Copper-rich yeast extract with anti-aging and repairing effects and preparation method thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a copper-rich yeast extract with anti-aging and repairing effects and a preparation method thereof. The present application provides a preparation method of the copper-rich yeast extract. The copper-rich yeast extract powder prepared by the preparation method has good solubility, stable color, anti-aging and repairing effects, and can be widely applied in the fields of medicine, feed, food and cosmetics. The preparation method is simple and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a copper-rich yeast extract with anti-aging and repairing effects and its preparation method. Background Technology

[0002] Copper is a coenzyme for many antioxidant enzymes, such as SOD. It also inhibits the inflammatory factor IL-17A, repairs damaged DNA within cells, and plays important roles in maintaining normal hematopoiesis, preserving the integrity of the central nervous system, promoting bone, blood vessel, and skin health, and participating in the body's antioxidant processes. Copper is an essential trace element for the human body, with an adult body containing 50-150 mg / kg.

[0003] Five thousand years ago, humans were already using the fermentation properties of yeast to improve the flavor and texture of food. Today, yeast is an indispensable element in the preparation of many foods. Furthermore, yeast can convert inorganic copper into organic copper; copper-rich yeast, rich in organic copper, has significantly lower toxicity compared to inorganic copper and is widely used in animal feed and food. However, the copper in copper-rich yeast is located inside the cells, making it difficult for humans and animals to fully utilize the copper within the yeast cells.

[0004] Existing preparation methods yield copper-rich yeast extracts with poor solubility and unstable color, which greatly limits their applications. Summary of the Invention

[0005] In view of this, the present invention provides a copper-rich yeast extract with anti-aging and repairing effects, and a method for preparing the same. The copper-rich yeast extract prepared by the method provided by the present invention exhibits good solubility, stable color, and anti-aging and repairing effects, and can be widely used in pharmaceuticals, animal feed, food, and cosmetics. This preparation method is simple and suitable for large-scale industrial production.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing copper-rich yeast extract, comprising the following steps:

[0008] Step 1: Prepare a copper-rich yeast solution with a mass fraction of 5-30% and perform heat shock treatment;

[0009] Step 2: Enzymatic hydrolysis with a protease at a concentration of 200-2000 U / mL; the protease includes any one or a combination of at least two of acidic protease, neutral protease, alkaline protease, or papain.

[0010] Step 3: Solid-liquid separation and decolorization to obtain a decolorized solution, which is then dried to obtain the copper-rich yeast extract.

[0011] In some specific embodiments of the present invention, step 3 is followed by drying the decolorized solution to obtain copper-rich yeast extract powder.

[0012] In some specific embodiments of the present invention, the copper-rich yeast extract includes the decolorized solution and / or the copper-rich yeast extract powder.

[0013] Preferably, the temperature of the heat shock treatment in step 1 is 80~95℃; and the time of the heat shock treatment is 5~50s.

[0014] Preferably, the copper-rich yeast in step 1 includes any one or a combination of at least two of the following: copper-rich yeast fermentation broth obtained by fermentation using copper-rich yeast strains, copper-rich yeast milk, copper-rich yeast sludge, and active or inactive copper-rich dry yeast obtained by drying.

[0015] As a preferred method, copper-enriched yeast fermentation specifically involves: at 10m 3 Copper-rich yeast fermentation was carried out in a fermenter with an initial fermentation volume of 3m³. 3 The tertiary seed yeast milk from (3) was inoculated into the fermenter, with an initial yeast wet weight of 70 g / L. Fermentation nutrient solution was fed in continuously during fermentation. Fermentation nutrient solution was fed in continuously during the culture period; at 960 min, the feeding of fermentation nutrient solution was stopped and ventilation was stopped, with a ventilation rate of 200 m³ / min. 3 / h. Adjust the pH to 5.0 and add copper sulfate solution continuously until the copper concentration in the fermentation broth reaches 550 mg / kg, then stop adding copper sulfate solution. Throughout the fermentation process, the fermentation temperature is 30℃, and the culture is carried out at 200-700 rpm until the total fermentation volume reaches 7 m³. 3 When the fermentation is complete, stop fermentation to obtain copper-rich yeast fermentation broth.

[0016] The copper-rich yeast fermentation broth was centrifuged and washed three times to obtain copper-rich yeast milk.

[0017] After using plate and frame filter press to increase the dry matter content to 35% (w:w), copper-rich yeast sludge is obtained.

