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

Abstract

The invention discloses a preparation method of chenopodium quinoa willd callus fermentation filtrate with whitening and oil control effects, which comprises the following steps: culturing to obtain chenopodium quinoa willd callus, and crushing to obtain chenopodium quinoa willd callus powder; then inoculating an MRS liquid culture medium with an activated bifidus yeast seed solution, dispersing the obtained quinoa callus powder in the MRS liquid culture medium, and carrying out enlarged culture at 37 DEG C to obtain a fermentation filtrate; and compared with the quinoa saponin extracted without treatment, the quinoa saponin component in the quinoa callus fermentation filtrate obtained by a plant tissue culture technology has the advantages of more stable source, easier absorption and utilization by skin, lower irritation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects. Background Art

[0002] Quinoa (Chenopodium quinoa Willd.), also known as South American quinoa, quinoa, quinoa, etc., is an annual dicotyledonous plant belonging to the genus Chenopodium of the Amaranthaceae family, with a long history of cultivation. Quinoa is native to the high altitude (2800-4200m) areas of the Andes Mountains in South America, and has a history of cultivation of more than 5,000 years in the local area. It is called the "mother of grains" for its rich nutrition and diverse health functions. Quinoa has good adaptability to biological and abiotic stresses such as salinity, drought, frost, pests and diseases, and is promoted for cultivation in many countries. However, quinoa has high requirements for planting climate and geography. Quinoa cultivation in China is still in its initial stage, with a narrow planting range, mainly distributed in Qinghai, Gansu and Shanxi. This makes quinoa of varying quality, and some excellent varieties are expensive, making it difficult to be widely used in the market. Quinoa has high economic and nutritional value, can meet the basic nutritional elements required by the human body, and is rich in protein, minerals, amino acids, cellulose and other trace elements, and its content is higher than other grain crops. Studies have shown that quinoa is not only rich in nutrients, but also contains polyphenols, saponins, flavonoids and other bioactive substances. It has strong antioxidant capacity and plays an important role in physiological effects such as anti-oxidation, lowering blood lipids, and enhancing immunity.

[0003] Plant tissue culture technology is a new asexual reproduction technology based on the theory that plant cells are omnipotent. This technology involves sterile manipulation of plant tissues or cells, inoculation on a culture medium containing various nutrients and plant hormones, and cultivation to obtain regenerated complete plants or produce other products with economic value. Plant tissue culture technology has many advantages, including the ability to rapidly propagate plants, not being restricted by natural growth, producing virus-free plants, and being able to carry out gene mutations and variety improvement.

[0004] Fermentation technology has a long history. In my country, the history of traditional fermented foods can be traced back to about 9,000 years ago. Fermentation technology uses the growth characteristics of cells and the biocatalytic reaction system to react with organic substrates to ultimately obtain the target product, that is, bioreaction synthetic substances or cells. This technology not only changes the original properties of the substrate, but also produces new functional substances. There are generally two forms of fermentation, one is liquid fermentation and the other is solid fermentation. Compared with chemical methods, fermentation technology has the advantages of better controllability, higher safety, energy saving, less by-products, and less environmental pollution. In addition, the decomposability of microorganisms can keep the biosynthesis of special structures highly uniform, ensuring the safety and efficacy of the product.

[0005] With the development of the economy and the improvement of the quality of life, people's demand for brightening and rejuvenating the skin is increasing, especially the demand for functional products such as whitening and oil control. At present, most of the skin care products with whitening effects on the market are achieved by adding chemical synthetic substances with whitening effects, such as hydroquinone, arbutin, kojic acid and BHT (2,6-di-tert-butyl-4-methylphenol), but the use of such compounds is often accompanied by problems such as skin irritation and cytotoxicity. Some oil control methods are also achieved through acid brushing, which has strict requirements on care methods and can damage the skin barrier due to acid brushing. Therefore, the safety of cosmetics has received increasing attention, and natural whitening and oil control ingredients derived from plants have received more attention due to their lower side effects. Summary of the invention

[0006] The present invention overcomes the deficiencies of the above-mentioned prior art, and the primary purpose of the present invention is to provide a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects. Compared with untreated quinoa extract, the quinoa active ingredient in the quinoa callus fermentation filtrate obtained by plant tissue culture technology has a more stable source, is more easily absorbed and utilized by the skin, and has lower irritation and other advantages.

