Calendula officinalis fermented product as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a calendula fermentation product. The preparation method comprises the step of fermenting calendula with saccharomycetes and lactobacillus in sequence. According to the invention, the calendula is subjected to secondary fermentation by adopting a method of firstly fermenting by using the saccharomycetes and then fermenting by using the lactobacillus, and the calendula is fermented through the synergistic effect of the two bacteria, so that the obtained calendula fermented product has the comprehensive effects of moisturizing, repairing and relieving.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation and cosmetics, and in particular relates to a marigold fermentation product and a preparation method and application thereof. Background Art

[0002] Calendula belongs to the Asteraceae family and is also known as marigold. Calendula is rich in vitamins, especially vitamin A and vitamin C. Calendula petals have beauty functions. The flowers contain carotenoids, lycoperdin, butterfly umexin, rubroxanthin, volatile oils, resins, mucilage, malic acid, etc. The ancient Egyptians believed that marigolds could delay aging, and the Indians revered it as a sacred flower. In the Middle Ages, people used marigolds to heal wounds and fight against plague and the Black Death.

[0003] Common extraction methods for calendula include hydrolysis extraction and microwave extraction, and high temperature can easily cause the inactivation of active ingredients. Therefore, in the process of water extraction of calendula, it is necessary to break the cell wall by heat energy, and ensure that the temperature is not too high to avoid the loss of the activity of the active ingredients. The conditions of hydrolysis extraction of calendula are harsh and not suitable for large-scale production. Microwave extraction changes the permeability of the cell wall by the transfer of heat energy at a specific wavelength, which can improve the extraction efficiency of calendula to a certain extent, but this method is only suitable for laboratory preparation of small samples, and the microwave extractor cannot operate continuously. Continuous operation will generate a lot of heat, which will cause the inactivation of active substances in calendula. Microwave extraction is not suitable for large-scale production in enterprises. In terms of cosmetic applications, the above extraction methods are all likely to cause the loss of active substances in calendula, making it difficult for calendula extracts to achieve skin care effects. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a fermented marigold. The obtained fermented marigold contains active substances and has moisturizing, repairing and soothing effects.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing a fermented product of marigold, comprising the following steps:

[0007] (1) using yeast to perform a first fermentation in a medium containing marigold to obtain a fermentation liquid A;

[0008] (2) performing a second fermentation on the fermentation liquid A using lactobacillus to obtain a fermentation liquid B;

[0009] (3) sterilizing and filtering the fermentation liquid B in sequence to obtain a marigold fermentation product.

[0010] The invention performs secondary fermentation on marigold by first fermenting with yeast and then with lactobacillus, and the obtained marigold fermentation product has the comprehensive effects of moisturizing, repairing and soothing through the synergistic fermentation of the two bacteria.

[0011] As a preferred embodiment of the first aspect, the inoculation amount of yeast seed liquid during yeast fermentation in step (1) is 3-7% (v / v); the inoculation amount of lactobacillus seed liquid during lactobacillus fermentation in step (2) is 2-4% (v / v).

[0012] As a preferred embodiment of the first aspect, the inoculation amount of yeast seed liquid during yeast fermentation can be 3%, 4%, 5%, 6%, 7% or any value therebetween; the inoculation amount of lactobacillus seed liquid during lactobacillus fermentation can be 2%, 3%, 4% or any value therebetween.

[0013] As a preferred embodiment of the first aspect, the yeast seed solution in step (1) has a cell concentration of 10 7 -10 9 CFU / mL, the bacterial concentration of the lactobacillus seed solution in step (2) is 10 7 -10 9 CFU / mL.

[0014] As a preferred embodiment of the first aspect, the yeast seed solution may have a cell concentration of 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL or any value in between.

[0015] As a preferred embodiment of the first aspect, the bacterial concentration of the lactobacillus seed solution can be 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL or any value in between.

[0016] As a preferred embodiment of the first aspect, in the step (1), the temperature of the first fermentation is 25-30°C, the culture time is 48-96h, the stirring paddle speed is 100-180r / min, and the sterile air supply volume is 0.3-0.5vvm; in the step (2), the temperature of the second fermentation is 34-40°C, the culture time is 36-60h, and the fermentation method is anaerobic fermentation.

[0017] As a preferred embodiment of the first aspect, the culture medium containing marigold in step (1) comprises marigold powder, a carbon source, a nitrogen source, an inorganic salt and water.

[0018] As a preferred embodiment of the first aspect, the carbon source is at least one of glucose, fructose, and α-glucan.

[0019] As a preferred embodiment of the first aspect, the nitrogen source is peptone.

