Preparation method of quinoa callus fermentation filtrate with whitening and oil control effects

Abstract

This invention discloses a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. First, quinoa callus tissue is cultured and then pulverized to obtain quinoa callus tissue powder. Then, activated Bifida ferment lysate is inoculated into MRS liquid culture medium, and the obtained quinoa callus tissue powder is dispersed in the MRS liquid culture medium. The mixture is then cultured at 37°C to obtain the fermentation filtrate. Compared with untreated quinoa saponins, the quinoa saponins obtained from the quinoa callus fermentation filtrate using plant tissue culture technology have a more stable source, are more easily absorbed and utilized by the skin, and have lower irritation.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. Background Technology

[0002] Quinoa (Chenopodium quinoa Willd.), also known as South American quinoa, is an annual dicotyledonous plant belonging to the genus Chenopodium in the family Amaranthaceae, with a long history of cultivation. Native to the high-altitude (2800-4200m) regions of the Andes Mountains in South America, quinoa has been cultivated there for over 5000 years and is known as the "mother of grains" due to its rich nutritional value and diverse health benefits. Quinoa exhibits good adaptability to biotic and abiotic stresses such as salinity, drought, frost, and pests, leading to its widespread cultivation in many countries. However, quinoa has relatively high requirements for climate and geographical conditions, and its cultivation in China is still in its initial stages, with a narrow planting area, mainly distributed in Qinghai, Gansu, and Shanxi provinces. This results in inconsistent quality, with some superior varieties commanding high prices and hindering widespread market application. Quinoa possesses high economic and nutritional value, meeting the basic nutritional needs of the human body. It is rich in protein, minerals, amino acids, fiber, and other trace elements, with higher content than other grain crops. Studies have shown that quinoa not only contains abundant nutrients, but also bioactive substances such as polyphenols, saponins, and flavonoids, which have strong antioxidant capabilities and play an important role in physiological functions such as anti-oxidation, lowering blood lipids, and enhancing immunity.

[0003] Plant tissue culture is a novel asexual reproduction technique based on the theory of totipotency of plant cells. This technique involves aseptically handling plant tissues or cells and inoculating them onto a culture medium containing various nutrients and plant hormones to obtain regenerated complete plants or produce other economically valuable products. Plant tissue culture offers many advantages, including rapid plant propagation, freedom from natural growth limitations, the ability to produce virus-free plants, and the capacity for gene mutation and variety improvement.

[0004] Fermentation technology has a long history; in my country, the history of traditional fermented foods can be traced back approximately 9,000 years. Fermentation technology utilizes the growth characteristics of cells and biocatalytic reaction systems to react with organic substrates, ultimately yielding the target product, namely, biosynthesized substances or cells. This technology not only alters the original properties of the substrate but also produces new functional substances. Fermentation generally takes two forms: liquid fermentation and solid-state fermentation. Compared to chemical methods, fermentation technology offers advantages such as better controllability, higher safety, energy efficiency, fewer byproducts, and less environmental pollution. Furthermore, the decomposition capabilities of microorganisms ensure highly uniform biosynthesis of specific structures, guaranteeing the safety and efficacy of the product.

[0005] With economic development and improved living standards, people's demand for skin brightening and rejuvenation is increasing, especially for products with whitening and oil-controlling effects. Currently, most skincare products on the market achieve whitening effects by adding chemically synthesized substances with whitening properties, such as hydroquinone, arbutin, kojic acid, and BHT (2,6-di-tert-butyl-4-methylphenol). However, these compounds often cause skin irritation and cytotoxicity during use. Some oil-control methods also rely on acid peels, which require strict skincare routines and can damage the skin barrier. Therefore, the safety of cosmetics is receiving increasing attention, and natural whitening and oil-controlling ingredients derived from plants are gaining popularity due to their lower side effects. Summary of the Invention

[0006] This invention overcomes the shortcomings of the prior art. The primary objective of this invention is to provide a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. Compared to unprocessed quinoa extract, the quinoa active ingredients obtained through plant tissue culture technology are more stable, more easily absorbed and utilized by the skin, and have lower irritation.

[0007] This invention provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects, comprising the following preparation steps:

[0008] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, and disinfect them with ethanol in a sterile environment. This step is to remove dust and bacteria from the surface of the seeds.

[0009] 2) Rinse the seeds that have been disinfected in 1) with sterile water, then treat them with sodium hypochlorite (NaClO), and finally rinse them with sterile water again. This step is to obtain sterile seeds.

[0010] 3) Under aseptic conditions, place the sterilized seeds neatly in sterilized petri dishes and culture them at 25°C with a 16h / 8h light cycle. This step is to obtain sterile seedlings for later use.

[0011] 4) The obtained sterile seedlings are cut into pieces to obtain quinoa explants.

[0012] 5) The explants obtained in 4) were spread evenly in the induction medium and cultured at 25℃ with a light cycle of 16h / 8h to obtain quinoa callus tissue, which was then pulverized to obtain quinoa callus tissue powder.