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

[0008] 1) Select mature and full-grained quinoa seeds, wash them with running water, and sterilize them with ethanol in a sterile environment. This step is to remove dust and bacteria on the surface of the seeds.

[0009] 2) The seeds disinfected in 1) are rinsed with sterile water, then treated with sodium hypochlorite (NaClO), and finally rinsed with sterile water again. This step is to obtain sterile seeds.

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

[0011] 4) Cut the obtained sterile seedlings into pieces to obtain quinoa explants.

[0012] 5) The explants obtained in 4) were respectively spread into induction medium, cultured at 25° C. and 16 h / 8 h photoperiod to obtain quinoa callus tissue, and crushed to obtain quinoa callus tissue powder.

[0013] 6) Take the bifid yeast frozen in a -80°C refrigerator, streak it on an MRS solid plate with an inoculation loop for purification, culture it anaerobically at 37°C, pick a single colony and transfer it to 5 mL of new liquid culture medium, grow it anaerobically at 37°C for 24 hours, then inoculate 4 vol% and subculture it to new liquid culture medium, culture it anaerobically at 37°C for 24 hours, and activate it several times continuously. This step is to obtain a highly active strain.

[0014] 7) The bacterial solution and the quinoa callus powder obtained in 5) were dispersed in MRS liquid culture medium, and cultured at 37° C. to obtain fermentation filtrate.

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

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

[0017] Optionally, the culture medium in step 3) can be MS culture medium, or 2 sterile filter papers, and then sterile water is poured into the filter papers to wet them.

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

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

[0020] Preferably, the induction medium in step 4) is MS medium plus 2,4-D at a 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 of 20-40 days, 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 drying and crushing. The freeze-drying time is 10-15 h, for example, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, etc., and the drying temperature is 40-60 ° C, for example, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, etc.

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

[0024] Preferably, the number of consecutive activations in step 6) is 3-5 times, for example, 3 times, 4 times, or 5 times.

[0025] Preferably, in step 7), the inoculation amount of the bifid yeast is 5-10 vol%, for example, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol%, 10 vol%, etc.

[0026] Preferably, quinoa callus powder is dispersed in the 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 the fermentation is completed, the quinoa tissue residue is removed by filtering, the bacterial cells are optionally broken again, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by filtration and sterilization.

[0028] The quinoa callus hair filtrate prepared by the above method can be applied in cosmetics.

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

[0030] (1) The present invention performs callus culture on quinoa, which greatly shortens the plant growth cycle compared to quinoa seed extract and the like, and can obtain higher active ingredients in a shorter time. In addition, the entire callus growth process is carried out in an artificial climate room, and the growth environment is more accurately controlled, thereby avoiding the problem that the active ingredients of quinoa are unstable due to the influence of the climate of the planting site, thereby reducing the efficacy, and improving the stability of the raw materials.

[0031] (2) The present invention unexpectedly discovered that the tissue filtrate obtained by combining quinoa callus with fermentation has stronger whitening and oil-control effects than the filtrate obtained by conventional plant extraction. It is speculated that the quinoa callus can degrade many macromolecular substances that are difficult to absorb into small molecular substances that are more easily absorbed under the fermentation of bifid yeast, thereby enhancing the whitening and oil-control effects of the raw material.

[0032] (3) The quinoa callus fermentation filtrate prepared by the present invention has good whitening and oil-control effects on the skin. Compared with traditional whitening and oil-control ingredients, it is safer and non-toxic, has no irritation to the skin, has high skin absorption and utilization, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a representative picture of the Oil Red O staining result of Test Example 4 of the present invention;

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

[0035] Figure 3 This is a representative diagram of the chick chorioallantoic membrane vascular test of Test Example 7 of the present invention. DETAILED DESCRIPTION

[0036] The following will further illustrate the technical solution designed in the present invention in conjunction with specific experimental examples. The present invention relates to the relevant embodiments, and the relevant changes and modifications made by those skilled in the art on this basis, if there is no creative work, will be regarded as the protection scope of the present invention.