[0018] Active copper-rich dry yeast was obtained by granulation and fluidized bed drying of copper-rich yeast sludge. The drying conditions were: drying at 70℃ with air temperature until the dry matter content was 95% (w:w).

[0019] Copper-enriched yeast milk was sterilized at 121℃ for 30 minutes and then spray-dried to obtain inactive copper-enriched yeast. The spray-drying conditions were: inlet air temperature 150℃ and outlet air temperature 90℃.

[0020] Preferably, the copper-rich yeast includes a yeast strain with accession number CCTCC NO:M 2017781.

[0021] Preferably, step 1 further includes preparing a 5-30 wt% solution of copper-rich yeast before the heat shock treatment.

[0022] Preferably, the pH value of the enzymatic hydrolysis in step 2 is 4.0~8.5; and the temperature of the enzymatic hydrolysis is 45~65℃.

[0023] Preferably, the enzymatic hydrolysis time in step 2 includes 10-20 hours.

[0024] Preferably, the combination in step 2 is selected from any one of the following: alkaline protease and neutral protease; neutral protease, acidic protease and papain; or acidic protease and neutral protease.

[0025] Preferably, the decolorization in step 3 includes a method of decolorization using any one or a combination of at least two of the following: gel, macroporous resin, activated carbon, or membrane separation.

[0026] Preferably, the drying in step 3 includes concentrating the decolorized solution to 10-30 wt% dry matter and then spray drying it.

[0027] Secondly, the present invention provides a copper-rich yeast extract prepared by the aforementioned preparation method.

[0028] In some specific embodiments of the present invention, the copper-rich yeast extract includes the decolorized solution and / or the copper-rich yeast extract powder;

[0029] Preferably, the copper content of the copper-rich yeast extract powder is 3000~30000 mg / kg.

[0030] Preferably, the protein content of the copper-rich yeast extract powder is 40-65 wt%.

[0031] Preferably, the solubility of the copper-rich yeast extract powder in water is 30-60%.

[0032] Thirdly, the present invention provides the application of the copper-rich yeast extract in the preparation of cosmetics with anti-UV, soothing, repairing, moisturizing, firming or anti-wrinkle properties.

[0033] Preferably, the application includes any of the following:

[0034] (I) Reduce the proportion of red area on the skin;

[0035] (II) Reduce skin F4 value;

[0036] (III) Reduce skin TWEL value;

[0037] (IV) Reduce the proportion of the area covered by crow's feet;

[0038] (V) Reduce the proportion of under-eye wrinkles;

[0039] (VI) Increase the moisture content of the stratum corneum of the skin.

[0040] Preferably, the product includes any one or a combination of at least two of the following: pharmaceuticals, animal feed, food, or cosmetics.

[0041] Fourthly, the present invention provides a composition comprising the copper-rich yeast extract described above.

[0042] Preferably, the composition further includes any one or a combination of at least two of the following: KAPPA MB, A-165, 1618 alcohol, GTCC, 122P beeswax, COSMOL ARV168, 2-EHP, DC-200 (350cs), 1,3-butanediol, glycerol, carbomer 941, PE9010, triethanolamine, or water.

[0043] Preferably, the composition, by weight percentage, comprises: 2% KAPPA MB, 1% A-165, 1.2% 1618 alcohol, 3% GTCC, 0.5% 122P beeswax, 1% COSMOL ARV 168, 4% 2-EHP, 2% DC-200 (350cs), 2% 1,3-butanediol, 3% glycerin, 0.15% Carbomer 941, 0.6% PE9010, 0.1% of the copper-rich yeast extract, and 0.15% triethanolamine, with the balance being water.

[0044] Fifthly, the present invention provides the use of the composition in the preparation of cosmetics.

[0045] Preferably, the cosmetic includes any one of skin cream, facial cleanser, lotion, facial mask, anti-wrinkle agent or skin lotion.

[0046] This invention provides the following beneficial effects:

[0047] 1. High solubility: Intracellular proteins are degraded into small peptides and amino acids through enzymatic hydrolysis by proteases, thereby increasing solubility (from 12% to 65%).

[0048] 2. Stable color and wide range of applications: The color of the extract changes from dark green to a stable blue after decolorization, and it can be widely used in medicine, feed, food, cosmetics and other fields.

[0049] 3. The copper-rich yeast extract obtained by this preparation method has significant anti-aging and repair effects.

