CN114891852B

CN114891852B - Method for obtaining high-content cyclamen saponin fermentation liquor, fermentation liquor and application thereof

Info

Publication number: CN114891852B
Application number: CN202210516585.7A
Authority: CN
Inventors: 周浩文; 徐禄清; 邓建科; 蔡永通
Original assignee: Guangzhou Cuipu Biotechnology Co ltd
Current assignee: Guangzhou Cuipu Biotechnology Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2023-10-03
Anticipated expiration: 2042-05-12
Also published as: CN114891852A

Abstract

The invention discloses a method for obtaining high-content cyclamen saponin fermentation liquor, which takes cyclamen endophyte Bacillus amyloliquefaciens as a strain, and takes cyclamen extract as a substrate to ferment in fermentation liquor containing urea and peptone as main nitrogen sources to obtain fermented organic matters; ultrasonic crushing the above fermented organic matter, centrifuging, and filtering to obtain high content cyclamen saponin fermentation liquid. The obtained fermented product has remarkably improved saponin content. The cyclamen fermentation liquid prepared by the invention is applied to cosmetics and has the functions of antioxidation and/or anti-aging.

Description

Method for obtaining high-content cyclamen saponin fermentation liquor, fermentation liquor and application thereof

Technical Field

The invention relates to the technical field of biological fermentation, in particular to a method for obtaining high-content cyclamen saponin fermentation liquor, the fermentation liquor and application thereof.

Background

Cyclamen (Cyclamen persicum Mill) is a perennial herb of the genus Cyclamen of the family primaceae, and is one of the important species in the current flower industry in northern China. At present, little research on active ingredients in cyclamen is carried out, and related documents only report chemical ingredients in two wild species corms of cyclamen (Cyclamen coum Miller) and cyclamen (Cyclamen mirabile Hidebr.) studied by Iis Calis et al in 1996. Iis Calis et al isolated from the corm of Cyclams Murraya, have 6 triterpenoid saponins, activity test shows that it has the function of promoting uterine contraction of test mice, and has obvious antifungal activity and weaker antibacterial activity.

Saponins, also known as saponins, are a relatively complex class of glycoside compounds found in the plant kingdom, which are known by the appearance of soap foam due to the shaking of their aqueous solutions, and can be classified into steroidal saponins and triterpenoid saponins according to their sapogenin structure. The saponin is an important secondary metabolite in the fermentation process of cyclamen and has the characteristics of good antioxidation, antivirus, antitumor and the like. Sabora et al prepared crude cyclamen saponins by Soxhlet extraction and found to have inhibitory effects on enterococcus faecalis, pseudomonas aeruginosa and Salmonella typhi.

Endophytes (endophytes) refer to micro-biological networks that are deposited in tissues, organs, cell gaps and cells of higher woody and herbaceous vegetation, monocotyledonous vegetation and dicotyledonous vegetation, and form a mutual win-win relationship with host plants, and the endophytes establish a unique relationship with the host plants in an evolutionary stage and have the same or similar source synthesis approaches. The evolution of the host plant genome is affected by its endophyte community, and the unique chemical environment of the host vegetation also affects the metabolites of the endophyte. The close synergy between plants and bacterial endophytes results in the ability of bacterial endophytes to synthesize a variety of potential secondary metabolites. The research shows that the inoculation of endophytes increases the content of vinca alkaloid in catharanthus roseus and benzylisoquinoline nitrogen-containing basic organic compounds in poppy flowers, and this finding effectively improves the problem of natural medicine source shortage. Endophytes play a vital role in the production of important pharmaceutical compounds such as taxanes, vincristine, hypericin and podophyllotoxin. Endophytes produce proteases, cellulases, esterases, etc. during the period of parasitism in plant tissues, and complex enzyme systems can convert the components in parasitic vegetation into new active substances through unique reaction metabolic processes, thereby generating new or higher active medicinal effects.