[0037] Preparation Example

[0038] This preparation example provides a bifid yeast strain (commonly available on the market), and the bifid yeast seed solution is activated by the following method:

[0039] Take the bifid yeast frozen in a -80°C refrigerator, streak inoculate and purify it on an MRS solid plate with an inoculation loop, culture it anaerobically at 37°C for 48 hours, pick a single colony and transfer it to 5 mL of new liquid culture medium, grow it anaerobically at 37°C for 24 hours, then inoculate 4 vol% and subculture it to a new liquid culture medium, culture it anaerobically at 37°C for 24 hours, and activate it three times in a row to obtain the activated bifid yeast seed liquid.

[0040] The quinoa not otherwise specified in the following embodiments 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-control effects, and the preparation method specifically comprises the following steps:

[0043] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0044] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 40 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0045] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0046] Example 2

[0047] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0048] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0049] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0050] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0051] Example 3

[0052] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0053] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0054] 2) Take the sterile seedlings cultured for 8 days and cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus that has grown for 20 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0055] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0056] Example 4

[0057] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0058] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0059] 2) Take the sterile seedlings cultured for 4 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0060] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0061] Example 5

[0062] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0063] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0064] 2) Take the sterile seedlings cultured for 6 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0065] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0066] Example 6

[0067] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0068] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0069] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0070] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 8 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0071] Example 7

[0072] This embodiment provides a method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, and the preparation method specifically comprises the following steps:

[0073] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0074] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them on the induction medium (MS+2,4-D (1.6 mg / L)), and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0075] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 5 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is centrifuged, and the quinoa callus fermentation filtrate is obtained by filtration and sterilization.

[0076] Comparative Example 1

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

[0078] 1) The quinoa seed sample was sieved, dried in an oven at 50°C to constant weight, and ground into powder. 2 g of quinoa powder was accurately weighed into a 250 mL round-bottom flask, and 30 mL of deionized water and enzyme preparation (the total enzyme dosage was 1.5%, based on the mass of quinoa powder, m (纤维素酶) :m (果胶酶) =3:2), the pH of the system was adjusted to 5.0, and the enzymatic hydrolysis was carried out at 50°C with electromagnetic stirring for 0.5 h.

[0079] 2) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 90°C for 30 seconds, 70 mL of anhydrous ethanol is added, and ultrasonic extraction is performed for 20 minutes. The extract is filtered under reduced pressure, and the residue is repeatedly extracted 3 times (50 mL of 70% ethanol solution by volume), and the filtrates are combined.

[0080] Comparative Example 2

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

[0082] 1) Crush and sieve quinoa seeds, weigh quinoa powder, add deionized water and anhydrous calcium chloride, stir, and heat for gelatinization; add α-amylase, keep warm at 70-85° C. for liquefaction, cool after liquefaction, adjust pH, heat to 55-60° C., add saccharifying enzyme, keep warm for saccharification for 2-5 hours, cool after saccharification, adjust pH, sterilize by high pressure, and then cool.

[0083] 2) The activated bifid yeast seed liquid of the above preparation example is inoculated into the sterile saccharification liquid of step 1) at an inoculation rate of 10 vol%, and cultured at 37° C. The fermentation is stopped when the pH value reaches a certain range; after filtering to remove the quinoa residue, the bacteria are crushed, the supernatant is obtained by high-speed centrifugation, and after filtering and sterilization, the quinoa fermentation filtrate is obtained.

[0084] Comparative Example 3

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

[0086] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish, add an appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and a 16h / 8h photoperiod.

[0087] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them flat in the induction MS+2,4-D (1.6 mg / L) medium, and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0088] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0089] Comparative Example 4

[0090] This comparison provides a method for preparing quinoa callus (dried and crushed) fermentation filtrate, and the preparation method specifically comprises the following steps:

[0091] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish and add appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and 16h / 8h photoperiod.

[0092] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them flat in the induction MS+2,4-D (1.6 mg / L) medium, and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, dry it at 60°C to constant weight, and crush and sieve it.

[0093] 3) The activated bifid yeast seed liquid of the above preparation example is inoculated into MRS liquid culture medium at an inoculation rate of 10 vol%, and the quinoa callus powder of step 2) is dispersed in the culture medium at a concentration of 30 g / L, and expanded culture is carried out at 37° C. After the fermentation is completed, the quinoa tissue residue is filtered out, the bacteria are optionally crushed, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by sterilization by filtration.