[0050] Biological Preservation Instructions

[0051] Classification and naming: Saccharomyces cerevisiae A3.12; deposit date: December 11, 2017; accession number: CCTCC NO: M 2017781; depositary institution: China Center for Type Culture Collection; address: Wuhan University, Wuhan, China. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0053] Figure 1 Indicates fibroblast survival rate;

[0054] Figure 2 Indicates COL-1 content;

[0055] Figure 3 This indicates the amount of IL-1α secreted;

[0056] Figure 4 Indicates HaCaT proliferation rate;

[0057] Figure 5 Indicates HaCaT cell adhesion levels;

[0058] Figure 6 Show the percentage of the red area on the skin;

[0059] Figure 7 Indicates skin F4 value;

[0060] Figure 8 Show skin TWEL value;

[0061] Figure 9 Indicates the moisture content of the stratum corneum of the skin;

[0062] Figure 10 The percentage of the area showing crow's feet;

[0063] Figure 11 The percentage of the area under the eyes is shown;

[0064] Figure 12 Method for preparing copper-rich yeast extract;

[0065] Figure 13 Comparison of copper sulfate solution (left) and yeast blue core solution (right);

[0066] Figure 14 Comparison of copper sulfate solution (left) and yeast blue core solution (right) after pH adjustment to alkalinity;

[0067] Figure 15 Comparison of copper sulfate solution and yeast blue core solution after centrifugation;

[0068] Figure 16 A comparison of copper sulfate light phase solution (left) and yeast blue core light phase solution (right). Detailed Implementation

[0069] This invention discloses a copper-rich yeast extract with anti-aging and repairing effects and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0070] This invention provides a method for preparing copper-enriched yeast extract. The copper-enriched yeast extract prepared by this method has good solubility, a blue color, and good stability. The copper content in the copper-enriched yeast extract is 3000~30000 mg / kg, the protein content is 40~65%, and the solubility is 30~60%.

[0071] The method for preparing copper-rich yeast extract provided by this invention ( Figure 12 This includes the following steps:

[0072] Step 1, Preparation: Prepare a solution of copper-rich yeast milk / copper-rich yeast slurry / active copper-rich yeast / non-active copper-rich yeast with a mass fraction of 5-30%.

[0073] Step 2, heat shock: Heat shock treatment at 80~95℃ for 5~50 seconds.

[0074] Step 3, Enzymatic hydrolysis: Adjust the pH to 4.0~8.5, raise the temperature to 45~65℃, and add 200~2000U / mL of protease for enzymatic hydrolysis for 10~20h. The protease consists of one or more of acidic protease, neutral protease, alkaline protease, and papain.

[0075] Step 4, solid-liquid separation: After enzymatic hydrolysis, solid and liquid are separated and the separated solution is collected.

[0076] Step 5, Decolorization: The separated solution is decolorized using one or more of the following methods: gelation, macroporous resin, activated carbon, and membrane separation, to obtain a decolorized solution.

[0077] Step 6, Drying: After concentrating the decolorized solution to 10%~30% dry matter, dry it to obtain copper-rich yeast extract powder.

[0078] The present invention also provides a copper-rich yeast extract obtained by the preparation method, which has significant anti-aging and repair effects.

[0079] The beneficial effects provided by this invention are as follows:

[0080] 1. High solubility: Intracellular proteins are degraded into small peptides and amino acids through enzymatic hydrolysis by proteases, thereby increasing solubility (from 12% to 65%).

[0081] 2. Stable color and wide range of applications: The color of the extract changes from dark green to a stable blue after decolorization, and it can be widely used in medicine, feed, food, cosmetics and other fields.

[0082] 3. The copper-rich yeast extract obtained by this preparation method has significant anti-aging and repair effects.

[0083] It should be noted that there are no explicit restrictions on the production strain of copper-enriched yeast used in this method. Any copper-enriched yeast strain used in the food, pharmaceutical, cosmetic, and feed industries can be used as the raw material for the copper-enriched yeast in this invention.

[0084] Unless otherwise specified, the raw materials and reagents used in the copper-rich yeast extract with anti-aging and repairing effects and its preparation method provided by this invention can all be purchased from the market.

[0085] The present invention will be further illustrated below with reference to the embodiments:

[0086] Example 1 Preparation of copper-enriched yeast

[0087] (1) Primary shake flask culture: Add 26g glucose, 4g yeast extract, 0.3g KH2PO4, and 0.3g MgSO4 to a 500mL Erlenmeyer flask, add purified water to make up to 200mL, adjust the pH to 5.0, and sterilize at 121℃ for 20min. Use an inoculation loop to scrape well-grown Saccharomyces cerevisiae A3.12 from the solid culture medium and inoculate it into the seed culture medium. Culture at 30℃ and 200rpm for 20h to obtain the primary seed culture.

