preparation of seed liquid

Yeast seed liquid: Take 0.5 mL of frozen capsule-coated yeast solution on a clean workbench and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture medium. Culture it at 28°C and 180 rpm for 16 hours. The bacteria are in the logarithmic growth phase. Seed culture medium ingredients: tryptone 20g/L, yeast extract 10g/L, glucose 20g/L, pH 5.5, 115°C, sterilized for 20 minutes.

Lactobacillus seed liquid: Take 0.5 mL of Lactobacillus rhamnosus cryopreservation liquid on the ultra-clean workbench and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture medium. Cultivate at 37°C for 16 hours. The bacteria are in the logarithmic growth phase. . Seed culture medium ingredients: tryptone 10g/L, yeast extract 5g/L, glucose 20g/L, beef extract powder 8g/L, ammonium citrate 2g/L, Tween80 1g/L, sodium acetate 5g/L, K ₂ HPO ₄ ·3H ₂ O, MgSO ₄ ·7H ₂ O 2g/L, MnSO ₄ ·H ₂ O 0.1g/L, pH 6.5, 115°C, sterilized for 20 minutes.

(2) Preparation of Rhodiola rosea yeast fermentation broth

Rhodiola rosea roots are dried, crushed, and passed through a 60-mesh sieve to obtain rhodiola rosea root powder for later use. Fermentation was carried out in a 5L automatic glass fermentation tank, with 2.5L of liquid in it. Rhodiola rosea root powder was added to the fermentation medium (containing tryptone 20g/L, yeast extract 10g/L, and glucose 20g) at a final concentration of 50g/L. /L, MgSO ₄ ·7H ₂ O 0.5g/L, KH ₂ PO ₄ 2g/L, pH 5.5). Sterilize at 115°C for 30 minutes, cool and then inoculate the yeast seed liquid in step (1). Fermentation process: stirring 300rpm, temperature 28°C, ventilation 1.5L/min, pressure 0.03MPa, fermentation cycle 36h. After fermentation, inactivate at 80°C for 20 minutes.

(3) Preparation of secondary fermentation broth of Lactobacillus rhamnosus

Add 50 ml of sterilized 70% (w/w) glucose solution to the fermentation broth after inactivation in step (2), inoculate the lactobacillus seed liquid in step (1), and perform secondary fermentation. Fermentation process: Stirring 50rpm, temperature 35℃, surface ventilation 0.5L/min, pressure 0.01MPa, alkali solution to control pH 6.0, fermentation cycle 28h, glucose exhaustion.

(4) Fermentation broth removal of impurities and bacteria

After the Rhodiola rosea secondary fermentation liquid obtained in step (3) was centrifuged at 10,000×g for 10 minutes and filtered and sterilized with a 0.22um membrane, the Rhodiola rosea fermentation product filtrate was obtained.

Example 2:

Rhodiola rosea root powder in Example 1 was changed from 50g/L to 25g/L.

Comparative Example 1: 50g/L Rhodiola rosea extract is not fermented

Rhodiola rosea roots are dried, crushed, and passed through a 60-mesh sieve to obtain rhodiola rosea root powder for later use. Weigh 125g Rhodiola rosea root powder into pure water, dilute to 2.5L, sterilize at 115°C for 20 minutes. After cooling, the rhodiola rosea extract was centrifuged at 10,000×g for 10 minutes and filtered with a 0.22um membrane for sterilization to obtain the rhodiola rosea extract filtrate.

Comparative Example 2: 25g/L Rhodiola rosea extract is not fermented

Rhodiola rosea roots are dried, crushed, and passed through a 60-mesh sieve to obtain rhodiola rosea root powder for later use. Weigh 62.5g Rhodiola rosea root powder into pure water, dilute to 2.5L, sterilize at 115°C for 20 minutes. After cooling, the rhodiola rosea extract was centrifuged at 10,000×g for 10 minutes and filtered with a 0.22um membrane for sterilization to obtain the rhodiola rosea extract filtrate.