[0020] As a preferred embodiment of the first aspect, the inorganic salt includes sulfate and / or phosphate.

[0021] As a preferred embodiment of the first aspect, the sulfate is magnesium sulfate.

[0022] As a preferred embodiment of the first aspect, the phosphate is at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0023] As a preferred embodiment of the first aspect, the culture medium containing calendula comprises, by mass percentage: 0.5-2% calendula powder, 2.2-4.5% carbon source, 0.8-1.5% nitrogen source, 0.4-1% inorganic salt and balance water; preferably, the mass percentage of calendula powder can be 0.5%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2% or any value therebetween; the mass percentage of the carbon source can be 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5% or any value therebetween; the mass percentage of the nitrogen source can be 0.8%, 1%, 1.2%, 1.3%, 1.4%, 1.5% or any value therebetween; the mass percentage of the inorganic salt can be 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or any value therebetween.

[0024] As a preferred embodiment of the first aspect, the carbon source is a composite carbon source of glucose and α-glucan.

[0025] As a preferred embodiment of the first aspect, the yeast in step (1) is Candida krusei. The present invention screens out the most suitable yeast for fermenting marigold from a large number of yeasts through a large number of strain screening tests, and the yeast is Candida krusei. Compared with other yeasts, the marigold fermentation product obtained by fermenting Candida krusei has the best effect. Therefore, Candida krusei is selected as the yeast fermentation strain of the present invention.

[0026] As a preferred embodiment of the first aspect, the lactobacillus in step (2) is Bifidobacterium adolescentis. The present invention screens out the most suitable lactobacillus for fermenting marigold from numerous lactobacilli through a large number of strain screening tests, and Bifidobacterium adolescentis is selected. Compared with other lactobacilli, the marigold fermentation obtained by fermentation with Bifidobacterium adolescentis has the best effect. Therefore, Bifidobacterium adolescentis is selected as the lactobacillus fermentation strain of the present invention, and it is found that Candida krusei and Bifidobacterium adolescentis have a synergistic effect, which can further improve the comprehensive effect of the fermentation.

[0027] As a preferred embodiment of the first aspect, the sterilization temperature in step (3) is 80-95°C, and the sterilization time is 15-30 minutes.

[0028] As a preferred embodiment of the first aspect, the filtration in step (3) is filtration using a diatom filter.

[0029] In a second aspect, the present invention provides a marigold fermented product, which is produced by the preparation method of the first aspect. The marigold fermented product has the comprehensive effects of moisturizing, repairing and soothing.

[0030] In a third aspect, the present invention provides a cosmetic composition comprising the marigold fermentation product according to the second aspect.

[0031] In a fourth aspect, the present invention provides the use of Candida krusei and / or Bifidobacterium adolescentis in the preparation of a fermented product of marigold. Using Candida krusei and / or Bifidobacterium adolescentis to ferment marigold can obtain active substances with skin care functions, so that the fermented product has a comprehensive effect of moisturizing, repairing and soothing. At the same time, there is a synergistic effect between the two, which can further improve the comprehensive effect of the fermented product.

[0032] In a fifth aspect, the present invention provides a culture medium for marigold fermentation, which comprises, by mass percentage, 0.5-2% marigold powder, 2.2-4.5% carbon source, 0.8-1.5% nitrogen source, 0.4-1% inorganic salt and the remainder water.

[0033] As a preferred embodiment of the fifth aspect, the culture medium containing marigold includes, by mass percentage, 1% marigold powder, 3% glucose, 0.2% α-glucan, 1% tryptone, 0.4% magnesium sulfate, 0.3% potassium dihydrogen phosphate and the remainder water.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention adopts yeast and lactobacillus to synergistically ferment to obtain marigold fermentation product. The two different fermentation steps can effectively extract the active substances in marigold. In addition, in addition to marigold, the culture medium also contains nutrients required for the growth of yeast and lactobacillus. The active substances produced by the metabolism of yeast and lactobacillus are mixed with the active substances in marigold, which has a good skin care effect.

[0036] (2) The fermentation and extraction conditions of the present invention are mild, which avoids high-temperature extraction, thereby reducing the problem of thermal inactivation of active substances, and the active substances can be effectively retained in the marigold fermentation product.

[0037] (3) The fermentation process of the present invention requires simple instruments and equipment, and the fermentation process does not need to be subject to strict high temperature and high pressure conditions. The fermentation and extraction process does not use organic solvents, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the marigold of the present invention;

[0039] Figure 2 This is a schematic diagram of the marigold fermentation product prepared by the preparation method of Example 1 of the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the examples. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit its content. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, if not otherwise specified, can all be obtained through commercial channels. Unless otherwise specified, the percentages mentioned in the present invention are all mass percentages.