At present, most of researches on cyclamen are focused on the aspect of preventing diseases and insect pests of cyclamen flowers, the separation of plant extracts and the identification of effective components are less, qian Renjuan and the like, the identification of cyclamen soft rot antagonistic bacteria and the biological control effect thereof are carried out, chinese journal of microecology, 24 rd edition of 2012, reported that authors collect cyclamen plants healthy in the region of Zea, gao Shanji th, and endophytic bacteria are separated and screened, so that an endophytic bacterium Y1 with stronger antagonism and genetic stability on bacterial soft rot bacteria is finally obtained, and the strain is identified from aspects of morphology, physiological biochemistry, 16S rDNA sequence, phylogenetic analysis and the like. The endophytic characteristics of the biocontrol bacterium Y1 are determined for the first time, the biocontrol effect of the biocontrol bacterium on the soft rot of cyclamen is explored, and scientific basis is provided for the deep research of the biocontrol mechanism and the production application thereof. However, at present, little research is done on the fermentation of the endophyte of cyclamen, no research on the change of the active ingredient and the action mechanism of the fermentation of cyclamen under the liquid condition is seen, and no report on the application of the endophyte of cyclamen to the fermentation of cyclamen and the application of the fermentation product in the field of cosmetics is seen.

Therefore, in order to widen the application of cyclamen, the inventor takes cyclamen endophyte Bacillus amyloliquefaciens as a strain, and takes cyclamen extract as a substrate to ferment in a fermentation broth containing urea and peptone as main nitrogen sources to obtain a fermented organic matter; ultrasonic crushing the above fermented organic matter, centrifuging, and filtering to obtain high content cyclamen saponin fermentation liquid. And the fermentation broth is used as an antioxidant active ingredient or an anti-aging ingredient to be applied to the cosmetic field.

Disclosure of Invention

In order to solve the technical problems, an object of the present invention is to provide a method for obtaining a high content cyclamen saponin broth:

the inventor researches and discovers that the fermentation organic matter is obtained by taking the cyclamen endophyte Bacillus amyloliquefaciens as a strain and taking 1-5g/L cyclamen extract as a substrate for fermentation in a fermentation liquid containing 2-8g/L urea and 4-10g/L peptone as main nitrogen sources; ultrasonic crushing at 80kHz-100kHz for 10-20min, centrifuging, and filtering to obtain high-content cyclamen saponin fermentation liquid.

The cyclamen endophyte Bacillus amyloliquefaciens is a currently known cyclamen endophyte, can be purchased in the market, and can also be separated from fresh cyclamen plant roots, stems, petioles and leaves, and the separation method of the endophyte specifically comprises the following steps: (1) Taking fresh cyclamen plants, washing the fresh cyclamen plants, and thoroughly disinfecting the surfaces of the plants to remove surface-attached microorganisms; (2) Cutting root, stem, leaf stalk, and leaf into small pieces of about 0.1cm×0.5 cm; (3) The small pieces are embedded into NA culture medium, cultured for 24 hours at 37 ℃, bacterial colonies with different forms are selected, streaked by a flat plate, purified and numbered for storage; (4) Identifying the strain Bacillus amyloliquefaciens of the cyclamen endophyte, carrying out morphological observation, physiological and biochemical characteristic measurement, gene extraction and PCR amplification on the strain, and identifying the strain as the cyclamen endophyte Bacillus amyloliquefaciens. The endophytes exist in the internal tissues of the plants, do not interfere with each other and form cooperative symbiosis in the growth process of the plants, and cannot cause harm to hosts. The cyclamen endophyte Bacillus amyloliquefaciens in the invention is from the subtropical crop institute of Zhejiang province. The separation and identification method of the cyclamen endophyte Bacillus amyloliquefaciens is shown in Qian Renjuan and the like, the identification of the cyclamen soft rot antagonistic bacteria and the biological control effect thereof, and the journal of China micro-ecology, 3 rd month, 24 rd period, 2012.