[0013] 6) Take Bifida ferment lysate frozen at -80℃, inoculate it on an MRS solid plate with an inoculation loop for purification, and culture it anaerobically at 37℃. Pick a single colony and transfer it to 5 mL of new liquid culture medium. Incubate it anaerobically at 37℃ for 24 h, and then subculture it into new liquid culture medium at 4 vol% and culture it anaerobically at 37℃ for 24 h. Repeat this activation several times to obtain a highly active strain.

[0014] 7) Take the above bacterial solution and disperse the quinoa callus powder obtained in 5) in MRS liquid culture medium, carry out large-scale culture at 37°C, and obtain fermentation filtrate.

[0015] Preferably, the alcohol in step 1) has a concentration of 60%-75%, for example, it can be 60%, 65%, 70%, 75%, etc.

[0016] Preferably, the sodium hypochlorite (NaClO) in step 2) has a concentration of 6%-12%, for example, it can be 6%, 8%, 10%, 12%, etc.

[0017] Optionally, the culture medium in step 3) can be MS culture medium, or two sterile filter papers, which are then soaked in sterile water.

[0018] Preferably, the sterile seedlings in step 3) are seedlings that are 4-8 days old, for example, the seedlings can be on the 4th, 5th, 6th, 7th, or 8th day.

[0019] Preferably, the plant tissue culture explants in step 4) include cotyledon explants, hypocotyl explants, and seed embryo explants.

[0020] Preferably, the induction medium in step 4) is MS medium with 2,4-D concentration of 0.4-2.0 mg / L, for example, 0.4 mg / L, 0.8 mg / L, 1.2 mg / L, 1.6 mg / L, 2.0 mg / L, etc.

[0021] Preferably, the callus tissue in step 4) is a callus tissue block that is 20-40 days old, for example, it can be 20 days, 25 days, 30 days, 35 days, 40 days, etc.

[0022] Preferably, in step 5), the powder is obtained by freeze-drying and grinding or by drying and pulverizing. The freeze-drying time is 10-15 hours, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, etc., and the drying temperature is 40-60 degrees Celsius, for example, 40 degrees Celsius, 45 degrees Celsius, 50 degrees Celsius, 55 degrees Celsius, 60 degrees Celsius, etc.

[0023] Preferably, the anaerobic culture time at 37°C in step 6) is 36-48 hours, for example, 36 hours, 40 hours, 44 hours, 48 ​​hours, etc.

[0024] Preferably, step 6) involves 3-5 consecutive activations, for example, 3, 4, or 5 times.

[0025] Preferably, the inoculation of Bifida ferment lysate in step 7) is 5-10 vol%, for example, it can be 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, etc.

[0026] Preferably, the quinoa callus powder is dispersed in a culture medium, and the concentration of quinoa is 12-30 g / L, for example, it can be 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, etc.

[0027] After fermentation is complete, filter to remove quinoa tissue residue, optionally break the cells, centrifuge to collect the supernatant, and filter to remove bacteria to obtain the quinoa callus tissue fermentation filtrate.

[0028] The quinoa callus filtrate obtained by the above method can be used in cosmetics.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses quinoa to culture callus tissue, which greatly shortens the plant growth cycle compared with quinoa seed extract, and can obtain higher active ingredients in a shorter time. Moreover, the entire callus tissue growth process is carried out in an artificial climate chamber, which allows for more precise control of its growth environment. This avoids the problem of unstable active ingredients due to the influence of climate such as the planting site on quinoa, thereby reducing efficacy and improving the stability of raw materials.

[0031] (2) The present invention unexpectedly discovered that the tissue filtrate obtained by combining quinoa callus tissue with the fermentation pathway has a stronger whitening and oil-controlling effect than the filtrate obtained by conventional plant extraction methods. It is speculated that under the fermentation action of Bifida Ferment Lysate, quinoa callus tissue can degrade many large molecules that are not easily absorbed into small molecules that are more easily absorbed, thereby improving the whitening and oil-controlling effects of the raw material.

[0032] (3) The quinoa callus fermentation filtrate prepared by the present invention has a good whitening and oil-controlling effect on the skin. Compared with traditional whitening and oil-controlling ingredients, it is safer and non-toxic, does not irritate the skin, has high skin absorption and utilization, and has broad application prospects. Attached Figure Description

[0033] Figure 1 This is a representative image of the Oil Red O staining results of Experiment Example 4 of the present invention;

[0034] Figure 2 This is a representative graph of the skin oil content test results from Experiment Example 6 of the present invention;

[0035] Figure 3 This is a representative diagram of the chicken embryo chorionic allantoic membrane vascular experiment in Experiment Example 7 of this invention. Detailed Implementation

[0036] The technical solutions designed in this invention will be further elaborated below with specific experimental examples. Any modifications or alterations made by those skilled in the art based on the relevant embodiments of this invention, without any inventive effort, will be considered within the scope of protection of this invention.