[0094] Comparative Example 5

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

[0096] Take the lactobacillus frozen in a -80°C refrigerator, inoculate and purify it on an MRS solid plate with an inoculation loop, culture it anaerobically at 37°C for 48 hours, pick a single colony and transfer it to 5 mL of new liquid culture medium, grow it anaerobically at 37°C for 24 hours, then inoculate 4 vol% and subculture it to a new liquid culture medium, culture it anaerobically at 37°C for 24 hours, activate it three times in a row, and then take out the activated lactobacillus seed liquid.

[0097] 1) Select mature and full-grained quinoa seeds, clean them with running water, and sterilize them with 75% ethanol in a sterile environment. Rinse the sterilized seeds with sterile water, treat them with 10% sodium hypochlorite (NaClO), and finally rinse them with sterile water again. Place the sterilized seeds neatly in a sterilized culture dish, add 2 filter papers in the culture dish and add appropriate amount of sterile water to soak the filter papers, and culture them at 25°C and 16h / 8h photoperiod.

[0098] 2) Take the sterile seedlings cultured for 8 days, cut the quinoa cotyledons, spread them flat in the induction MS+2,4-D (1.6 mg / L) medium, and culture them at 25°C and 16h / 8h photoperiod. Take the quinoa callus grown to 30 days, freeze it at -80°C for 12h, and grind it in liquid nitrogen.

[0099] 3) Inoculating the lactobacillus seed liquid into an MRS liquid culture medium at an inoculation rate of 10 vol%, and taking the quinoa callus powder of step 2), dispersing it in the culture medium at a concentration of 30 g / L, and expanding the culture at 37° C. After the fermentation is completed, filtering to remove the quinoa tissue residue, optionally breaking the bacteria, centrifuging to obtain the supernatant, and filtering and sterilizing to obtain the quinoa callus fermentation filtrate.

[0100] Test Example 1

[0101] Tyrosinase activity inhibition assay

[0102] L-tyrosine solution, sample solution (Example 1-7 and Comparative Example 1-5), and PBS buffer were added to a 96-well plate in sequence, mixed thoroughly, and incubated at 37°C for 10 min. Then, 20 μL of tyrosinase solution (500 U / mL) was added to each well in sequence. After mixing and reacting at 37°C for 5 min±5 s, the plate was immediately placed in a microplate reader (the time from the addition of the tyrosinase solution to the absorbance measurement of each well was kept consistent), and the plate was measured at a wavelength of 475 nm.

[0103] In a 96-well ELISA plate, set up solvent background wells (T1), solvent reaction wells (T2), sample background wells (T3), and sample reaction wells (T4). Among them, group T1 is a solvent background group, without adding substrate L-tyrosine solution and sample solution; group T2 is a solvent reaction group, with substrate L-tyrosine solution but no sample solution; group T3 is a sample background group, without adding substrate L-tyrosine solution and adding sample solution; group T4 is a sample reaction group, with both substrate L-tyrosine solution and sample solution. Three replicate wells are made for each group.

[0104] Table 1. Tyrosinase activity inhibition test reagent addition table

[0105]

[0106]

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

[0108]

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

[0110] The specific test results are as follows:

[0111] Table 2. Tyrosinase activity inhibition rate

[0112]

[0113] It can be seen from the test results in Table 2 that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application can well inhibit tyrosinase activity, with an inhibition rate of more than 79%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate of tyrosinase activity is about 1.5 times higher, and its inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is first increased and then decreased. Through research, it is found that the callus that has grown for about 30 days has the best inhibitory effect on tyrosinase activity after fermentation, and it is unexpectedly found that changes in quinoa origin, callus crushing method, fermentation strain, etc. will reduce the inhibition of tyrosinase activity by fermentation products. The experiment proves that the quinoa callus fermentation filtrate of the present invention has a good whitening effect.

[0114] Test Example 2

[0115] Melanin synthesis inhibition test

[0116] B16 melanoma cells were cultured at 2.5×10 3 The cells were inoculated at a density of 100 / mL in a 6-well plate, 2 mL per well, and incubated in a 37°C, 5% CO2 incubator for 24 hours. Then, 3 mL of culture solution containing samples (Examples 1-7 and Comparative Examples 1-5) was added to each well. A blank control group without culture medium and cells was set up at the same time.