[0088] (2) Secondary shake flask culture: 550g glucose, 100g yeast extract, 10g KH2PO4, 10g MgSO4, bring to a final volume of 5L, adjust pH to 5.5, and sterilize at 121℃ for 20min. Inoculate 2% of the seed culture from (1) at 30℃ and 200 rpm for 20h to obtain the secondary seed culture solution;

[0089] (3) Tertiary seed culture: Seed fermentation is carried out in a 500L fermenter. The secondary seed culture solution in (3) is inoculated into the fermenter. During the culture, fermentation nutrient solution is fed in and fermented at 30℃ until the yeast wet weight is above 150g / L. Fermentation is stopped, and the yeast is centrifuged and concentrated to obtain tertiary seed yeast milk.

[0090] (4) Copper-enriched yeast fermentation: at 10m3 Copper-rich yeast fermentation was carried out in a fermenter with an initial fermentation volume of 3m³. 3 The tertiary seed yeast milk from (3) was inoculated into the fermenter, with an initial yeast wet weight of 70 g / L. Fermentation nutrient solution was fed in continuously during fermentation. Fermentation nutrient solution was fed in continuously during the culture period; at 960 min, the feeding of fermentation nutrient solution was stopped and ventilation was stopped, with a ventilation rate of 200 m³ / min. 3 / h. Adjust the pH to 5.0 and add copper sulfate solution continuously until the copper concentration in the fermentation broth reaches 550 mg / kg, then stop adding copper sulfate solution. Throughout the fermentation process, the fermentation temperature is 30℃ and the rpm is 200-700 until the total fermentation volume reaches 7 m³. 3 Fermentation was stopped at a certain time, yielding a copper-rich yeast fermentation broth. The broth was centrifuged and washed three times to obtain a copper-rich yeast milk. The dry matter content was reduced to 35% (w:w) using plate and frame filter press to obtain a copper-rich yeast sludge. The sludge was granulated and then fluidized bed dried to obtain active copper-rich dry yeast. Drying conditions: 70℃ air temperature to 95% (w:w) dry matter. The copper-rich yeast milk was sterilized at 121℃ for 30 minutes and then spray-dried to obtain inactive copper-rich yeast. Spray drying conditions: inlet air temperature 150℃, outlet air temperature 90℃.

[0091] The fermentation nutrient solution includes: 3.2 m³ of molasses with a fermentable sugar content of 30% (960 kg of fermentable sugar), 90 L of 16% ammonia water, and 8 kg of NH₄H₂PO₄. The alcohol content in the fermentation broth is controlled at 0.1-0.3% during the fermentation process, and the flow rate of the nutrient solution is adjusted according to the alcohol content.

[0092] The copper content (on dry matter) and protein content (on dry matter) were determined by atomic absorption spectrometry and Kjeldahl nitrogen determination, respectively. The results are shown in Table 1.

[0093] Table 1

[0094]

[0095] Example 2 Preparation of copper-rich yeast extract (enzymatic hydrolysis with 200 U / mL alkaline protease for 15 h, decolorization with macroporous resin column)

[0096] Preparation: The copper-rich yeast milk from Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 80°C for 50 seconds.

[0097] Enzymatic hydrolysis: Adjust pH to 8.5, heat to 60℃, add 200 U / mL alkaline protease, and hydrolyze for 15 h.

[0098] Solid-liquid separation: Collect the filtrate after filtering the diatomaceous earth.

[0099] Decolorization and drying: The solution was decolorized using a macroporous resin column until it turned blue. The eluent (copper-rich yeast extract solution) was collected and then concentrated under vacuum to 10% dry matter. The copper-rich yeast extract powder was obtained by spray drying.

[0100] Example 3 Preparation of copper-enriched yeast extract (enzymatic hydrolysis with 800 U / mL alkaline protease and 800 U / mL neutral protease for 12 h, followed by gel column decolorization)

[0101] Preparation: The copper-rich yeast sludge from Example 1 was prepared into a 30% (w / w) solution, and then subjected to heat treatment at 95°C for 5 seconds.

[0102] Enzymatic hydrolysis: Adjust pH to 7.5, heat to 45℃, add 800 U / mL alkaline protease and 800 U / mL neutral protease, and hydrolyze for 12 h.