[0037] Preparation Example

[0038] This preparation example provides a Bifida ferment lysate (commercially available), which is obtained by activation of the Bifida ferment lysate seed culture through the following method:

[0039] Take Bifida Ferment Lysate frozen at -80℃, inoculate and purify it on an MRS solid plate using an inoculation loop, and culture it anaerobically at 37℃ for 48h. Pick a single colony and transfer it to 5mL of new liquid culture medium, and grow it anaerobically at 37℃ for 24h. Then, subculture it into new liquid culture medium at 4vol% and culture it anaerobically at 37℃ for 24h. After three consecutive activations, the activated Bifida Ferment Lysate seed culture can be obtained.

[0040] Unless otherwise specified, the quinoa used in the following examples and comparative examples all originated from Qinghai.

[0041] Example 1

[0042] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0043] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0044] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a 16h / 8h light cycle. Take the quinoa callus tissue that has grown for 40 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0045] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0046] Example 2

[0047] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0048] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0049] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a light cycle of 16h / 8h. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0050] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0051] Example 3

[0052] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0053] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0054] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a 16h / 8h light cycle. Take the quinoa callus tissue that has grown for 20 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0055] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0056] Example 4

[0057] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0058] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0059] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 4 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a 16h / 8h light cycle. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0060] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0061] Example 5

[0062] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0063] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0064] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 6 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a light cycle of 16h / 8h. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0065] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0066] Example 6

[0067] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0068] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0069] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a light cycle of 16h / 8h. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0070] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 8 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After filtration to remove bacteria, the quinoa callus fermentation filtrate was obtained.

[0071] Example 7

[0072] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects. The preparation method specifically includes the following steps:

[0073] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0074] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them flat on the induction medium (MS+2,4-D(1.6mg / L)), and culture them at 25℃ with a light cycle of 16h / 8h. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80℃ for 12h, and grind it in liquid nitrogen.

[0075] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 5 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. The supernatant was then filtered to remove bacteria, and the quinoa callus fermentation filtrate was obtained.

[0076] Comparative Example 1

[0077] This comparative example provides a method for preparing quinoa seed extract, the preparation method specifically including the following steps:

[0078] 1) Sift the quinoa seed sample, dry it in an oven at 50℃ to constant weight, grind it into powder, accurately weigh 2g of quinoa powder into a 250mL round-bottom flask, add 30mL of deionized water and enzyme preparation (total enzyme dosage is 1.5%, based on the mass of quinoa powder, m (纤维素酶) :m (果胶酶) =3:2), adjust the pH of the system to 5.0, and enzymatically hydrolyze at 50℃ for 0.5h with electromagnetic stirring.

[0079] 2) After enzymatic hydrolysis is complete, inactivate the enzyme at 90℃ for 30 seconds, add 70 mL of anhydrous ethanol, and extract by sonication for 20 minutes. Filter the extract under reduced pressure, and repeat the extraction three times with 50 mL of 70% ethanol solution. Combine the filtrates.

[0080] Comparative Example 2

[0081] This comparative example provides a method for preparing quinoa fermentation filtrate, the preparation method specifically including the following steps:

[0082] 1) Crush quinoa seeds, sieve them, weigh quinoa flour, add deionized water and anhydrous calcium chloride, stir, and heat to gelatinize; add α-amylase, keep warm at 70-85℃ to liquefy, after liquefaction, cool, adjust pH, heat to 55-60℃, add saccharifying enzyme, keep warm to saccharify for 2-5 hours, after saccharification, cool, adjust pH, autoclave and cool.

[0083] 2) The activated Bifida yeast seed liquid prepared in the above example was inoculated into the sterile saccharification liquid in step 1) at an inoculation amount of 10 vol%. It was cultured at 37°C and fermentation was stopped when the pH value reached a certain range. After filtering to remove quinoa residue, the cells were broken up, the supernatant was collected by high-speed centrifugation, and after filtration to remove bacteria, the quinoa fermentation filtrate was obtained.

[0084] Comparative Example 3

[0085] This comparative example provides a method for preparing quinoa (Shanxi) callus fermentation filtrate, the preparation method specifically including the following steps:

[0086] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ under a 16h / 8h light cycle.

[0087] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them evenly in MS medium with 1.6 mg / L for induction, and culture them at 25°C with a 16 h / 8 h light cycle. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80°C for 12 h, and grind it in liquid nitrogen.

[0088] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0089] Comparative Example 4

[0090] This comparative study provides a method for preparing fermentation filtrate from quinoa callus tissue (dried and pulverized), the preparation method specifically including the following steps:

[0091] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ with a 16h / 8h light cycle.

[0092] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them evenly in MS medium with 1.6 mg / L for induction, and culture them at 25°C with a 16 h / 8 h light cycle. Take the quinoa callus tissue that has grown for 30 days, dry it at 60°C to constant weight, and then crush and sieve it.