[0117] After the culture plate was placed in the incubator for 72 hours, the supernatant was discarded, the cells were washed twice with 1×PBS, the PBS was aspirated, 200μL of 0.25% trypsin was added to each well to digest the cells, and the cells were placed in a CO2 incubator for digestion for 5 minutes. 2mL PBS was added twice to blow the cells, and the cells were collected into a centrifuge tube and centrifuged at 4000rpm for 5 minutes. Try to suck out the residual PBS in the centrifuge tube, add 200μL of melanin extract (1mol / L NaOH containing 10% DMSO) to each tube, shake evenly, and heat in an 80℃ water bath for 1h. After cooling, moderately remove the droplets from the tube wall, blow evenly, aspirate 150μL of solution in each centrifuge tube, transfer to a 96-well plate, and detect the absorbance of each well at 405nm with an ELISA reader.

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

[0119]

[0120] Where: T-absorbance of the test sample well; C-average of the absorbance of the negative control group for 3 times; C0-background absorbance of the melanin extract.

[0121] The specific test results are as follows:

[0122] Table 3. Melanin synthesis inhibition rate

[0123] Test samples 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] It can be seen from the test results in Table 3 that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application has a good inhibitory effect on melanin synthesis, with an inhibition rate of more than 80%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate of melanin synthesis is about 1.6 times higher, and its inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is first increased and then decreased. Through research, it is found that the callus that has grown for about 30 days has the most significant inhibitory effect on melanin synthesis after fermentation, and changes in quinoa origin, callus crushing method, fermentation strain and other conditions will also affect the inhibitory activity of fermentation products on pigments. The experiment proves that quinoa callus fermentation filtrate can inhibit melanin synthesis, thereby reducing pigment deposition and promoting skin metabolism, further proving that it can achieve whitening effects from multiple aspects.

[0125] Test Example 3

[0126] DPPH free radical scavenging activity test

[0127] The test was conducted by referring to the method specified in Larrauri JA, and a 0.2 mM DPPH solution was prepared; the test sample solution was diluted 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 and reacted for 30 minutes, and the absorbance was measured at 517 nm, with anhydrous ethanol as a blank control.

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

[0129]

[0130] Wherein: 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, A2 is the absorbance of 2.0 mL of DPPH solution and 2.0 mL of anhydrous ethanol, and the average value is taken after three parallel tests.

[0131] The specific test results are as follows:

[0132] Table 4. DPPH free radical inhibition rate

[0133] Test samples 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] It can be seen from the test results in Table 4 that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application has a good inhibitory effect on DPPH free radicals, and the inhibition rate can reach more than 79%. Compared with the traditional plant extraction method (Comparative Example 1), the inhibition rate of DPPH free radicals is about 1.4 times higher, and its inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is first increased and then decreased. Through research, it is found that the callus that has grown for about 30 days has the most significant inhibitory effect on DPPH free radicals after fermentation. The same quinoa origin, callus crushing method, and fermentation strain also significantly reduce the inhibition rate of free radicals. The experiment proves that the quinoa callus fermentation filtrate has a good scavenging effect on DPPH free radicals, can enhance the antioxidant capacity of skin tissue, reduce the free radical content of skin tissue, and reduce skin dullness caused by lipid peroxidation.

[0135] Test Example 4

[0136] Oil Red O staining method to test the oil control effect in vitro

[0137] When the cell plating rate in the 24-well plate reaches 40-60%, group administration is performed, with 3 replicate wells in each group. 1 mL of MEM culture fluid is added to each well of the blank control group, 1 mL of culture fluid containing 50 μM isotretinoin is added to each well of the positive control group, and 1 mL of culture fluid containing 5% sample (Example 1-7 and Comparative Example 1-5) is added to each well of the experimental group. After administration, the cells are placed in a CO2 incubator (37°C, 5% CO2) and cultured for 72 hours, with the fluid changed and administration performed every day during the culture period.

[0138] The old solution was discarded, the cells were washed with PBS, and the cells were fixed with paraformaldehyde. 300 μL of Oil Red O working solution was added to each well. The cells were stained for 15 min in a dark environment, washed with PBS, and observed with a microscope. The results were as follows: Figure 1 After taking the photo, discard the waste liquid, add 200 μL of isopropanol to each well, and incubate on a microplate shaker at 800 rpm for 5 minutes at room temperature to fully dissolve Oil Red O in isopropanol. Then take 20 μL from each well and transfer to a 96-well plate to dilute 10 times, and then read the absorbance at 510 nm using an enzyme reader.