[0041] The Candida krusei used in the present invention is numbered CICC 1683 and the Bifidobacterium adolescentis is numbered CICC 6070, both of which are from the China Industrial Microbiological Culture Collection Center.

[0042] The Saccharomyces cerevisiae used in the present invention is numbered BNCC 336054 and is derived from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., the Lactobacillus brevis used in the present invention is numbered CICC 20269 and is derived from Beijing Yuwei Technology Co., Ltd., the Bifidobacterium animalis subsp. lactis used in the present invention is numbered ATCC 700541 and is derived from Shanghai Xuanke Biotechnology Co., Ltd., the Candida maltosa used in the present invention is numbered BMZ111806, the Candida rugosa is numbered BMZ118960, the Candida carpophila is numbered B98036, the Pichia fermentans is numbered BMZ339612, and the lactic acid yeast Kluyveromyces lactis numbered B80850, Bifidobacterium infantis numbered BMZ20090, Bifidobacterium numbered BMZ118374, Bifidobacterium longum BMZ132587, and Bifidobacterium pseudoseudocatenulatum numbered BMZ014603 were all from Ningbo Mingzhou Biotechnology Co., Ltd.

[0043] The Saccharomyces cerevisiae boulardii used in the present invention was purchased from Shanghai Collection Biotechnology Center with the serial number SHBCCD25151.

[0044] In the present invention, the cell concentration of the seed solution of each strain is 10 7 -10 9 CFU / mL, the preparation methods of various sub-liquids can be prepared by the common seed liquid preparation methods in the prior art, and the present invention does not make any specific limitation on the preparation method of the seed liquid.

[0045] The preparation method of the culture medium of the present invention is as follows: each material in the culture medium is added in sequence according to a proportion and fully dissolved in water, sterilized at 115° C. for 30 minutes, and then cooled for standby use.

[0046] Example 1

[0047] This embodiment provides a method for preparing a fermented product of marigold, which specifically comprises the following steps:

[0048] (1) Add Candida krusei seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into a medium containing marigold at an inoculum size of 5% (v / v), and the first fermentation was carried out at 28°C, a stirring blade speed of 140 r / min, and a sterile air flow rate of 0.4 vvm for 72 hours to obtain fermentation liquid A;

[0049] (2) Add Bifidobacterium adolescentis seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into fermentation liquid A at an inoculum amount of 3% (v / v), and a second fermentation was carried out at 37°C in an anaerobic fermentation manner for 48 hours to obtain fermentation liquid B;

[0050] (3) The fermentation liquid B was kept at 90° C. for 20 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0051] Wherein, the culture medium containing marigold includes, by mass percentage, 1% marigold powder, 3% glucose, 0.2% α-glucan, 1% tryptone, 0.4% magnesium sulfate, 0.3% potassium dihydrogen phosphate and the balance water.

[0052] The preparation method of marigold powder is as follows: dry marigolds are placed in a powder grinder and crushed, and the powder is passed through a 200-mesh sieve to obtain the lower layer of marigold powder. The preparation methods of marigold powder in other embodiments and comparative examples are the same as those in embodiment 1, and will not be described in detail later.

[0053] Example 2

[0054] This embodiment provides a method for preparing a fermented product of marigold, which specifically comprises the following steps:

[0055] (1) Add Candida krusei seed solution (cell concentration is about 10 7 CFU / mL) was inoculated into a medium containing marigold at an inoculum size of 7% (v / v), and the first fermentation was carried out at 30°C, a stirring blade speed of 100 r / min, and a sterile air flow rate of 0.5 vvm for 48 hours to obtain a fermentation liquid A;

[0056] (2) Add Bifidobacterium adolescentis seed solution (cell concentration is about 10 7 CFU / mL) was inoculated into fermentation liquid A at an inoculation rate of 4% (v / v), and a second fermentation was carried out at 40°C in an anaerobic fermentation manner for 60 hours to obtain fermentation liquid B;

[0057] (3) The fermentation liquid B was kept at 80° C. for 30 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0058] Wherein, the culture medium containing marigold includes, by mass percentage, 0.5% marigold powder, 3.5% glucose, 1% α-glucan, 0.8% tryptone, 0.2% magnesium sulfate, 0.2% dipotassium hydrogen phosphate and the balance water.