[0093] 3) The activated Bifida Ferment Lysate seed culture prepared in the above example was inoculated into MRS liquid medium at an inoculation rate of 10 vol%. Quinoa callus powder from step 2) was dispersed in the medium at a concentration of 30 g / L and cultured at 37°C. After fermentation, the quinoa tissue residue was removed by filtration. The cells were optionally broken up again, and the supernatant was collected by centrifugation. After sterilization by filtration, the quinoa callus fermentation filtrate was obtained.

[0094] Comparative Example 5

[0095] This comparative study provides a method for preparing quinoa callus fermentation (lactic acid bacteria) filtrate, the preparation method specifically including the following steps:

[0096] Take Lactobacillus frozen at -80℃, inoculate and purify it on an MRS solid plate using an inoculation loop, and culture it anaerobically at 37℃ for 48h. Pick a single colony and transfer it to 5mL of new liquid culture medium, and grow it anaerobically at 37℃ for 24h. Then, subculture it into a new liquid culture medium at 4vol% and culture it anaerobically at 37℃ for 24h. After three consecutive activations, the activated Lactobacillus seed culture can be obtained.

[0097] 1) Select mature and plump quinoa seeds, wash them thoroughly with running water, disinfect them with 75% ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Arrange the disinfected seeds neatly in a sterilized petri dish, add two sheets of filter paper to the petri dish and add an appropriate amount of sterile water to moisten the filter paper, and incubate them at 25℃ with a 16h / 8h light cycle.

[0098] 2) Take the quinoa cotyledons from the sterile seedlings that have been cultured for 8 days, spread them evenly in MS medium with 1.6 mg / L for induction, and culture them at 25°C with a 16 h / 8 h light cycle. Take the quinoa callus tissue that has grown for 30 days, freeze it at -80°C for 12 h, and grind it in liquid nitrogen.

[0099] 3) Inoculate the Lactobacillus seed culture into MRS liquid medium at an inoculation rate of 10 vol%, and take the quinoa callus powder from step 2) and disperse it in the medium at a concentration of 30 g / L. Carry out the culture at 37°C. After the fermentation is completed, filter to remove the quinoa tissue residue, optionally break the cells again, centrifuge to collect the supernatant, and filter to remove bacteria to obtain the quinoa callus fermentation filtrate.

[0100] Experimental Example 1

[0101] Tyrosinase activity inhibition test

[0102] Add L-tyrosine solution, sample solutions (Examples 1-7 and Comparative Examples 1-5), and PBS buffer sequentially to a 96-well plate, mix thoroughly, and incubate at 37°C for 10 min. Then, add 20 μL of tyrosinase solution (500 U / mL) to each well sequentially, mix at 37°C for 5 min ± 5 s, and immediately place the plate into a microplate reader (keeping the time from adding tyrosinase solution to measuring absorbance consistent for each well), and measure at a wavelength of 475 nm.

[0103] A 96-well microplate was configured with solvent background wells (T1), solvent reaction wells (T2), sample background wells (T3), and sample reaction wells (T4). T1 was the solvent background group, containing neither L-tyrosine substrate solution nor sample solution; T2 was the solvent reaction group, containing L-tyrosine substrate solution but no sample solution; T3 was the sample background group, containing neither L-tyrosine substrate solution nor sample solution; and T4 was the sample reaction group, containing both L-tyrosine substrate solution and sample solution. Each group was run in triplicate.

[0104] Table 1. Dosage of reagents added for tyrosinase activity inhibition test

[0105]

[0106]

[0107] Calculate the tyrosinase activity inhibition rate (Y):

[0108]

[0109] In the formula: T a —Solvent background absorbance; T b —Absorbance of solvent reaction group; T c —Sample background absorbance; T d —Absorbance of the sample reaction group.

[0110] The specific test results are shown below:

[0111] Table 2. Tyrosinase activity inhibition rate

[0112]

[0113] As shown in Table 2, the Bifida ferment filtrate of quinoa callus prepared using the method of this application can effectively inhibit tyrosinase activity, with an inhibition rate of over 79%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate of tyrosinase activity is increased by about 1.5 times. The inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is that it first increases and then decreases. The study found that the callus that has grown for about 30 days has the best inhibitory effect on tyrosinase activity after fermentation. It was also unexpectedly found that changes in quinoa origin, callus crushing method, and fermentation strain can reduce the inhibition of tyrosinase activity by the fermentation product. This experiment proves that the quinoa callus ferment filtrate of this invention has a good whitening effect.

[0114] Experimental Example 2

[0115] Melanin Synthesis Inhibition Test

[0116] B16 melanoma cells were injected at a rate of 2.5 × 10⁻⁶. 3 Cells were seeded at a density of 2 mL / mL in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 3 mL of culture medium containing the samples (Examples 1-7 and Comparative Examples 1-5) was added to each well. A blank control group without sample solution and without sample solution was also set up.