[0139] Calculate the oil secretion inhibition rate:

[0140]

[0141] The specific test results are as follows:

[0142] Table 5. Oil secretion inhibition rate

[0143]

[0144] It can be seen from the test results in Table 5 that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application has a good inhibitory effect on oil secretion, and the inhibition rate can reach more than 70%, which is about 1.2 times that of isotretinoin, and about 1.3 times that of the traditional plant extraction method (Comparative Example 1). The inhibitory effect is positively correlated with the seedling growth time, but the relationship with the callus growth time is first increased and then decreased. Through research, it is found that the fermentation of callus tissue that has grown for about 30 days has the best inhibitory effect on oil secretion. After changes in the origin of quinoa, the method of callus crushing, the fermentation strain, etc., the effect on oil secretion becomes smaller. The experiment proves that the quinoa callus fermentation filtrate of the present invention has a better clearing effect on oil secretion, which can achieve effective oil control.

[0145] Combined with the above-mentioned test examples 1-4, it was found that the culturing time of quinoa seedlings was positively correlated with the efficacy of quinoa callus fermentation filtrate, which may be related to the growth of some effective ingredients over time, but not positively correlated with the callus development time. The analysis may be because the accumulation of relevant components stagnates after the callus grows to 40 days, and the tissue cells only have the continuous expansion of volume, but no longer secrete effective ingredients, and even consume some of the original nutrients, resulting in a decrease in efficacy. In the experiment, it was unexpectedly found that the callus fermentation liquid efficacy of quinoa from different origins also has certain differences. The quinoa from Qinghai origin is affected by various factors such as long light, large temperature difference between day and night, and fertile soil. Its active substance content may be richer, so the fermentation product efficacy is better, and the application of callus can well reduce the influence of these environmental factors. The effect of different crushing methods on quinoa callus fermentation filtrate may be due to different temperatures under different crushing methods. High temperature causes some components related to whitening and oil control to be inactivated, thereby affecting its efficacy, and the process of ultra-low temperature grinding can retain the activity of the effective ingredients to the greatest extent. Finally, we also found that if we want to obtain more ideal efficacy, we must preferably use the bifid yeast of the present invention as the strain for fermentation.

[0146] Test Example 5

[0147] Human body efficacy evaluation - whitening

[0148] Test recipe

[0149] Table 6. Test essence formula

[0150] INCI Experimental Group Control group Blank Group Butanediol 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 glycyrrhizinate 0.1 0.1 0.1 Example 1 1 0 0 ascorbic acid 0 5 0 water 92.42 88.42 93.42

[0151] 30 healthy subjects were recruited. Inclusion criteria: 18-59 years old, healthy male or female; serious and positive attitude, able to use the product correctly according to regulations and return visits on time; have accurate writing and language expression ability, and can truly reflect the feelings after using the product.

[0152] Exclusion criteria: pregnant or breastfeeding women, or those who are planning to get pregnant in the near future; those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, etc.; those who have taken oral or topical anti-inflammatory drugs such as corticosteroids in the past month; those who have taken oral or topical products or drugs that affect skin color (such as hydroquinone preparations) in the past 2 months; those who have participated in similar trials in the past 3 months or participated in similar trials 3 months ago.

[0153] Test method: Divide the subjects into 3 groups (experimental group, control group and blank group, 10 persons in each group) for blind test. The subjects used the sample solution once in the morning and evening every day for 4 consecutive weeks. No other whitening skin care products were used during the test. Tests were conducted on the 0th day (before use), 14th day and 28th day of the experiment.

[0154] Before the test, the subjects were required to clean their faces with clean water, wipe off the moisture on their faces, sit quietly for 20 minutes in a constant temperature and humidity environment (20±2℃, relative humidity 50±5%), and then take the test.