[0059] Example 3

[0060] This embodiment provides a method for preparing a fermented product of marigold, which specifically comprises the following steps:

[0061] (1) Add Candida krusei seed solution (cell concentration is about 10 9 CFU / mL) was inoculated into a medium containing marigold at an inoculum size of 3% (v / v), and the first fermentation was carried out at 28°C, a stirring blade speed of 180 r / min, and a sterile air flow rate of 0.3 vvm for 96 hours to obtain a fermentation liquid A;

[0062] (2) Add Bifidobacterium adolescentis seed solution (cell concentration is about 10 9 CFU / mL) was inoculated into fermentation liquid A at an inoculum amount of 2% (v / v), and a second fermentation was carried out at 35°C in an anaerobic fermentation manner for 36 hours to obtain fermentation liquid B;

[0063] (3) The fermentation liquid B was kept at 95° C. for 15 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0064] Wherein, the culture medium containing marigold includes, by mass percentage, 2% marigold powder, 1.8% glucose, 0.4% α-glucan, 1.5% soy peptone, 0.3% magnesium sulfate, 0.7% sodium dihydrogen phosphate and the balance water.

[0065] Example 4

[0066] The difference between Example 4 and Example 1 is that in Example 4, the 3% glucose and 0.2% α-glucan in the culture medium containing marigold in step (1) are changed to 3.2% α-glucan, and the remaining steps and parameters are the same as in Example 1.

[0067] Example 5

[0068] The difference between Example 5 and Example 1 is that in Example 5, the 3% glucose and 0.2% α-glucan in the culture medium containing marigold in step (1) are changed to 1.6% glucose and 1.6% α-glucan, and the remaining steps and parameters are the same as in Example 1.

[0069] Example 6

[0070] The difference between Example 6 and Example 1 is that in Example 6, the inoculation volume ratio of the Candida krusei seed solution in step (1) is changed from 5% (v / v) to 7% (v / v).

[0071] Comparative Example 1

[0072] The difference between Comparative Example 1 and Example 1 is that step (2) of Example 1 is deleted, and the marigold fermented product of Comparative Example 1 is obtained only by fermenting Candida krusei seed liquid, and the rest is the same as Example 1, and the specific steps are as follows:

[0073] (1) Add Candida krusei seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into a medium containing marigold at an inoculation volume ratio of 5%, and fermented at 28°C, a stirring blade speed of 140r / min, and a sterile air flow rate of 0.4vvm for 72h to obtain a fermentation liquid;

[0074] (2) The fermentation liquid was kept at 90° C. for 20 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0075] Wherein, the culture medium containing marigold is the same as that in Example 1.

[0076] Comparative Example 2

[0077] The difference between Comparative Example 2 and Example 1 is that step (1) of Example 1 is deleted, and the marigold fermented product of Comparative Example 2 is obtained only by fermenting Bifidobacterium adolescentis seed liquid, and the rest is the same as Example 1, and the specific steps are as follows:

[0078] (1) Add Bifidobacterium adolescentis seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into a medium containing marigold at an inoculum amount of 3% (v / v), and fermented at 37°C in an anaerobic fermentation manner for 48 hours to obtain a fermentation liquid;

[0079] (2) The fermentation liquid was kept at 90° C. for 20 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0080] Wherein, the culture medium containing marigold is the same as that in Example 1.

[0081] Comparative Example 3

[0082] The difference between Comparative Example 3 and Example 1 is that the order of steps (1) and (2) of Example 1 is reversed, Bifidobacterium adolescentis is used for the first fermentation, and then Candida krusei is used for the second fermentation. The rest is the same as Example 1. The specific steps are as follows:

[0083] (1) Add Bifidobacterium adolescentis seed solution (cell concentration is about 10 8CFU / mL) was inoculated into a medium containing marigold at an inoculum amount of 3% (v / v), and the first fermentation was carried out at 37°C by anaerobic fermentation for 48 hours to obtain fermentation liquid A;

[0084] (2) Add Candida krusei seed solution (cell concentration is about 10 8 CFU / mL) was inoculated into fermentation liquid A at an inoculum amount of 5% (v / v), and the second fermentation was carried out at 28°C, a stirring blade speed of 140r / min, and a sterile air flow rate of 0.4vvm for 72h to obtain fermentation liquid B;

[0085] (3) The fermentation liquid B was kept at 90° C. for 20 min for sterilization, and then filtered through a diatom filter to obtain a marigold fermentation product.

[0086] Wherein, the culture medium containing marigold is the same as that in Example 1.