[0117] After incubating the culture plate in an incubator for 72 hours, discard the supernatant, wash the cells twice with 1×PBS, aspirate the PBS, add 200 μL of 0.25% trypsin to each well to digest the cells, and incubate in a CO2 incubator for 5 minutes. Add 2 mL of PBS in two portions and gently pipette the cells into centrifuge tubes. Centrifuge at 4000 rpm for 5 minutes. Aspirate as much PBS residue as possible from the centrifuge tubes, add 200 μL of melanin extraction solution (1 mol / L NaOH containing 10% DMSO) to each tube, shake well, and heat in an 80°C water bath for 1 hour. After cooling, gently remove droplets from the tube wall, gently pipette to mix, and transfer 150 μL of solution from each centrifuge tube into a 96-well plate. Measure the absorbance at 405 nm in each well using a microplate reader.

[0118] Calculate the melanin synthesis inhibition rate (X):

[0119]

[0120] Where: T - absorbance of the test sample well; C - average of three absorbance measurements of the negative control group; C0 - background absorbance of the melanin extract.

[0121] The specific test results are shown below:

[0122] Table 3. Melanin Synthesis Inhibition Rate

[0123] Test sample Melanin synthesis inhibition rate (%) Example 1 88.54±0.35 Example 2 89.03±0.73 Example 3 82.4±1.08 Example 4 81.78±0.93 Example 5 83.25±0.18 Example 6 84.67±0.71 Example 7 80.18±1.27 Comparative Example 1 51.2±1.28 Comparative Example 2 52.9±0.39 Comparative Example 3 61.09±0.34 Comparative Example 4 58.06±1.55 Comparative Example 5 57.99±0.86

[0124] As shown in Table 3, the quinoa callus fermentation filtrate prepared using the method of this application exhibits a good inhibitory effect on melanin synthesis, with an inhibition rate of over 80%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate on melanin synthesis is increased by approximately 1.6 times. The inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is one of initial increase followed by decrease. The study found that the inhibitory effect on melanin synthesis is most significant after fermentation of callus tissue grown for about 30 days. Furthermore, changes in quinoa origin, callus tissue pulverization method, and fermentation strain can also affect the inhibitory activity of the fermentation product on pigments. This experiment demonstrates that the quinoa callus fermentation filtrate can inhibit melanin synthesis, thereby reducing pigmentation and promoting skin metabolism, further proving that it can achieve whitening effects from multiple aspects.

[0125] Experimental Example 3

[0126] DPPH free radical scavenging activity test

[0127] The experiment was conducted according to the method specified in Larrauri JA. A 0.2 mM DPPH solution was prepared. The test sample solution was prepared by diluting the quinoa callus fermentation filtrate provided in Examples 1-7 and the fermentation filtrate provided in Comparative Examples 1-5 with ultrapure water to obtain a solution with a mass concentration of 0.1%. 2.0 mL of the test sample solution and 2.0 mL of the 0.2 mM DPPH solution were placed in a test tube, mixed thoroughly, and reacted for 30 min. The absorbance was measured at 517 nm. Anhydrous ethanol served as a blank control.

[0128] Calculate the DPPH radical inhibition rate (Z):

[0129]

[0130] In the formula: A0 is the absorbance of 2.0 mL of DPPH solution and 2.0 mL of test sample solution, A1 is the absorbance of 2.0 mL of test sample solution and 2.0 mL of anhydrous ethanol, and A2 is the absorbance of 2.0 mL of DPPH solution and 2.0 mL of anhydrous ethanol. The average value is taken from three parallel tests.

[0131] The specific test results are shown below:

[0132] Table 4. DPPH free radical inhibition rate

[0133] Test sample DPPH free radical inhibition rate (%) Example 1 89.32±1.38 Example 2 91.02±0.72 Example 3 84.77±2.01 Example 4 81.46±1.48 Example 5 85.19±1.73 Example 6 84.41±0.94 Example 7 79.48±1.09 Comparative Example 1 54.91±2.37 Comparative Example 2 57.06±0.79 Comparative Example 3 66.67±1.25 Comparative Example 4 64.47±1.46 Comparative Example 5 62.28±0.49

[0134] As shown in Table 4, the Bifida ferment filtrate from quinoa callus prepared using the method described in this application has a good inhibitory effect on DPPH free radicals, with an inhibition rate of over 79%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate of DPPH free radicals is increased by about 1.4 times. The inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is that it first increases and then decreases. The study found that the inhibitory effect of fermentation on DPPH free radicals on callus tissue with a growth time of about 30 days is the most significant. The same quinoa origin, callus pulverization method, and fermentation strain also significantly reduced the inhibition rate of free radicals. This experiment proves that the fermentation filtrate from quinoa callus tissue has a good scavenging effect on DPPH free radicals, can enhance the antioxidant capacity of skin tissue, reduce the content of free radicals in skin tissue, and reduce skin dullness caused by lipid peroxidation.