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

[0156] ITA° is calculated as follows:

[0157]

[0158] The specific test results are as follows:

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

[0160]

[0161]

[0162] It can be seen from the test results in Table 7 that compared with the blank group, all the people in the experimental group showed significant improvement 2 weeks and 4 weeks after using the bifid yeast fermentation filtrate prepared by adding quinoa callus tissue (P < 0.05). Compared with ascorbic acid, quinoa callus fermentation filtrate has the characteristics of low effective concentration and strong efficacy, and none of the above subjects reported any discomfort symptoms such as allergies, which proves that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application has a whitening effect.

[0163] Test Example 6

[0164] Human efficacy evaluation - oil control

[0165] Table 8. Test essence formula

[0166] INCI Formula ratio Butanediol 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 glycyrrhizinate 0.1 Example 1 1 water 92.42

[0167] Thirty healthy subjects were recruited. Inclusion criteria: 19-35 years old, healthy male; people with oily skin, skin oil content on the forehead ≥ 120 μg / cm 2 ; Have a serious and positive attitude, be able to use the product correctly as required and make return visits on time; have accurate written and verbal expression skills, and be able to truly reflect the feelings after using the product.

[0168] Exclusion criteria: patients who have used antihistamines in the past week or immunosuppressants or immunomodulatory biological agents and small molecule drugs in the past month; patients who have used any anti-inflammatory drugs in the test area in the past two months; patients with clinically uncured inflammatory skin diseases; patients with insulin-dependent diabetes; patients who have had an attack and treatment of allergic diseases or used anti-inflammatory drugs such as hormones in the past month; patients whose skin scars, pigmentation, atrophy, port-wine stains or other blemishes on the test area may affect the determination of the test results.

[0169] Test method: The subjects used the essence of formula in Table 8 once in the morning and once in the evening for 4 consecutive weeks. No other whitening skin care products were used during the test period. The test was carried out on the 0th day (before use) and the 28th day of the experiment.

[0170] Before the test, the subjects were required to clean their faces with clean water, wipe off the moisture on their faces, sit quietly for 20 minutes in a constant temperature and humidity environment (20±2℃, relative humidity 50±5%), and then take the test.

[0171] The skin oil content tester Sebumeter (SM815, Courage and Khazaka, Germany) uses the world-recognized SEBUMETER method to characterize the oil content of the skin by measuring the change in light transmittance of a 0.1mm thick special matte tape before and after absorbing oil. During the test, the tape is in contact with the test area and becomes translucent after absorbing the oil on the human skin. The light transmittance also changes accordingly. The more oil absorbed, the greater the light transmittance. Measuring the light transmittance of the tape can measure the oil content of the skin (measuring the skin oil of 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, T2 is after 2 weeks of use; SPSS Statistics 25 was used for statistical analysis, two-tailed test, test level α = 0.05; measurement data were analyzed based on the normal distribution test results, and the difference analysis method was selected: if the measurement value was normally distributed, the t test method was used for statistical analysis; if it was non-normally distributed, the rank sum test method was used for statistical analysis.

[0175] The specific test results are as follows:

[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): the difference is not statistically significant; “***” (P<0.001): the difference is statistically significant.

[0181] The representative figure of skin oil content test results is as follows Figure 2 shown.

[0182] From Table 9 and Figure 2 The test results show that the oil content of the volunteers was significantly reduced by 41.08% (P<0.001) 4 weeks after using the bifid yeast fermentation filtrate prepared with quinoa callus tissue, and no allergic symptoms were reported, proving that the quinoa callus bifid yeast fermentation filtrate prepared by the method of the present application has the effect of controlling oil.

[0183] Test Example 7

[0184] Chicken embryo chorioallantoic membrane vascular test

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

[0186] Place the fertilized chicken embryo in a constant temperature and humidity chamber and incubate it at (37±1)℃ and 50%-70% humidity for about 9 days. Take it out and place it on a homemade egg rack with the air chamber end facing upward. Use the toothed forceps to gently break the eggshell and carefully remove the eggshell at the air chamber end to completely expose the eggshell membrane. Add 1mL of physiological saline to moisten the eggshell membrane, absorb the physiological salt, and carefully peel off the eggshell membrane with curved forceps to expose the chorioallantoic membrane (CAM).