Comparative Example 3: 50g/L Rhodiola rosea fermented with yeast only

In the fermentation method of Example 1, the third step "preparation of secondary fermentation liquid of Lactobacillus rhamnosus" is omitted, which is the preparation method of Rhodiola rosea fermentation liquid.

Comparative Example 4: 25g/L Rhodiola rosea fermented with yeast only

In the fermentation method of Example 2, the third step "preparation of secondary fermentation liquid of Lactobacillus rhamnosus" is omitted, which is the preparation method of Rhodiola rosea fermentation liquid.

Comparative Example 5: 50g/L Rhodiola rosea fermented only with Lactobacillus rhamnosus

(1) Preparation of seed liquid

Lactobacillus seed liquid: Take 0.5 mL of Lactobacillus rhamnosus cryopreservation liquid on the ultra-clean workbench and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture medium. Cultivate at 37°C for 16 hours. The bacteria are in the logarithmic growth phase. . The seed medium contains tryptone 10g/L, yeast extract 5g/L, glucose 20g/L, beef extract powder 8g/L, ammonium citrate 2g/L, Tween80 1g/L, sodium acetate 5g/L, K ₂ HPO ₄ ·3H ₂ O, MgSO ₄ ·7H ₂ O 2g/L, MnSO ₄ ·H ₂ O 0.1g/L, pH 6.5, 115℃, sterilized for 20 minutes.

(2) Preparation of Rhodiola rosea and Lactobacillus rhamnosus fermentation broth

Rhodiola rosea roots are dried, crushed, and passed through a 60-mesh sieve to obtain rhodiola rosea root powder for later use. Fermentation was carried out in a 5L automatic glass fermentation tank, with 2.5L of liquid. Rhodiola rosea root powder was added to the fermentation medium (containing tryptone 10g/L, yeast extract 5g/L, and glucose 20g) at a final concentration of 50g/L. /L, beef dip powder 8g/L, ammonium citrate 2g/L, Tween801g/L, sodium acetate 5g/L, K ₂ HPO ₄ ·3H ₂ O, MgSO ₄ ·7H ₂ O 2g/L, MnSO ₄ ·H ₂ O 0.1g/L, pH6.5). Sterilize at 115°C for 20 minutes, cool and then inoculate the Lactobacillus rhamnosus seed liquid in step (1). Fermentation process: stirring 50rpm, temperature 35°C, surface ventilation 0.5L/min, pressure 0.01MPa, alkali liquid control pH 6.0, fermentation cycle 28h.

(3) Fermentation broth removal of impurities and bacteria

After the Rhodiola rosea fermentation liquid obtained in step (2) was centrifuged at 10,000×g for 10 minutes and filtered and sterilized with a 0.22um membrane, the Rhodiola rosea fermentation product filtrate was obtained.

Comparative Example 6: The addition amount of Rhodiola rosea root powder in Comparative Example 5 was changed to 25g/L.

Experimental results

Rhodiola rosea extract without yeast fermentation has obvious inhibition on Lactobacillus. After inoculation with Lactobacillus rhamnosus, Lactobacillus rhamnosus is inhibited and the number of viable bacteria is greatly reduced. Therefore, rhamnosus can hardly grow and metabolize. consume glucose. However, the antibacterial effect of Rhodiola rosea extract after yeast fermentation on Lactobacillus rhamnosus is weakened. Lactobacillus rhamnosus can grow and metabolize in the culture medium and consume the glucose in the culture medium. The fermented Rhodiola rosea extract has improved whitening and antioxidant capabilities.

1) The two-step fermentation method reduces the antibacterial effect of Rhodiola rosea fermentation extract on Lactobacillus rhamnosus

In order to compare the antibacterial effect of Rhodiola rosea on Lactobacillus rhamnosus before and after yeast fermentation, the changes in the number of viable bacteria in the Lactobacillus rhamnosus fermentation broth were detected. The results are shown in Table 1 below.