According to the invention, single-factor experimental examination is carried out on the culture medium components through experiments, and the fact that the content of saponin produced by the cyclamen endophyte Bacillus amyloliquefaciens in the corn starch culture medium with the concentration of 20-40g/L is obviously increased is probably because the corn starch is a composite carbon source, contains a small amount of nitrogen source and other growth factors, promotes specific growth, and increases the yield of saponin. Considering that corn flour is relatively inexpensive, has the potential for mass fermentation, corn starch is selected as the optimal carbon source. Screening of nitrogen sources, the saponin content in the culture medium containing 2-8g/L urea and 4-10g/L peptone as main nitrogen sources is remarkably increased, and the inventor unexpectedly found that if peptone or urea alone is not high in saponin content, particularly if urea alone is used as the main nitrogen source, the saponin content is very low, which means that the strain is very weak in the ability to utilize inorganic nitrogen, but when urea and peptone are used as the composite nitrogen source, the saponin content is remarkably increased, probably because peptone is used as the main nitrogen source, the growth of thallus can be satisfied, but after microorganism growth and metabolism, the pH is reduced, the cell membrane density is increased, the permeability is reduced, and the transportation of intracellular saponin is not facilitated, but when urea is added, the pH is not reduced, but rather is increased, and the transportation of intracellular saponin is facilitated. The obtained fermented product has remarkably improved saponin content.

The inventors of the present inventionIt was also found that the addition of 0.5-5mg/L of manganese chloride to the medium significantly increased the saponin content, probably due to Mn ²⁺ At a certain concentration, the production of spores can be stimulated, and the saponin substance produced by bacillus may be related to the number of spores.

The inventor of the invention also discovers that when the fermentation organic matter is obtained after the fermentation is finished, the ultrasonic extraction has a great influence on the dissolution of the intracellular saponin of the fermentation organic matter, and the ultrasonic breaking frequency is 80kHz-100kHz, and the time is 10-20min, thereby being most beneficial to the dissolution of the intracellular saponin.

In a preferred embodiment of the invention, the culture medium is 40g/L of corn meal, 6.7g/L of peptone and 5mg/L of manganese chloride, the optimal culture condition is that the pH of the initial fermentation liquid is 5.0, the liquid loading amount is 30mL, the inoculation amount is 4%, the culture temperature is 30 ℃, the rotation speed of a shaking table is 200r/min, and the culture time is 144 hours. The ultrasonic crushing frequency is 90kHz, and the saponin content is 16.6mg/mL when the ultrasonic crushing time is 10min, which is 500 times higher than that of the unfermented cyclamen extract.

Another object of the present invention is to provide a cyclamen fermentation broth prepared by the above method for obtaining cyclamen saponin fermentation broth with high content.

A further object of the present invention is the use of the above-mentioned cyclamen fermentation broth as an antioxidant active ingredient or as an anti-aging ingredient in the preparation of cosmetics.

Further, the amount of the high-content cyclamen saponin fermentation liquor in the cosmetics is 10-20wt%, based on the total weight percentage of cyclamen saponin fermentation liquor in the cosmetics raw materials.

The cosmetics include, but are not limited to, lotions, essences, creams, masks and the like containing the high content cyclamen saponin fermentation broth. In addition to the high level cyclamen saponin broth, moisturizers, skin conditioners, thickeners, preservatives, and the like, as known in the art, may be included.

Drawings

FIG. 1 shows thin layer chromatograms of crude saponin (A) supplemented with urea and crude saponin (B) without urea medium with oleanolic acid as control (C).

FIG. 2 shows the DPPH-active urea-added crude saponin (A) and the urea-free medium crude saponin (B) of the cyclamen saponin-containing fermentation broth and the control VC.

FIG. 3 shows the anti-aging activity of cyclamen saponin broth measured using mouse embryo fibroblasts.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: preparation of cyclamen extract:

the experimental cyclamen plants are healthy cyclamen plants in the growing period of the flower base of the mountain of the Zea mays in the Zhejiang province subtropical crop institute. Cutting the root, stem, leaf and flower of cyclamen into root segments with proper size, and sun drying. Taking sun-dried crushed cyclamen plants, soaking and extracting with methanol solution, standing for one night after ultrasonic treatment for 1h, performing ultrasonic treatment again for 1h to obtain full extraction, removing cyclamen plant residues, collecting extractive solution, and repeating the above methanol soaking ultrasonic extraction operation for three times until the color of the extractive solvent becomes light. Concentrating the collected extractive solution under reduced pressure to obtain extract. The extractum is dissolved in hot water, the water-soluble part is strong polar components such as inorganic salt, amino acid, glycoside and the like, and the impurities which are insoluble in water are discarded. The ester with larger polarity is removed by diethyl ether extraction for three times, and the water layer is reserved. Extracting with saturated n-butanol suitable for dissolving medium and large polar components to obtain extract, and collecting cyclamen extract.