[0187] Chicken embryos were randomly divided into groups, with the negative control group dripping with 0.9% NaCl solution, the positive control group dripping with 1% SDS solution, and the experimental group dripping with the sample of the present invention (Example 1), with at least 3 chicken embryos in each group. 0.3 mL of the tested agent was respectively drawn with a pipette and dripped on the surface of the chorioallantoic membrane, and the changes in the blood vessels within 5 minutes were recorded, and the bleeding and coagulation conditions of the outflowing blood vessels were observed, and photographs and videos were taken.

[0188] Representative picture of chicken embryo chorioallantoic membrane vascular test Figure 3 shown.

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

[0190] The above examples are only illustrative and are used to explain some features of the method of the present invention. The protection scope of this application is not limited to the above examples. All technical solutions under the concept of this application belong to the protection scope of this application. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of this application should also be regarded as the protection scope of this application.

Claims

1. A method for preparing quinoa callus fermentation filtrate with whitening and oil-control effects, comprising the following preparation steps: 1) Select mature quinoa seeds with full grains, wash and disinfect them to obtain sterile seeds for later use; 2) Under sterile conditions, the sterilized seeds are neatly placed in a sterilized culture dish and cultured at 25°C with a 16h / 8h photoperiod. This step is to obtain sterile seedlings for later use; 3) When the sterile seedlings are cultured to at least have cotyledons, the obtained sterile seedlings are cut into pieces to obtain quinoa explants; 4) The explants obtained in 3) were respectively spread into induction medium, cultured at 25° C. and 16 h / 8 h photoperiod to obtain quinoa callus, and crushed to obtain quinoa callus powder; 5) The activated bifid yeast seed solution is inoculated into MRS liquid culture medium, and the quinoa callus powder obtained in 4) is dispersed into MRS liquid culture medium, and expanded culture is performed at 37° C. to obtain fermentation filtrate.

2. The method for preparing a quinoa callus fermentation filtrate having 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, then treat with sodium hypochlorite, and finally rinse with sterile water again.

3. The method for preparing a quinoa callus fermentation filtrate having whitening and oil-controlling effects according to claim 1, characterized in that: The sterile seedlings in 3) are 4-8 days old seedlings; the quinoa explants include cotyledon explants, hypocotyl explants and embryo explants.

4. The method for preparing the quinoa callus fermentation filtrate with whitening and oil-controlling effects according to claim 1, characterized in that: Step 4) The induction medium is MS medium plus 2,4-D, the concentration of 2,4-D is 0.4-2.0 mg / L, and the callus is a callus block cultured for 20-40 days.

5. The method for preparing the quinoa callus fermentation filtrate with whitening and oil-control effects according to claim 1, characterized in that: Step 4) The pulverization is carried out by freeze-drying and grinding in liquid nitrogen, and the freeze-drying time is 10-15 hours.

6. The method for preparing the quinoa callus fermentation filtrate with whitening and oil-control effects according to claim 1, characterized in that: In step 5), the bifid yeast seed solution is activated by the following method: Take the bifid yeast frozen in a -80°C refrigerator, streak inoculate and purify it on an MRS solid plate with an inoculation loop, culture it anaerobically at 37°C for 48 hours, pick a single colony and transfer it to 5 mL of new liquid culture medium, grow it anaerobically at 37°C for 24 hours, then inoculate 4 vol% and subculture it to a new liquid culture medium, culture it anaerobically at 37°C for 24 hours, and activate it three times in a row to obtain the activated bifid yeast seed liquid.

7. The method for preparing the quinoa callus fermentation filtrate having whitening and oil-controlling effects according to claim 1, characterized in that: Step 5) The inoculation amount of the bifid yeast is 5-10 vol%, the culture time is 37° C., and the culturing time is 36-48 h. Quinoa callus powder is dispersed in the culture medium, and the concentration of quinoa is 12-30 g / L.

8. The method for preparing the quinoa callus fermentation filtrate having whitening and oil-controlling effects according to claim 1, characterized in that: In step 5), after the fermentation is completed, the quinoa tissue residue is filtered out, the bacterial cells are optionally broken again, the supernatant is obtained by centrifugation, and the quinoa callus fermentation filtrate is obtained by filtration and sterilization.

9. A cosmetic with whitening and oil-control effects, characterized in that: The cosmetic comprises at least the fermentation filtrate according to any one of claims 1 to 8.

10. Use of the quinoa callus hair filtrate obtained according to claims 1-8 in the preparation of cosmetics.

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