[0135] Test Example 4

[0136] Oil Red O staining method for testing in vitro oil-controlling efficacy

[0137] When the cell deposition rate in the 24-well plates reached 40-60%, the cells were divided into groups and administered the drug, with three replicates per group. The blank control group received 1 mL of MEM culture medium per well, the positive control group received 1 mL of culture medium containing 50 μM isotretinoin per well, and the experimental groups received 1 mL of culture medium containing 5% of the sample (Examples 1-7 and Comparative Examples 1-5) per well. After drug administration, the cells were incubated in a CO2 incubator (37°C, 5% CO2) for 72 hours, with daily medium changes and drug administration during the incubation period.

[0138] Discard the old solution, wash with PBS, fix cells with paraformaldehyde, add 300 μL of Oil Red O working solution to each well, stain in the dark for 15 min, wash with PBS, and observe under a microscope. Results are as follows. Figure 1 As shown. After taking the photo, discard the waste liquid, add 200 μL of isopropanol to each well, and treat at 800 rpm for 5 minutes on a microplate shaker at room temperature to ensure that Oil Red O is fully dissolved in isopropanol. Then, take 20 μL from each well and transfer it to a 96-well plate to dilute it 10 times. Then, read the absorbance at 510 nm using a microplate reader.

[0139] Calculate the sebum secretion inhibition rate:

[0140]

[0141] The specific test results are shown below:

[0142] Table 5. Sebum secretion inhibition rate

[0143]

[0144] As shown in Table 5, the quinoa callus fermentation filtrate prepared using the method of this application has a good inhibitory effect on oil secretion, with an inhibition rate of over 70%. This is approximately 1.2 times more effective than isotretinoin and about 1.3 times more effective than traditional plant extraction methods (Comparative Example 1). The inhibitory effect is positively correlated with seedling growth time, but the relationship with callus growth time is initially increased and then decreased. The study found that callus that has grown for about 30 days exhibits the best inhibitory effect on oil secretion after fermentation. Changes in quinoa origin, callus crushing method, and fermentation strain have a smaller impact on oil secretion. This experiment demonstrates that the quinoa callus fermentation filtrate of this invention has a superior removal effect on oil secretion and can effectively control oil production.

[0145] Combining the above experimental examples 1-4, it was found that the quinoa seedling cultivation time was positively correlated with the efficacy of quinoa callus fermentation filtrate. This may be related to the increasing activity of some active ingredients over time, but not positively correlated with the callus development time. This may be because the accumulation of related components stagnates after 40 days of callus growth; the cells only continue to expand in size but no longer secrete effective components, and may even consume some of the original nutrients, thus reducing efficacy. Unexpectedly, the experiment also revealed some differences in the efficacy of quinoa callus fermentation filtrate from different origins. Quinoa from Qinghai, influenced by factors such as longer daylight hours, larger diurnal temperature range, and fertile soil, may have a richer content of active substances, resulting in a more effective fermentation product. The application of callus tissue can effectively mitigate the influence of these environmental factors. The effect of different grinding methods on quinoa callus fermentation filtrate may be due to the different temperatures associated with each grinding method. High temperatures cause some components related to whitening and oil control to become inactive, thus affecting efficacy, while ultra-low temperature grinding can retain the activity of active ingredients to the greatest extent. Finally, we also found that if we want to obtain more ideal efficacy, we must choose the bifida ferment lysate of the present invention as the starter culture for fermentation.

[0146] Experimental Example 5

[0147] Human efficacy evaluation - skin whitening

[0148] Test formula

[0149] Table 6. Test Serum Formulas

[0150] INCI experimental group control group Blank group Butylene glycol 2 2 2 glycerin 2 2 2 Sodium hyaluronate 1 1 1 Allantoin 0.1 0.1 0.1 Carbomer 0.2 0.2 0.2 Tromethamine 0.18 0.18 0.18 p-Hydroxyacetophenone 0.5 0.5 0.5 1,2-Hexanediol 0.5 0.5 0.5 Dipotassium glycyrrhizate 0.1 0.1 0.1 Example 1 1 0 0 ascorbic acid 0 5 0 water 92.42 88.42 93.42

[0151] We are recruiting 30 healthy participants. Inclusion criteria: 18-59 years old, healthy male or female; serious and positive attitude, able to use the product correctly as instructed and follow up on time; have accurate written and verbal communication skills, and be able to truthfully reflect their feelings after using the product.

[0152] Exclusion criteria: pregnant or breastfeeding women, or those planning to conceive in the near future; those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, or severe acne; those who have taken orally or used anti-inflammatory drugs such as corticosteroids within the past month; those who have taken orally or used any products or drugs that affect skin color (such as hydroquinone preparations) within the past two months; and those who have participated in similar trials within the past three months or more ago.

[0153] Test method: The subjects were divided into 3 groups (experimental group, control group and blank group, 10 people in each group) and blinded test. The subjects used the sample solution once in the morning and once in the evening for 4 consecutive weeks. During the test, they did not use it with other whitening skin care products. The test was conducted on day 0 (before use), day 14 and day 28 of the experiment.