Example 2: fermentation

(1) Preparing seed liquid:

picking up fresh cyclamen endophyte Bacillus amyloliquefaciens single colony with toothpick to LB culture solution (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7.0), shaking at 28deg.C for 20 hr at 200r/min, and culturing to obtain seed solution.

(2) Preparation of the culture medium:

20.0g/L of corn starch, 8g/L of peptone, 3.5mg/L of manganese chloride, 2g/L of urea, 3.5g of cyclamen extract in example 1 were dissolved in 2000ml of medium and sterilized at 121℃for 20min. The saponin content of the sample before fermentation was determined to be 0.0267mg/mL.

(3) Inoculating and fermenting:

according to the pH 4.0 of the initial fermentation liquor, the liquid loading amount is 20 (v/v), the inoculation amount is 2%, the culture temperature is 25 ℃, the rotation speed of a shaking table is 200r/min, the culture time is 70h, the fermentation culture is stopped, and the fermented organic matter is obtained.

(4) Crushing, centrifugal filtration

Crushing the fermented organic matters in 100kHz ultrasonic for 10min, and centrifuging at 7000rpm for 30min by adopting a butterfly centrifuge to obtain the final high-content cyclamen saponin fermentation liquor.

(5) Filtering the high content cyclamen saponin fermentation broth with gauze, and measuring its saponin content 8.9753mg/mL. The method for measuring the content of the saponin in the fermentation product comprises the following steps:

drawing an oleanolic acid standard curve: accurately weighing oleanolic acid standard product dried at 105deg.C to constant weight, adding a small amount of absolute ethanol for dissolving, fixing volume to scale, and shaking. Respectively sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of oleanolic acid standard solution into a 10mL volumetric flask, using absolute ethyl alcohol to fix volume scale, sucking 1mL of the standard solution into a test tube, sequentially dripping 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid into the test tube, carrying out water bath for 15 minutes in a water bath kettle at 60 ℃, cooling to room temperature, dripping 3mL of glacial acetic acid for reaction, using absolute ethyl alcohol instead of a sample as a blank reference, and measuring the absorbance at 570nm by using an ultraviolet spectrophotometer. And (3) taking an OD value as an abscissa and the concentration of oleanolic acid as an ordinate, making a standard curve of oleanolic acid, and establishing a regression equation.

Determination of the saponin content of the fermentation broth:

filtering the fermentation liquor by 8 layers of gauze, sucking 25mL of filtrate into a 50mL triangular flask, putting into a 60 ℃ oven for drying to the rest third, adding 25mL of 75% ethanol, carrying out water bath for 4 hours in a 75 ℃ water bath, pouring into a 50mL centrifuge tube, centrifuging for 10min at 5000r/min, and reserving supernatant for later use. Sucking 1mL of supernatant into a 10mL test tube, sequentially adding 0.2mL of vanillin-glacial acetic acid, 0.8mL of perchloric acid, carrying out water bath at 60 ℃ for 15min, cooling to room temperature, adding 3mL of glacial acetic acid, mixing uniformly, and measuring the absorbance at 571 nm.

Example 2:

the difference from example 1 is that the composition ratio of the culture medium is different, the fermentation conditions are different, the culture medium is prepared by dissolving 40.0g/L of corn starch, 6g/L of peptone, 2mg/L of manganese chloride, 5g/L of urea, 2.0g of cyclamen extract in example 2 in 2000ml of culture medium, and sterilizing at 121 ℃ for 20min. According to the pH value of the initial fermentation liquor of 5.0, the liquid loading quantity of 50 (v/v), the inoculation quantity of 6 percent, the culture temperature of 30 ℃, the rotation speed of a shaking table of 200r/min, the culture time of 100h, and the fermentation culture and stopping to obtain the fermentation organic matter. Crushing the fermented organic matters in 80kHz ultrasonic for 15min, and centrifuging at 8000rpm for 30min to obtain final high-content cyclamen saponin fermentation broth. Filtering the high content cyclamen saponin fermentation broth with gauze, and measuring its saponin content 14.5699mg/mL.