[0087] Comparative Example 4

[0088] The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, the Candida krusei in step (1) is replaced by Saccharomyces cerevisiae (the cell concentration of the Saccharomyces cerevisiae seed liquid is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0089] Comparative Example 5

[0090] The difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, the Candida krusei in step (1) is replaced by Candida maltosa (the cell concentration of the Candida maltosa seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0091] Comparative Example 6

[0092] The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, the Candida krusei in step (1) is replaced by Candida rugosa (the cell concentration of the Candida rugosa seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0093] Comparative Example 7

[0094] The difference between Comparative Example 7 and Example 1 is that in Comparative Example 7, the Candida krusei in step (1) is replaced by Candida aurantifolia (the cell concentration of the Candida aurantifolia seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0095] Comparative Example 8

[0096] The difference between Comparative Example 8 and Example 1 is that in Comparative Example 8, the Candida krusei in step (1) is replaced by Saccharomyces boulardii (the cell concentration of the Saccharomyces boulardii seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0097] Comparative Example 9

[0098] The difference between Comparative Example 9 and Example 1 is that in Comparative Example 9, the Candida krusei in step (1) is replaced by Pichia pastoris (the cell concentration of the Pichia pastoris seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0099] Comparative Example 10

[0100] The difference between Comparative Example 10 and Example 1 is that in Comparative Example 10, the Candida krusei in step (1) is replaced by lactic acid yeast (the bacterial concentration of the lactic acid yeast seed liquid is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0101] Comparative Example 11

[0102] The difference between Comparative Example 11 and Example 1 is that in Comparative Example 11, the Bifidobacterium adolescentis in step (2) is replaced by Bifidobacterium infantis (the cell concentration of the seed solution of Bifidobacterium infantis is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0103] Comparative Example 12

[0104] The difference between Comparative Example 12 and Example 1 is that in Comparative Example 12, the Bifidobacterium adolescentis in step (2) is replaced by Bifidobacterium bifidum (the cell concentration of the Bifidobacterium bifidum seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0105] Comparative Example 13

[0106] The difference between Comparative Example 13 and Example 1 is that in Comparative Example 13, the Bifidobacterium adolescentis in step (2) is replaced by Bifidobacterium longum (the cell concentration of the Bifidobacterium longum seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0107] Comparative Example 14

[0108] The difference between Comparative Example 14 and Example 1 is that in Comparative Example 14, the Bifidobacterium adolescentis in step (2) is replaced by Bifidobacterium pseudocatenulatum (the cell concentration of the seed solution of Bifidobacterium pseudocatenulatum is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0109] Comparative Example 15

[0110] The difference between Comparative Example 15 and Example 1 is that in Comparative Example 15, the Bifidobacterium adolescentis in step (2) is replaced by Lactobacillus brevis (the cell concentration of the Lactobacillus brevis seed solution is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0111] Comparative Example 16

[0112] The difference between Comparative Example 16 and Example 1 is that in Comparative Example 16, the adolescent Bifidobacterium in step (2) is replaced by animal Bifidobacterium lactis subsp. (the bacterial concentration of the animal Bifidobacterium lactis subsp. seed liquid is about 10 8 CFU / mL), and the rest is the same as in Example 1.

[0113] Comparative Example 17

[0114] The difference between Comparative Example 17 and Example 1 is that in Comparative Example 17, the 3% glucose and 0.2% α-glucan in the culture medium containing marigold in step (1) of Example 1 are replaced with 3.2% sucrose, and the remaining steps and parameters are the same as in Example 1.

[0115] Comparative Example 18

[0116] The difference between Comparative Example 18 and Example 1 is that in Comparative Example 18, 1% trypsin in the culture medium containing marigold in step (1) of Example 1 is replaced with 1% beef extract, and the remaining steps and parameters are the same as in Example 1.

[0117] Comparative Example 19

[0118] The difference between Comparative Example 19 and Example 1 is that in Comparative Example 19, the amount of sterile air introduced in step (1) of Example 1 is replaced by 1.0 vvm, and the remaining steps and parameters are the same as Example 1.

[0119] Comparative Example 20

[0120] This comparative example provides a calendula extract, and the preparation method is as follows:

[0121] 1% of marigold and 99% of water were mixed evenly, heated and extracted at 60°C for 12 hours, then heated to 90°C and kept for 20 minutes, and filtered through a diatom filter to obtain a marigold extract. The difference between Comparative Example 20 and Example 1 is that Comparative Example 20 uses a water extraction method to extract the active substances of marigold.

[0122] Effect Example 1 Moisturizing Effect Test

[0123] Experimental principle: The main structural basis of the skin barrier function is the stratum corneum formed by the most superficial keratinocytes, lipids between keratinocytes, natural protective factors (NMF), etc. Keratinocytes undergo nuclear division, differentiation, degeneration and disappearance, passing through the basal layer, spinous cell layer, and granular cell layer, and finally become the stratum corneum of the outermost layer of the epidermis, forming its unique multi-layer structure. Keratinocytes are stacked in the continuous matrix of intercellular lipids, forming a classic "brick wall" structural system.