[0103] Solid-liquid separation: The light phase solution is collected after separation by centrifugation.

[0104] Decolorization and drying: The blue solution (copper-rich yeast extract solution) was separated and collected using a gel column chromatography method, and then concentrated under vacuum to 30% dry matter. The solution was then freeze-dried to obtain copper-rich yeast extract powder.

[0105] Example 4 Preparation of copper-enriched yeast extract (700 U / mL neutral protease, 700 U / mL acidic protease, 600 U / mL papain, enzymatic hydrolysis for 10 h, decolorization with activated charcoal)

[0106] Preparation: The active copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 85°C for 30 seconds.

[0107] Enzymatic hydrolysis: Adjust pH to 6.0, heat to 50℃, add 700 U / mL neutral protease, 700 U / mL acidic protease, and 600 U / mL papain, and hydrolyze for 20 h.

[0108] Solid-liquid separation: Collect the filtrate after ceramic membrane filtration.

[0109] Decolorization and drying: Decolorization was performed using an activated carbon column, and the blue solution (copper-rich yeast extract solution) was collected. Then, it was concentrated under vacuum to 15% dry matter. Spray drying was then performed to obtain copper-rich yeast extract powder.

[0110] Example 5 Preparation of copper-enriched yeast extract (2000 U / mL acidic protease, enzymatic hydrolysis for 10 h, gel column decolorization)

[0111] Preparation: The inactive copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 90°C for 15 seconds.

[0112] Enzymatic hydrolysis: Adjust pH to 4.0, heat to 65℃, add 2000 U / mL of acidic protease, and hydrolyze for 10 h.

[0113] Solid-liquid separation: After enzymatic hydrolysis, the light phase solution is collected after diatomaceous earth filtration.

[0114] Decolorization and drying: Decolorization was performed using a gel column chromatography method, and the blue solution (copper-rich yeast extract solution) was collected. Then, it was concentrated under vacuum to 20% dry matter. Spray drying was then performed to obtain copper-rich yeast extract powder.

[0115] Example 6 Preparation of copper-enriched yeast extract (800 U / mL acidic protease, 1200 U / mL neutral protease, enzymatic hydrolysis for 20 h, decolorization with activated charcoal)

[0116] Preparation: The active copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 85°C for 40 seconds.

[0117] Enzymatic hydrolysis: Adjust pH to 5.5, heat to 55℃, add 800 U / mL acidic protease and 1200 U / mL neutral protease, and hydrolyze for 20 h.

[0118] Solid-liquid separation: After enzymatic hydrolysis, the light phase solution is collected after diatomaceous earth filtration.

[0119] Decolorization and drying: Decolorization was performed using an activated carbon column, and the blue solution (copper-rich yeast extract solution) was collected. Then, it was concentrated under vacuum to 30% dry matter. Spray drying was then performed to obtain copper-rich yeast extract powder.

[0120] Example 7 Test experiment on free copper content in copper-enriched yeast extract

[0121] 1. Experimental Background and Objectives

[0122] Experimental Background

[0123] Free copper ions form an insoluble Cu(OH)₂ precipitate under alkaline conditions. In organic copper compounds, the copper is more firmly bound to organic matter, and no precipitate forms even under alkaline conditions. Therefore, the free copper content in a solution can be detected by alkaline precipitation.

[0124] Experimental Objective

[0125] The pH of the yeast blue core sample and copper sulfate solution was adjusted to above 10.0, causing free copper ions to form a water-insoluble precipitate. After centrifugation, the light phase solution was collected, and the copper content was determined to calculate the ratio of free copper in the yeast blue core and copper sulfate solutions.

[0126] 2. Experimental Procedure

[0127] Experimental materials

[0128] Yeast Blue Core Solution: 30g of copper-rich yeast extract powder (copper content 30000mg / kg) from Example 6 of the patent "A Copper-Rich Yeast Extract with Anti-Aging and Repairing Effects and Its Preparation Method" is added to purified water and weighed down to 600g. At this point, the copper content in the solution is 1500mg / kg, which is the yeast blue core solution. Figure 13 ).

[0129] Copper sulfate solution: 5.8940g of copper sulfate pentahydrate was diluted with purified water to a final volume of 1L to prepare a solution with a copper content of 1500mg / kg. Figure 13 ).

[0130] 4N NaOH solution: Add 160g of AR grade flake NaOH solid to purified water to a final volume of 1L, and cool to room temperature to obtain a 4N NaOH solution.