Example 3:

the difference from example 1 is that the composition ratio of the culture medium is different, the fermentation conditions are different, the culture medium is prepared by dissolving 40.0g/L of corn starch, 6.7g/L of peptone, 5mg/L of manganese chloride and 4g/L of urea, 3.5g of cyclamen extract in example 3 in 2000ml of culture medium, and sterilizing at 121 ℃ for 20min. According to the pH value of the initial fermentation liquor of 5.0, the liquid loading amount of 30 (v/v), the inoculation amount of 4 percent, the culture temperature of 30 ℃, the rotation speed of a shaking table of 200r/min, the culture time of 144 hours, and the fermentation culture and stopping to obtain the fermentation organic matter. Crushing the fermented organic matters in 90kHz ultrasonic for 10min, and centrifuging at 8000rpm for 30min to obtain final high-content cyclamen saponin fermentation broth. Filtering the high content cyclamen saponin fermentation broth with gauze, and measuring its saponin content 16.6937mg/mL.

Example 4:

the difference from example 1 is that the composition ratio of the culture medium is different, the fermentation conditions are different, the preparation of the culture medium comprises 33.3g/L of corn starch, 4g/L of peptone, 3.5mg/L of manganese chloride, 8g/L of urea, 1.0g of cyclamen extract in example 3 is dissolved in 2000ml of culture medium, and the sterilization is carried out at 121 ℃ for 20min. According to the pH value of the initial fermentation liquor of 6.0, the liquid loading quantity of 70 (v/v), the inoculation quantity of 8 percent, the culture temperature of 35 ℃, the rotation speed of a shaking table of 200r/min, the culture time of 120 hours, and the fermentation culture, and stopping, thus obtaining the fermentation organic matter. Crushing the fermented organic matters in 80kHz ultrasonic for 10min, and centrifuging at 8000rpm for 30min to obtain final high-content cyclamen saponin fermentation broth. Filtering the high content cyclamen saponin fermentation broth with gauze, and measuring its saponin content 8.5264mg/mL.

Examples 5 to 8:

the difference from example 1 is the composition ratio of the culture medium, the fermentation conditions, the ultrasonic frequency and the crushing time, and the specific reference is shown in Table 1.

Example 9-example 11:

the same composition ratio of the medium as in example 3 and the same fermentation conditions as in example 3 were different in the frequency of ultrasonication, as shown in Table 1.

Example 12:

the same composition ratio and fermentation conditions as in example 3 were used, except that the fermentation broth containing cyclamen saponin was obtained by direct centrifugation without ultrasonication, as shown in Table 1.

Example 13:

the difference from example 3 is that urea is not contained in the medium, peptone is used only as the main nitrogen source, and the other conditions are the same as those of example 3, specifically shown in Table 1.

Example 14:

the difference from example 3 is that peptone is not contained in the medium, urea is used only as the main nitrogen source, and other conditions are the same as in example 3, specifically shown in Table 1.

Example 15:

the saponin content of the extract of cyclamen without fermentation is shown in Table 1.

TABLE 1 Saponin content in cyclamen fermentation broth

As can be seen from the above Table 1, the differences between the examples 2-8 and 1 are the different composition ratio of the culture medium, the fermentation conditions are different, the optimal fermentation conditions of the example 3 are finally determined, the corn starch is 40.0g/L, the peptone is 6.7g/L, the manganese chloride is 5mg/L, the urea is 4g/L, the pH of the initial fermentation liquor is 5.0, the liquid loading amount is 30 (v/v), the inoculation amount is 4%, the culture temperature is 30 ℃, the shaking table rotation speed is 200r/min, the culture time is 144h, the fermentation culture is stopped, and the fermentation product is obtained.