[0124] Aquaporin (AQP), also known as water pores, is a protein located on the cell membrane (intrinsic membrane protein) that can form "pores" on the cell membrane to control the flow of water in and out of the cell. Aquaporin 3 (AQP3) is expressed in the epidermis and basal layer of the skin. It is a natural moisturizing factor that can promote the structural water circulation of organelles, ensure the continuous water content of the epidermis, regulate the water balance of keratinocytes, maintain the hydration function of the skin, promote skin absorption and metabolism, and play a key role in skin hydration and moisturizing. By experimentally detecting the effect of samples on the expression of the AQP3 gene in keratinocytes, it is possible to evaluate whether the samples have the effect of promoting moisturizing.

[0125] The higher the AQP3 gene expression, the better the sample's moisturizing effect, and vice versa.

[0126] Test sample: the fermented marigolds of Examples 1-6 and Comparative Examples 1-20 were prepared into a 1% by mass solution using DMEM culture medium containing 10% serum.

[0127] Test steps: HaCaT human keratinocytes were inoculated in a 6-well plate (200,000 cells / well) and cultured in an incubator at 37°C for 24 hours. After the culture was completed, the original culture medium in the well was discarded, and DMEM culture medium containing 10% serum (referred to as culture medium) was added to the negative control group. Cell culture medium containing the test sample was added to each well of the sample group and cultured for 24 hours. After the culture was completed, the supernatant was discarded. The expression of AQP3 mRNA in HaCaT human keratinocytes was detected by qRT-PCR technology. The relative expression of the remaining groups was calculated based on the expression of AQP3 mRNA in the negative control group. Four parallel samples were set for each group, and the test results were averaged. Using the negative control group as a reference, the significant difference of each group of test samples was calculated using SPSS statistical analysis software. The test results are shown in Table 1, and the significant difference analysis is shown in Table 2.

[0128] Table 1: Moisturizing effect of fermented products of Examples and Comparative Examples

[0129]

[0130]

[0131] The analysis of significant differences is shown in the following table:

[0132] Table 2: Analysis of significant differences

[0133] p>0.05 p≤0.05 p≤0.01 p≤0.001 No statistically significant difference There is a statistical difference There is a statistically significant difference There is a statistically significant difference

[0134] The above test results show that the marigold fermented product of the embodiment of the present invention can increase the relative expression of the cell AQP3 gene, indicating that the marigold fermented product of the embodiment of the present invention can promote the expression of aquaporin 3 in the basement membrane, and show the effect of regulating the water balance of stratum corneum cells and maintaining the hydration function of the skin. The marigold fermented product has a moisturizing effect, while the moisturizing effect of the comparative example is worse than that of the embodiment.

[0135] Effect Example 2 Repair Effect Test

[0136] Test principle: Silent information regulator Sirtuin 1 (SIRT1) is a group of type III protein deacetylases, which is considered to be a new anti-aging protein involved in the regulation of cell aging and inflammation. SIRT1 levels and activity are reduced in cell aging caused by oxidative stress. SIRT1-mediated anti-inflammatory aging mechanisms are related to inflammation, premature cell aging, telomere loss, aging-related secretory phenotypes and regulation of DNA damage response. SIRT1 activation has the potential to attenuate cellular oxidative aging by downregulating oxidative stress-mediated cell aging.

[0137] In this experiment, H2O2-induced L929 cells were used as an in vitro oxidative damage cell model. RNA was extracted from the cells using an RNA extraction kit and reverse transcribed into cDNA. Finally, fluorescent quantitative PCR was performed to detect the expression level of the silent information regulator (SIRT1) of the cell gene, thereby evaluating the repair effect of the test sample.

[0138] The higher the expression level of SIRT1, the faster the cell oxidation is reduced and the better the repair effect of the test sample.

[0139] Test sample: the fermented products of marigolds of Examples 1-6 and Comparative Examples 1-20 were prepared into a solution with a mass concentration of 0.5% using H-DMEM medium.