[0131] 10% H2SO4 solution: Dilute 50 mL of AR grade sulfuric acid with purified water to 500 mL to obtain a 10% H2SO4 solution.

[0132] Experimental content

[0133] (1) Adjusting pH to alkalinity: The pH of 80g of yeast blue core solution and copper sulfate solution was adjusted to above 10.0 using 4N NaOH solution and 10% H2SO4 solution, respectively. The measured pH of the yeast blue core solution was 11.63, and the pH of the copper sulfate solution was 11.08. After adjusting the pH to alkalinity, a blue precipitate appeared in the copper sulfate solution, while the yeast blue core solution remained clear and transparent without obvious precipitation. Figure 14 ).

[0134] (2) Centrifugation: After adjusting the pH of the yeast blue core solution and copper sulfate solution to alkaline, put them into a 50mL centrifuge tube and centrifuge at 5000rpm for 5min.

[0135] (3) After centrifugation, collect the light phase solution (e.g.) Figure 15 and Figure 16 (As shown), add purified water to a final volume of 100g.

[0136] The copper content in yeast blue core solution, copper sulfate solution, yeast blue core light phase solution and copper sulfate light phase solution was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to GB 5009.13, and the free copper content was calculated.

[0137] Free copper percentage (%) = 100% - (Weight of light phase solution * Copper content in light phase solution) / (Weight of solution * Copper content in solution) * 100%

[0138] 3. Experimental Results

[0139] The copper content detection results of yeast blue core solution, copper sulfate solution, yeast blue core light phase solution, and copper sulfate light phase solution are shown in Table 2:

[0140] When the pH of the copper sulfate solution is adjusted to 11.08, almost all the copper in the solution precipitates, making it impossible to detect the copper content in the light phase solution. Therefore, the proportion of free copper in the copper sulfate solution is >99.996%.

[0141] After adjusting the pH of the yeast blue core solution to 11.63, no obvious precipitation occurred in the solution, and the copper content in the light phase of the yeast blue core solution was not lost. Therefore, the proportion of free copper in the yeast blue core solution was 0%.

[0142] Table 2. Detection results of copper ions in different test samples

[0143]

[0144] 4. Discussion

[0145] a) Copper sulfate solution was used as a control group, which proved that free copper would form a precipitate at pH 11.0, thus verifying the authenticity and reliability of this method.

[0146] b. The free copper content in the yeast blue core solution (Patent Example 6: Copper-rich yeast extract powder diluted with purified water to a copper content of 1500 mg / kg) is 0%, and the organic copper content is 100%.

[0147] Comparative Example 1: Preparation of copper-enriched yeast extract (without enzymatic hydrolysis or decolorization)

[0148] Preparation: The active copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 85°C for 30 seconds.

[0149] Solid-liquid separation: The light phase solution is collected after diatomaceous earth filtration.

[0150] Drying: The collected light phase solution was concentrated under vacuum to 15% dry matter. Spray drying yielded copper-rich yeast extract powder.

[0151] Comparative Example 2: Preparation of copper-rich yeast extract (200 U / mL alkaline protease, enzymatic hydrolysis for 8 h, decolorization with macroporous resin).

[0152] Preparation: The active copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 80°C for 50 seconds.

[0153] Enzymatic hydrolysis: Adjust pH to 8.5, heat to 60℃, add 200 U / mL alkaline protease, and hydrolyze for 8 hours.

[0154] Solid-liquid separation: After enzymatic hydrolysis, the diatomaceous earth is filtered and the filtrate is collected.

[0155] Decolorization and drying: Decolorization was performed using a macroporous resin column, and the blue solution (copper-rich yeast extract solution) was collected. Then, it was concentrated under vacuum to 15% dry matter. Spray drying was then performed to obtain copper-rich yeast extract powder.

[0156] Comparative Example 3: Preparation of copper-enriched yeast extract (1500 U / mL acidic protease, 2000 U / mL neutral protease, enzymatic hydrolysis for 30 h, gel column color).

[0157] Preparation: The active copper-rich dry yeast in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 85°C for 40 seconds.

[0158] Enzymatic hydrolysis: Adjust pH to 5.5, heat to 55℃, add 1500 U / mL acidic protease and 2000 U / mL neutral protease respectively, and hydrolyze for 30 h.

[0159] Solid-liquid separation: After enzymatic hydrolysis, the diatomaceous earth is filtered and the filtrate is collected.