Examples 9 to 11, which are identical in the composition ratio of the medium of example 3 and the fermentation conditions, are identical in the difference from example 3 in the frequency of ultrasonic disruption, and the ultrasonic cavitation is enhanced due to the increase of the ultrasonic frequency, and cavitation bubbles generated inside the cells continuously shrink and expand the cells, and the cells continuously grow until they are ruptured to a certain extent. Under the ultrasonic frequency treatment of 80-100K Hz, the cell structure has larger cell distortion degree due to the increase of the ultrasonic frequency, and even part of cells are broken, so that the dissolution of intracellular saponins is further increased. In comparison, 80kHz sonication resulted in a smaller degree of cell disruption than 100kHz sonication. However, as the ultrasonic frequency increases, cells start to be gradually broken, and under the action of centrifugation, the broken cells are likely to be blocked in the intercellular spaces, so that the dissolution rate of the saponins after centrifugation is reduced as the ultrasonic frequency increases to a certain extent, and therefore, the ultrasonic frequency treatment of 80-100k Hz is more suitable.

Example 12: the same composition ratio and fermentation conditions as those of the culture medium of example 3 were used, and the difference from example 3 was that the high-content cyclase saponin broth was obtained by direct centrifugation without ultrasonication. Ultrasonic extraction has a greater effect on the dissolution of intracellular saponins than examples 1-11. Therefore, ultrasonication is a necessary step to obtain a high content of cyclamen saponin broth.

Example 13: the difference from example 3 is that urea is not contained in the medium, peptone is used only as the main nitrogen source, and the other conditions are the same as those of example 3. Example 14: the difference from example 3 is that peptone is not contained in the medium, urea is used only as the main nitrogen source, and other conditions are the same as in example 3. From examples 13 and 14, it can be seen that the utilization of inorganic nitrogen urea by cyclamen endophyte Bacillus amyloliquefaciens is very weak, and that the culture medium using peptone as the main nitrogen source is helpful for the growth of cyclamen endophyte Bacillus amyloliquefaciens, but we have studied the extracellular saponin content. The inventors have unexpectedly found that the addition of urea to a peptone-containing medium not only satisfies the growth of cells, but also promotes the transport of intracellular saponins to the outside of the cells. Peptone is used as a main nitrogen source, the pH is reduced after the growth and metabolism of microorganisms, the cell membrane density is increased, the permeability is reduced, and the pH is not reduced but is increased after the use of urea, so that the transport of saponins is facilitated.

EXAMPLE 16 thin layer chromatography

Taking cyclamen fermented liquid fermented in example 3 and example 13 (culture medium without adding urea), concentrating under reduced pressure to remove most of water, uniformly mixing concentrated liquid and absolute ethyl alcohol according to the proportion of 1:25, putting into a water bath kettle at 80 ℃ to extract for 4 hours, collecting extract, centrifuging, collecting filtrate, concentrating under reduced pressure to remove absolute ethyl alcohol, and freeze-drying to obtain crude saponin (A) with urea and crude saponin (B) without urea in example 3 respectively.

Dissolving 0.25g of the above A and B with a small amount of water, extracting with n-butanol for 3 times, concentrating under reduced pressure to dry, dissolving with methanol, dissolving oleanolic acid as control (C) with methanol, activating silica gel sheet, sampling with capillary, and developing to dissolve into chloroform: methanol: ethyl acetate: the water ratio is 25:10:5:2, and the unfolding effect is shown in figure 1.

As can be seen from FIG. 1, the crude saponin (A) with urea added and the crude saponin (B) without urea added medium were the same as the most of the saponin components of oleanolic acid as control (C).