[0140] Test steps: Add 2×10 5L929 cells were added with H-DMEM culture medium (referred to as culture medium) and cultured for 24 hours at 37°C, 5% CO2 and saturated humidity. The 6-well plate was taken out, the original culture medium in the well was discarded, and the test sample group and the model control group were added with culture medium containing 0.1μg / mL H2O2 solution as a stimulus and cultured for 1 hour, and then the H2O2 solution was removed. The negative control group was not induced by adding H2O2 solution; the test sample group was added with the test sample, and the negative control group and the model control group were added with culture medium, and cultured in the incubator for 24 hours. The cells in each group were crushed and RNA was collected, reverse transcribed into cDNA, and the expression of SIRT1 gene was determined by fluorescence quantitative PCR. The relative expression of the remaining groups was calculated according to the expression of SIRT1 mRNA in the negative control group. Four parallel samples were set for each group, and the test results were averaged. The model control group was used as a reference, and the significant difference of the test sample group was calculated by SPSS statistical analysis software. The results are shown in Table 3:

[0141] Table 3: Repairing effect of fermented products of Examples and Comparative Examples

[0142]

[0143]

[0144] The above test results show that the marigold fermented product of the embodiment of the present invention can promote the expression of SIRT1 gene in cells, indicating that the marigold fermented product of the embodiment of the present invention can weaken the oxidation of cells. The marigold fermented product shows a repairing effect, while the repairing effect of the comparative example is poor.

[0145] Effect Example 3: Soothing Effect Test

[0146] Test principle: Inflammatory pain caused by skin sensitivity is a reaction produced under the stimulation of various inflammatory and pain-causing factors. The mitochondrial function of skin cells is impaired, lysosomes and lipid membranes are destroyed, and tissue and organ damage is aggravated, so the pain is caused. Prostaglandins (PGs) are a class of strong inflammatory mediators. Among them, prostaglandin E2 (PGE2) has a stronger inflammatory effect, which can induce chemotaxis and aggregation of inflammatory cells, dilate capillaries to increase their permeability, promote inflammatory exudation, cause fever and increase the inflammatory and pain-causing effects of other inflammatory mediators, induce explosive inflammatory cascade reactions, and further enhance and amplify inflammatory reactions. In this experiment, SDS-induced mouse fibroblasts L929 were used as an in vitro sensitive cell model, and the relative expression of PGE2 gene was measured to evaluate whether the test sample has a soothing effect. If the expression of the inflammatory factor PGE2 gene decreases after the test sample is used, it indicates that the test sample can reduce the inflammatory factor PGE2 in the cell, and the test sample has a soothing and anti-allergic effect.

[0147] Test sample: the fermented products of marigolds of Examples 1-6 and Comparative Examples 1-20 were prepared into a solution with a mass concentration of 0.5% using H-DMEM medium.

[0148] Test steps: L929 cells were seeded in 6-well plates (2×10 5 cells), added H-DMEM culture medium (referred to as culture medium), and cultured for 24 hours at 37°C, 5% CO2, and saturated humidity. After the culture was completed, the culture medium was aspirated and discarded, 1 mL of H-DMEM culture medium was added to the blank control group, and 1 mL of 1% SDS solution was added to the model control group and the test sample group to induce modeling for 1 hour, and the culture medium was aspirated and discarded. Subsequently, 1 mL of culture medium was added to the blank control group and the model control group, and 1 mL of test sample was added to the test sample group, and culture was continued for 24 hours. After the culture was completed, the expression of PGE2 mRNA was detected by real-time qPCR. The relative expression multiple of PGE2 gene in the blank control group was F=1, and 2 -ΔΔCT The F value of the model control group and each sample was calculated by the SPSS statistical analysis software, with the model control group as the reference. The test results are shown in Table 4.

[0149] Table 4: Soothing effects of fermented products of Examples and Comparative Examples

[0150]

[0151] The above test results show that after using the marigold fermented product of the embodiment of the present invention, the relative expression of the PGE2 gene is significantly reduced, indicating that the marigold fermented product of the present invention can reduce the level of inflammatory factors in cells and show a soothing effect, while the soothing effect of the comparative example is poor.

[0152] It can be seen from Tables 1-4 that the moisturizing, repairing and soothing effects of Examples 1-6 are better than those of Comparative Examples 1-20, indicating that the fermentation product obtained by sequentially fermenting calendula with Candida krusei and Bifidobacterium adolescentis has better comprehensive moisturizing, repairing and soothing effects.

[0153] Comparative Examples 1 and 2 only use one of the bacteria for fermentation, and Comparative Example 3 replaces the fermentation order of the two bacteria, resulting in poor moisturizing, repairing, and soothing effects of the fermentation products obtained by the methods of Comparative Examples 1-3. This indicates that fermenting Candida krusei and Bifidobacterium adolescentis in a certain order can play a synergistic role and improve the moisturizing, repairing, and soothing effects of the final fermentation product.