Table 1

It can be seen from the viable bacteria detection data that the presence of Rhodiola rosea in the culture medium has a significant antibacterial effect on Lactobacillus rhamnosus (Comparative Examples 4 and 5 vs. the control group without Rhodiola rosea). After using the two-step fermentation method ( Examples 1 and 2), the inhibitory effect of Rhodiola rosea fermentation broth on Lactobacillus rhamnosus after yeast fermentation was significantly reduced (Example 1 vs Comparative Example 4; Example 2 vs Comparative Example 5), in which the two-step fermentation Compared with Comparative Example 4 of single-step fermentation, the inhibitory effect of Example 1 on Lactobacillus rhamnosus was significantly reduced by about 5 orders of magnitude ((1.9±1.3)×10 ⁶ vs (8.7±2.1)×10). Compared with Comparative Example 5 of single-step fermentation, the inhibitory effect of Example 2 on Lactobacillus rhamnosus was significantly reduced by about 4 orders of magnitude ((7.1±0.7)×10 ⁶ vs (6.9±1.3)×10 ² ). This may be because yeast degrades some components in Rhodiola rosea that inhibit the growth of Lactobacillus, and the rich nutrients in yeast metabolites just meet the high nutritional requirements of Lactobacillus.

2) The two-step fermentation method improves the whitening effect of Rhodiola rosea fermentation extract

Tyrosinase is the key enzyme for the formation of melanin. Many whitening and freckle removal products achieve whitening effects by inhibiting tyrosinase. The strength of the inhibitory effect on tyrosinase is the main indicator for evaluating the whitening ability. The results of the tyrosinase inhibition IC50 measured for Examples 1-2 and Comparative Examples 1-6 are shown in Table 2 below.

Table 2

* indicates comparison with before fermentation, p＜0.05; ** indicates comparison with before fermentation, p＜0.01

It can be seen from the results in Table 2 above that the tyrosinase inhibition rate IC50 of the fermentation broth after secondary fermentation of 50g/L Rhodiola rosea root powder was reduced to 7.67% (volume %), which is comparable to Comparative Example 1 and Comparative Example 3. Compared with Comparative Example 5, there are significant differences (p values are 0.015, 0.013, and 0.002 respectively). The IC50 of the fermentation broth tyrosinase inhibition rate of 25g/L Rhodiola rosea root powder after secondary fermentation was reduced to 14.72% (volume %), which was significantly different from Comparative Examples 2 and 6 (p The values are 0.001 and 0.005 respectively), and the whitening ability is greatly improved.

3) The two-step fermentation method improves the antioxidant capacity of Rhodiola rosea fermentation extract.

The DPPH method is a commonly used method to determine the antioxidant properties of food, pharmaceutical and other samples. The principle is based on the reaction between DPPH free radicals and antioxidants, and the antioxidant capacity of the sample is evaluated by comparing the absorbance changes before and after DPPH free radical reduction. The results of the DPPH scavenging IC50 measured for Examples 1-2 and Comparative Examples 1-6 are shown in Table 3 below.

table 3

** means compared with before fermentation, p<0.01

After secondary fermentation of 50g/L Rhodiola rosea root powder, the DPPH clearance IC50 decreased from 0.214% to 0.121% (volume fraction). Compared with Comparative Example 1, Comparative Example 3 and Comparative Example 5, there was a significant difference ( The p values are 0.007, 0.003, 0.002) respectively. The DPPH clearance IC50 of 25g/L Rhodiola rosea root powder after secondary fermentation was reduced to 0.209% (volume fraction). Compared with Comparative Example 2, Comparative Example 4 and Comparative Example 6, there was a significant difference (p value 0.003, 0.008, 0.001) respectively. The antioxidant capacity of Rhodiola rosea root extract after secondary fermentation has been greatly improved, and the second Lactobacillus fermentation has a greater improvement.