[0160] Decolorization and drying: Decolorization was performed using a gel column chromatography method, and the blue solution (copper-rich yeast extract solution) was collected. Then, it was concentrated under vacuum to 15% dry matter. Spray drying was then performed to obtain copper-rich yeast extract powder.

[0161] Comparative Example 4: Preparation of copper-enriched yeast extract (200 U / mL alkaline protease, enzymatic hydrolysis for 8 h, no decolorization).

[0162] Preparation: The copper-rich yeast milk in Example 1 was prepared into a 20% (w / w) solution and then subjected to heat shock treatment at 95°C for 10 seconds.

[0163] Enzymatic hydrolysis: Adjust pH to 8.5, heat to 60℃, add 200 U / mL alkaline protease, and hydrolyze for 8 hours.

[0164] Solid-liquid separation: Collect the filtrate after filtering the diatomaceous earth.

[0165] Drying: The light phase solution (copper-rich yeast extract solution) was concentrated under vacuum to 20% dry matter. The solution was then freeze-dried to obtain copper-rich yeast extract powder.

[0166] Example 1: Determination of protein content, solubility, and copper content in copper-enriched yeast extract.

[0167] The copper-rich yeast extract powder prepared using the method in Example 2 was blue in color and had a solubility of 30%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 3000 mg / kg and 40 g / 100 g, respectively.

[0168] The copper-rich yeast extract powder prepared using the method in Example 3 was blue in color and had a solubility of 43%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 16000 mg / kg and 47 g / 100g, respectively.

[0169] The copper-rich yeast extract powder prepared using the method in Example 4 was blue in color and had a solubility of 55%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 24000 mg / kg and 58 g / 100g, respectively.

[0170] The copper-rich yeast extract powder prepared using the method in Example 5 was blue in color and had a solubility of 57%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 28000 mg / kg and 60 g / 100 g, respectively.

[0171] The copper-rich yeast extract powder prepared using the method in Example 6 was blue in color and had a solubility of 60%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 30000 mg / kg and 65 g / 100g, respectively.

[0172] The copper-rich yeast extract powder prepared using the method of Comparative Example 1 was green in color and had a solubility of 12%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 400 mg / kg and 16 g / 100 g, respectively.

[0173] The copper-rich yeast extract powder prepared using the method of Comparative Example 2 was blue in color and had a solubility of 17%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 1400 mg / kg and 21 g / 100g, respectively.

[0174] The copper-rich yeast extract powder prepared using the method of Comparative Example 3 was blue in color and had a solubility of 60%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 30000 mg / kg and 65 g / 100g, respectively.

[0175] The copper-rich yeast extract powder prepared using the method in Comparative Example 4 was green in color and had a solubility of 11%. The copper and protein contents, determined according to GB 5009.13 (Method II) and GB 5009.5 (Method I), were 1800 mg / kg and 23 g / 100 g, respectively.

[0176] The results of the above examples and comparative experiments show that the preparation method of copper-enriched yeast extract of the present invention can significantly improve the protein content, solubility, and copper content of the copper-enriched yeast extract. The copper-enriched yeast extract has a stable blue color, which better meets consumers' color requirements for products. The preparation method is simple, increases product recovery rate, and significantly reduces production costs. Among them, Example 5 shows the best effect, with combined enzymatic hydrolysis using 800 U / mL acidic protease and 1200 U / mL neutral protease, resulting in a protein content, copper content, and solubility of 65 g / 100 g, 30000 mg / kg, and 60%, respectively.

[0177] Table 3. Protein content, solubility, and copper content of copper-enriched yeast extract

[0178]

[0179] Example 2: Effects of copper-enriched yeast extract on fibroblast survival rate, IL-1α and COL-1 levels after UV irradiation

[0180] 1. Sample pretreatment

[0181] The copper-rich yeast extract solution (copper-rich yeast extract powder) prepared in Example 5 was subjected to sterile filtration pretreatment using a 0.22 μm filter, and then diluted to the required concentration using BASICH-DEME medium.

[0182] 2. UV damage - cell viability test

[0183] Fibroblast plating

[0184] After cell counting, the cells were diluted to the desired concentration and the cell suspension was added to 96-well plates. After 24 hours of plating, the cell confluence rate was 40-60%.

[0185] The three test groups are set up as follows:

[0186] Table 4. Experimental Groups

[0187]

[0188] According to the above test groups, the samples were irradiated and treated with sample loading.