Example 17: DPPH activity eliminating saponin

DPPH radical scavenging Activity was as described in Chun-Lin Ye et al. To 4mL of a 0.1mM DPPH methanol solution were added 0.1, 0.2, 0.3, 0.4, 0.5, 0.6mg/ml crude saponin (A) added with urea and crude saponin (B) not added with urea medium, and the reaction was carried out in the dark for 30min. Absorbance was measured at 517 nm. Vc is used as a positive control. All systems were repeated three times. The DPPH scavenging activity is calculated as follows:

clearance activity (%) = (OD _{Blank space} -(OD _{Treatment group} -OD _{Sample of} ))/OD _{Blank space} ×100％

As can be seen from FIG. 2, the clearance of DPPH with increasing concentration of crude saponin (A) added with urea and crude saponin (B) without urea in the culture medium is also increased, and the clearance of crude saponin (A) added with urea is obviously larger than that of crude saponin (B) without urea in the culture medium, and when the addition amount of the crude saponin (A) and the crude saponin (B) added with urea is 0.3mg/ml, the clearance of crude saponin (A) added with urea is more stable with increasing concentration, and the clearance of crude saponin (B) without urea in the culture medium is 65%. The clearance of the crude saponin (A) added with urea before 0.2mg/ml compared with vitamin C is basically the same as that of vitamin C, reaches 80%, and the clearance of the vitamin C only exceeds the clearance of the crude saponin (A) added with urea after the addition amount is more than 0.2 mg/ml.

Example 18: anti-aging Activity assay

The mouse embryo fibroblasts (NIH-3T 3) used in this experiment were cultured in DMEM complete medium with 10% FBS (fetal bovine serum and 1% PS (penicillin-streptomyin)) and containing 5% CO at 37 ℃ ₂ Culturing in an incubator. Will be 1X 10 ⁵ adding/mL NIH-3T3 cell suspension into 96-well plate, culturing for 24 hr, adding high-content cyclamen saponin fermentation liquid with different concentrations into 96-well plate, pre-treating for 1 hr, and adding sublethal concentration of H ₂ O ₂ (600. Mu.M) treating NIH-3T3 cells for 5h, and finally analyzing the cell (NIH-3T 3) viability of the mouse fibroblast cells by using a WST (water-soluble cytotoxicity detection kit), and measuring the absorbance at 450 nm. All reaction systems were repeated three times. As shown in FIG. 3, in the anti-aging activity, control group 1 was not H-passed ₂ O ₂ The activity of the treated mouse embryo fibroblast (NIH-3T 3) is basically normal as can be seen in the figure, the control group 2 is a blank experiment group, and the fermentation liquor with high content of cyclase saponin is not added, but the fermentation liquor is subjected to H ₂ O ₂ Treatment, we can see that the cell viability of the blank group 2 is significantly reduced; the cell viability of the cyclamen fermentation broth of examples 3 to 5 was significantly higher than that of control group 2 when 0.2, 0.4, 0.6, 0.8, 1.0mg/ml was added, and the cell viability of example 3 was 95.67% very close to that of the normal group when cyclamen fermentation broth was added in an amount of 0.8 mg/ml. It is explained that fermentation of the extract of cyclamen with the endophyte bacillus amyloliquefaciens can increase the anti-aging activity of the cyclamen extract.

Example 19: anti-aging toning lotion for cyclamen fermentation liquor

The cyclamen fermentation broth in example 1 is used for preparing anti-aging toning lotion, and the formula is as follows:

composition of the components	Content (mass%)
		Deionized water	82.70％
EXAMPLE 1 Cyclamen fermentation broth	10％
		Glycerol	3％
Butanediol (butanediol)	4％
		Potassium sorbate	0.30％

Adding deionized water into an emulsifying pot, heating to 85 ℃, adding butanediol into the deionized water in advance, heating to 85-90 ℃, preserving heat for 30min, cooling to below 45 ℃, adding the cyclamen fermentation broth of example 1, uniformly mixing, and adding potassium sorbate to obtain the cyclamen fermentation broth toning lotion.