[0154] Before the test, the subject needs to clean their face with water, dry their face, and sit quietly for 20 minutes in a constant temperature and humidity environment (20±2℃, relative humidity 50±5%) before the measurement is performed.

[0155] The skin color test (Chromameter) uses a spectrophotometer (CM-700D, Konica Minolta, Japan) and employs the colorimetric system (Lab colorimetric system) specified by the International Commission on Illumination (CIE) to represent changes in skin color. The ITA° value is the individual skin type angle, which is a value related to L* and b* that characterizes the brightness of the skin. The larger the ITA° value, the lighter the skin color, and vice versa.

[0156] The formula for calculating ITA° is as follows:

[0157]

[0158] The specific test results are shown below:

[0159] Table 7. ITA° values ​​before and after product trial

[0160]

[0161]

[0162] As shown in Table 7, compared with the blank group, the experimental group showed significant improvement after 2 and 4 weeks of using the Bifida Ferment Lysate prepared with added quinoa callus tissue (P < 0.05). Compared with ascorbic acid, the quinoa callus tissue fermentation filtrate has the characteristics of low effective concentration and strong efficacy. Moreover, none of the subjects reported any allergic or other discomfort symptoms, proving that the quinoa callus tissue Bifida Ferment Lysate prepared by the method of this application has whitening effect.

[0163] Experimental Example 6

[0164] Human efficacy evaluation - oil control

[0165] Table 8. Test Serum Formula

[0166] INCI Formula proportion Butylene glycol 2 glycerin 2 Sodium hyaluronate 1 Allantoin 0.1 Carbomer 0.2 Tromethamine 0.18 p-Hydroxyacetophenone 0.5 1,2-Hexanediol 0.5 Dipotassium glycyrrhizate 0.1 Example 1 1 water 92.42

[0167] Thirty healthy participants will be recruited. Inclusion criteria: healthy males aged 19-35; individuals with oily skin, and facial oil content ≥120 μg / cm² on the forehead. 2 A serious and proactive attitude, able to use the product correctly according to regulations and conduct timely follow-up visits; possessing accurate written and verbal communication skills, and able to truthfully reflect the user's experience after using the product.

[0168] Exclusion criteria: Patients who have used antihistamines within the past week or immunosuppressants or immunomodulatory biological agents and small molecule drugs within the past month; patients who have applied any anti-inflammatory drugs to the test site within the past two months; patients with clinically unhealed inflammatory skin diseases; patients with insulin-dependent diabetes mellitus; patients who have experienced an allergic reaction or treatment within the past month or have used hormones or other anti-inflammatory drugs; and patients whose test results are affected by scars, pigmentation, atrophy, port-wine stains, or other blemishes at the skin test site.

[0169] Test method: Subjects used the essence formulated in Table 8 twice a day, morning and evening, for 4 consecutive weeks. During the test, the essence was not used with other whitening skin care products. Tests were conducted on day 0 (before use) and day 28 of the experiment.

[0170] Before the test, the subject needs to clean their face with water, dry their face, and sit quietly for 20 minutes in a constant temperature and humidity environment (20±2℃, relative humidity 50±5%) before the measurement is performed.

[0171] The Sebumeter (SM815, Courage and Khazaka, Germany) skin oil content analyzer uses the internationally recognized SEBUMETER method. It characterizes skin oil content by measuring the change in light transmittance of a 0.1mm thick special matte adhesive tape before and after absorbing oil. During the test, the tape contacts the test area, absorbing oil from the skin and becoming translucent. The amount of light transmitted changes accordingly; the more oil absorbed, the greater the light transmittance. Measuring the light transmittance of the tape allows for the determination of skin oil content (measured in the experimental group).

[0172] The calculation formula is as follows:

[0173]

[0174] Where: average value Where x = individual parameter measurement value, n = number of valid data, T1 is before use, and T2 is after 2 weeks of use; SPSS Statistics 25 was used for statistical analysis, with a two-tailed test and a significance level of α = 0.05; for the measurement data, the difference analysis method was selected based on the normality test results: if the measurement values ​​are normally distributed, the t-test method was used for statistical analysis; if they are not normally distributed, the rank-sum test method was used for statistical analysis.

[0175] The specific test results are shown below:

[0176] Table 9. Descriptive statistics of skin oil content (n=30)

[0177]

[0178] Table 10. Statistical analysis results of skin oil content (n=30)

[0179]

[0180] Statistical analysis results: "-" (P≥0.05): no statistically significant difference; "***" (P<0.001): statistically significant difference.

[0181] The results of the skin oil content test are shown in the figure below. Figure 2 As shown.

[0182] From Table 9 and Figure 2 The test results showed that after using the Bifida Ferment Lysate fermented filtrate prepared with added quinoa callus tissue, the oil content of the volunteers decreased significantly by 41.08% (P<0.001) after 4 weeks, and no allergic reactions or other discomfort were reported. This proves that the Bifida Ferment Lysate fermented filtrate prepared by the method of this application has oil-controlling effects.