Claims (17)

1. A method for preparing Rhodiola rosea fermentation extract through two-step fermentation, wherein the method includes the following steps: (a) using yeast to ferment Rhodiola rosea raw materials for the first time to obtain the first fermentation extract; (b) Use Lactobacillus to ferment the first fermentation liquid obtained in step (a) for a second time to obtain a second fermentation extract. 2. The method according to claim 1, wherein after the first fermentation ends and before the second fermentation starts, the first fermentation extract is inactivated, preferably by heating, more preferably at 80 Inactivation is performed at ~100°C. 3. The method according to claim 2, wherein inactivation is carried out from 5 minutes to 1 hour, preferably from 5 minutes to 40 minutes, more preferably from 10 minutes to 30 minutes, most preferably about 20 minutes. 4. The method according to any one of claims 1-3, wherein during the second fermentation, nutrients required for lactobacillus fermentation are additionally added to the fermentation liquid, preferably carbon sources, more preferably glucose and fructose. , sucrose, arabinose, maltose, most preferably glucose. 5. The method according to claim 4, wherein the amount of glucose added is such that the final concentration of glucose in the yeast fermentation broth after addition is 10-20 g/L. 6. The method of any one of the preceding claims, wherein the method optionally further comprises the steps of: (c) Centrifuge and sterile filter the second fermentation extract obtained in step (b). 7. The method according to any one of the preceding claims, wherein the raw material of Rhodiola rosea in step (a) is Rhodiola rosea root, preferably Rhodiola rosea root powder, more preferably Rhodiola rosea with a particle size of 40-60 mesh. Sedum root powder. 8. The method according to any one of the preceding claims, wherein in step (a), Rhodiola rosea raw materials are added to the fermentation culture broth, and the content of Rhodiola rosea raw materials in the fermentation culture broth is 10～50g/L. 9. The method of claim 8, wherein the fermentation broth contains a nitrogen source, a carbon source and growth factors, optionally also containing inorganic salts and water. 10. The method of claim 9, wherein the fermentation broth contains tryptone, yeast extract and glucose, optionally also _MgSO4 · _7H2O , _KH2PO4 and _water . 11. The method according to any one of the preceding claims, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces cerevisiae, Candida, Hansenula sporogenes, Pichia pastoris, Kluyveromyces or the like. combination, and the Lactobacillus is selected from Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus salivarius or a combination thereof. 12. The method according to any one of the preceding claims, wherein the fermentation time for fermentation with yeast is 24 to 48 h, and the fermentation time for fermentation with lactobacilli is 18 to 32 h. 13. The method according to any one of the preceding claims, wherein in the first fermentation of step (a), the amount of yeast added is 1.0×10 ⁵ to 1.0×10 ⁶ CFU/ml; in step In the second fermentation of (b), the amount of Lactobacillus added is 1.0×10 ⁵ to 1.0×10 ⁶ CFU/ml. 14. The method according to any one of the preceding claims, wherein in the second fermentation of step (b), Lactobacillus is added in the form of Lactobacillus seed liquid, the bacterial concentration of the Lactobacillus seed liquid is 1.0×10 ⁷ to 1.0×10 ⁸ CFU/ml, and the inoculation amount of Lactobacillus seed liquid is 0.5-2% of the Rhodiola rosea yeast fermentation liquid, and the pH is controlled at 5.5-6.5. 15. A Rhodiola rosea fermentation extract, the Rhodiola rosea fermentation extract is prepared by the method of any one of claims 1-14. 16. A pharmaceutical preparation or cosmetic composition comprising the rhodiola rosea fermentation extract according to claim 15. 17. Use of the rhodiola rosea fermentation extract according to claim 15 in the preparation of pharmaceutical or cosmetic compositions with whitening and/or antioxidant capabilities.