[0154] In Comparative Examples 4-16, different yeasts and lactobacilli were used to ferment marigolds, and the moisturizing, repairing, and soothing effects of the fermented products finally obtained were not as good as those in the examples. Due to the diversity of microbial fermentation, the components of the fermented products obtained by fermenting the same substrate with different microorganisms are also different. However, the present invention selects Candida krusei and Bifidobacterium adolescentis from many yeasts and lactobacilli for fermentation of marigolds, and the obtained fermented products have the comprehensive effects of moisturizing, repairing, and soothing. This shows that fermenting marigolds with Candida krusei and Bifidobacterium adolescentis has certain specificity.

[0155] In Comparative Examples 17-19, the types of carbon and nitrogen sources for bacterial fermentation and the ventilation volume of sterile air during yeast fermentation were changed. This shows that the types of carbon and nitrogen sources and sterile air affect the growth and reproduction process of the bacterial strains, and affect the moisturizing, repairing and soothing effects of the final fermentation product. Compared with Example 1, the ventilation volume of Comparative Example 19 is greatly improved. In the early stage of fermentation, Candida krusei multiplies in large quantities, and uses the nutrients in the culture medium to produce a large number of bacteria without separating metabolic active substances. In the later stage of fermentation, Candida krusei will undergo autolysis of bacteria due to large-scale reproduction, further consuming the nutrients in the culture medium, resulting in poor comprehensive moisturizing, repairing and soothing effects of the final product.

[0156] Comparative Example 20 uses water extraction to extract the active substances of marigold to obtain marigold extract. From the above table, it can be seen that the comprehensive effect of the extract is not as good as that of the embodiment. It can be seen that the active substances of the marigold extract prepared by water extraction are not as good as those of the embodiment of the present invention, thus affecting its final efficacy.

[0157] In summary, the present invention screens out two types of bacteria that are most suitable for marigold fermentation, namely Candida krusei and Bifidobacterium adolescentis, and adopts the order of first using yeast fermentation and then using lactobacillus fermentation. The marigold is fermented through the synergistic effect of the two bacteria, and the final marigold fermentation product obtained has the comprehensive effects of moisturizing, repairing and soothing.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. 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 solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a fermented product of marigold, characterized in that: The steps include: (1) using yeast to perform a first fermentation on a medium containing marigolds to obtain a fermentation liquid A; (2) fermenting the fermentation liquid A for a second time using lactobacillus to obtain fermentation liquid B; (3) sterilizing and filtering the fermentation liquid B in sequence to obtain a marigold fermentation product.

2. The preparation method according to claim 1, characterized in that In the step (1), the inoculation amount of the yeast seed liquid during the yeast fermentation is 3-7% (v / v); in the step (2), the inoculation amount of the lactobacillus seed liquid during the lactobacillus fermentation is 2-4% (v / v); the bacterial concentration of the yeast seed liquid is 10 7 -10 9 CFU / mL, the bacterial concentration of the lactobacillus seed solution is 10 7 -10 9 CFU / mL.

3. The preparation method according to claim 1, characterized in that: In step (1), the first fermentation temperature is 25-30°C, the fermentation time is 48-96h, the stirring paddle speed is 100-180r / min, and the sterile air intake is 0.3-0.5vvm; in step (2), the second fermentation temperature is 34-40°C, the fermentation time is 36-60h, and the second fermentation method is anaerobic fermentation.

4. The preparation method according to claim 1, characterized in that: The culture medium containing marigold in step (1) comprises marigold powder, a carbon source, a nitrogen source, an inorganic salt and water; Preferably, the carbon source is at least one of glucose, fructose and α-glucan; the nitrogen source is peptone; the inorganic salt includes sulfate and / or phosphate; Preferably, the carbon source is a composite carbon source of glucose and α-glucan; Preferably, the sulfate is magnesium sulfate; and the phosphate is at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

5. The preparation method according to claim 4, characterized in that: Measured in percentage by mass, the culture medium containing marigolds comprises: 0.5-2% marigold powder, 2.2-4.5% carbon source, 0.8-1.5% nitrogen source, 0.4-1% inorganic salt and the balance water.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The yeast in step (1) is Candida krusei; the lactobacillus in step (2) is Bifidobacterium adolescentis.

7. A fermented product of marigold, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. A cosmetic composition, characterized in that The invention comprises the marigold fermentation product as claimed in claim 7.

9. Use of Candida krusei and / or Bifidobacterium adolescentis in preparing marigold fermentation product.

10. A culture medium for marigold fermentation, characterized in that: The invention comprises, by mass percentage, 0.5-2% of marigold powder, 2.2-4.5% of a carbon source, 0.8-1.5% of a nitrogen source, 0.4-1.0% of an inorganic salt and the balance of water.

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