[0183] Experimental Example 7

[0184] Chicken embryo chorionic allantoic membrane vascular test

[0185] The chicken embryo chorioallantoic membrane vascular test is commonly used as a standard for evaluating the eye irritation of cosmetic formulations. This invention uses the chicken embryo chorioallantoic membrane vascular test to detect the irritation of the sample (Example 1) involved in this invention.

[0186] Fertilized chicken embryos were placed in a constant temperature and humidity chamber and incubated for about 9 days at (37±1)℃ and 50%~70% humidity. They were then removed and placed on a homemade egg rack with the air cell end facing upwards. The eggshell was gently broken with the tip of toothed tweezers and the air cell end of the eggshell was carefully removed to fully expose the eggshell membrane. 1mL of physiological saline was added to the eggshell membrane to moisten it. The saline was then absorbed, and the eggshell membrane was carefully peeled off with curved tweezers to expose the chorioallantoic membrane (CAM).

[0187] Chicken embryos were randomly divided into groups: a negative control group was given 0.9% NaCl solution, a positive control group was given 1% SDS solution, and the experimental group was given the sample described in this invention (Example 1). Each group contained at least 3 chicken embryos. 0.3 mL of the test reagent was pipetted onto the surface of the chorioallantoic membrane, and changes in blood vessels were recorded over 5 minutes. Bleeding and coagulation were observed, and photographs and videos were taken.

[0188] A representative image of the chicken embryo chorionic allantoic membrane vascular test is shown below. Figure 3 As shown.

[0189] Depend on Figure 3The test results show that the Bifida ferment filtrate prepared from quinoa callus tissue in this invention did not produce any vascular bleeding or coagulation, confirming that the Bifida ferment filtrate prepared from quinoa callus tissue by the method of this application has the characteristics of being mild and non-irritating.

[0190] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The scope of protection of this application is not limited to the above embodiments; all technical solutions falling within the scope of this application's concept are within the scope of protection of this application. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of this application should also be considered within the scope of protection of this application.

Claims

1. A method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects, comprising the following preparation steps: 1) Select mature, plump quinoa seeds from Qinghai, wash and disinfect them to obtain sterile seeds for later use; 2) Under aseptic conditions, the sterilized seeds are neatly arranged in sterilized petri dishes and cultured at 25℃ with a 16 h / 8 h light cycle. This step is to obtain sterile seedlings for later use. 3) Take quinoa cotyledons from sterile seedlings cultured for 4-8 days as explants, spread them evenly in induction medium (MS + 2,4-D, 1.6 mg / L), and culture at 25℃ with a 16 h / 8 h photoperiod. Take quinoa callus tissue that has grown for 20-40 days, freeze it at -80℃ for 12 h, and grind it in liquid nitrogen to obtain quinoa callus tissue powder. 4) The activated Bifida Ferment Lysate seed culture was inoculated into MRS liquid medium, and the quinoa callus powder obtained in 3) was dispersed in MRS liquid medium and cultured at 37°C to obtain fermentation filtrate.

2. The method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects according to claim 1, characterized in that: The cleaning and disinfection steps in 1) are as follows: clean with running water, disinfect with ethanol in a sterile environment, rinse the disinfected seeds with sterile water, treat with sodium hypochlorite, and finally rinse with sterile water again.

3. The method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects according to claim 1, characterized in that: In step 4), the Bifida ferment lysate seed culture is activated using the following method: Take Bifida Ferment Lysate frozen at -80 ℃, inoculate it on an MRS solid plate with an inoculation loop for purification, and culture it anaerobically at 37 ℃ for 48 h. Pick a single colony and transfer it to 5 mL of new liquid culture medium, and grow it anaerobically at 37 ℃ for 24 h. Then, subculture it into a new liquid culture medium at 4 vol% and culture it anaerobically at 37 ℃ for 24 h. After three consecutive activations, the activated Bifida Ferment Lysate seed culture can be obtained.

4. The method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects according to claim 1, characterized in that: Step 4) The inoculation of Bifida Ferment Lysate is 5-10 vol%, cultured at 37℃ for 36-48 h, and the quinoa callus powder is dispersed in the culture medium at a concentration of 12-30 g / L.

5. The method for preparing quinoa callus fermentation filtrate with whitening and oil-controlling effects according to claim 1, characterized in that: In step 4), after fermentation is complete, filter to remove quinoa tissue residue, optionally break the cells again, centrifuge to collect the supernatant, and filter to remove bacteria to obtain the quinoa callus tissue fermentation filtrate.

6. A cosmetic product with whitening and oil-controlling effects, characterized in that, The cosmetic product comprises the fermentation filtrate as described in any one of claims 1-5.

7. The use of the quinoa callus filtrate prepared according to any one of claims 1-5 in the preparation of cosmetics.

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