Example 20: emulsion for resisting aging of cyclamen fermentation liquor

The cyclamen fermentation broth of example 3 was used to prepare an anti-aging emulsion having the following formulation:

composition of the components	Content (mass%)
		Deionized water	73.70％
Glycerol	4％
		Butter tree (BUTYROSPERMUM PARKII) fruit oil	5％
EXAMPLE 3 Cyclamen fermentation broth	15％
		Biological sugar gum-1	2％
Methyl paraben	0.30％

Adding deionized water and glycerol into an emulsifying pot, heating to 85deg.C, preheating butter oil of Butyrospermum parkii (BUTYROSPERMUM PARKII) to 85deg.C, adding into the emulsifying pot, homogenizing and emulsifying (3200R/min) for 5min, stirring (25R/min), vacuumizing to 0.4 atm, cooling water slowly to 45deg.C, adding dissolved biological gum-1 and methyl hydroxybenzoate, stirring for 15min to completely uniformity, discharging, and standing.

Example 21:

recruiting 31 cases of female subjects with standard inclusion age of 34-55 years old, healthy female subjects with non-sensitive facial skin (through a sensitive skin screening questionnaire), all facial skin types (including oiliness, mixed oiliness, normal, mixed dryness, dryness) without problems of large-area or long-term frequent growth of vaccinia, acne marks, desquamation, stinging and the like, self-evaluating facial skin to be rough, loose, lack of elasticity, facial forehead wrinkles (head lines) to be more than or equal to 3 (according to Asian skin aging pattern Vol.02 (English edition), P32-33), facial side eye wrinkles (fish tail lines) to be more than 2 (refer to Asian skin aging pattern Vol.02 (English edition), P40-41), facial side nose and lip wrinkles (French lines) to be 2-5 (refer to Asian skin aging Vol.02 (edition), P54-55), testing area skin to be free from obvious redness, skin damage, scars and the like, and all contents of a person's can read and understand self-agreements.

After the face of a test subject is cleaned every day, firstly, using cyclamen to ferment anti-aging toning lotion of the liquid, then using cyclamen to ferment anti-aging emulsion of the liquid, controlling the temperature and the relative humidity, testing the skin water loss rate (test instrument tewamet TM 300), the skin elasticity (test instrument Cutometer dual MPA 580), the facial image acquisition and the skin texture analysis (test instrument Visioscan VC20 plus), the facial image acquisition and the skin wrinkle analysis (test instrument Primos-CR) of the face of the test subject after the face of the test subject is slightly cleaned, and performing test analysis: after 42 days of application of the sample, the number of skin wrinkles was reduced by 20%, the average volume of skin wrinkles was reduced by 20.73%, the average area of skin wrinkles was reduced by 17.49%, and the average length of skin wrinkles was reduced by 15.67%.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.

Claims

1. A method for obtaining a high content cyclamen saponin broth, comprising the steps of:

(1) Taking cyclamen endophyte Bacillus amyloliquefaciens as a strain, and fermenting in a fermentation broth containing urea and peptone as main nitrogen sources by taking cyclamen extract as a substrate to obtain a fermented organic matter;

the fermentation broth contains: 40.0g/L of corn starch, 6.7g/L of peptone, 5mg/L of manganese chloride, 4g/L of urea, 5.0 of initial fermentation liquor pH and 30 (v/v) of liquid loading, wherein the inoculation amount of the cyclamen endophyte Bacillus amyloliquefaciens is 4%, the culture temperature is 30 ℃, the rotation speed of a shaking table is 200r/min, the culture time is 144 hours, and fermentation culture is stopped, so that a fermented organic matter is obtained;

(2) Ultrasonically crushing the fermented organic matters, centrifuging and filtering to obtain a high-content cyclamen saponin fermentation liquid;

the ultrasonic crushing frequency is 80-100kHZ.

2. The cyclamen saponin broth of claim 1 prepared by the method of obtaining cyclamen saponin broth with high content.

3. The cyclamen saponin fermentation broth of claim 2, wherein the cyclamen saponin content in the cyclamen saponin fermentation broth is not less than 16.6mg/mL.

4. Use of a cyclamen saponin broth as claimed in claim 2 as an antioxidant active ingredient or an anti-aging ingredient for the preparation of cosmetics.

5. The use according to claim 4, wherein the amount of the high content cyclamen saponin broth in the cosmetic is 10-20wt% based on the total weight of cyclamen saponin broth as raw material of the cosmetic.