[0189] Cell viability test

[0190] Remove the waste liquid, add DEME containing 10% cck-8, incubate for 1~1.5h, and then use a microplate reader set to OD450nm to take the reading.

[0191] 3. Determination of IL-1α and COL-1 content

[0192] Collect 200 μL of cell culture supernatant from each well into a 1.5 mL sterile centrifuge tube and perform the assay according to the instructions for use of the IL-1α and COL-1 ELISA kit.

[0193] IL-1α: Sangon Biotech (Shanghai) Co., Ltd., D711047-0096, Human Interleukin-1α (IL-1α) ELISA Kit [KY]; COL-1: Wuhan Huamei Biotechnology Co., Ltd., CSB-E08082h, Human Type I Collagen (ColⅠ) ELISA Kit [KY].

[0194] Survival rate calculation

[0195] Survival rate (%) = (OD) Cn -OD VCb ) / (OD C0 -OD VCb )*100%

[0196] In the formula:

[0197] Cn—Contains the test substance and has cells;

[0198] C0—solvent control, no test substance, cells present;

[0199] VCb—solvent blank control, no test substance, no cells.

[0200] Calculation of IL-1α and COL-1 content

[0201] A curve was plotted with absorbance (OD) value on the ordinate (Y) and the corresponding concentration of the analyte standard on the abscissa (X). The concentration of the analyte in the sample could be calculated from the standard curve based on its OD value. The final result of the analyte concentration was taken as the average value of the three replicates for each group.

[0202] 4. Experimental Results

[0203] Cell viability decreased from 100% to 50.55% after UV irradiation, while cell viability increased to 72.06% in the copper-enriched yeast experimental group, demonstrating that copper-enriched yeast extract can significantly improve cell viability under UV irradiation. Figure 1 ).

[0204] The COL-1 content decreased from 100% to 49.12% after UV irradiation, while the COL-1 content in the experimental group increased to 168.59%, demonstrating that copper-rich yeast extract can significantly increase the COL-1 content under UV irradiation. Figure 2 ).

[0205] The IL-1α content increased from 100% to 171.16% after UV irradiation, while the IL-1α content in the experimental group decreased to 44.83%, demonstrating that copper-enriched yeast extract can significantly reduce IL-1α content during UV irradiation. Therefore, copper-enriched yeast extract can reduce UV-induced cell damage and has significant photodamage repair and anti-aging effects. Figure 3 ).

[0206] Example 3: Effect of copper-enriched yeast extract on HaCaT cell adhesion ability

[0207] 1. Sample pretreatment

[0208] The copper-rich yeast extract (copper-rich yeast extract powder) prepared in Example 6 was formulated into a copper-rich yeast extract solution with a copper content of 200 mg / kg. The copper-rich yeast extract solution was subjected to sterilization filtration pretreatment using a 0.22 μm filter.

[0209] 2. HaCaT cell proliferation test

[0210] After cell counting, the cells were diluted to the desired concentration and added to 96-well plates. After 24 hours of plating, when the cell confluence rate was 40-60%, the test substance was added using a pipette to achieve the set concentration. A control solution was prepared, and five replicates were set up for each experimental group. 48 hours after sample addition, the waste liquid was removed, and DMEM containing 10% CCK-8 was added. After incubation for 1-1.5 hours, the cells were detected using a microplate reader with an OD of 450 nm.

[0211] The experimental group was set up as follows:

[0212] Table 5. Experimental group setup

[0213]

[0214] 3. Cell adhesion test

[0215] 3.1 Sample addition

[0216] The test group settings are as follows:

[0217] Table 6. Experimental Groups

[0218]

[0219] Add 100 μL of the above-prepared reagent to a 24-well plate without TC treatment, with 3 controls per group.

[0220] Incubate overnight in a cell culture incubator until dried.

[0221] 3.2 Cell Plating

[0222] Healthy keratinocytes were digested, counted, and plated onto 24-well plates as described above. 2 mL was collected per well, with a cell density of 1 × 10⁻⁶ cells. 5 per mL.

[0223] 3.3 Detection

[0224] After 4 hours, tilt the culture plate to remove the culture medium and unattached cells.

[0225] Fix with 4% paraformaldehyde for at least 10 minutes, then wash twice with distilled water for 2 minutes each time.

[0226] Use a crystal violet staining kit to stain cells in the culture plate at room temperature for 10-20 minutes, depending on the number of cells in the sample well.

[0227] After thoroughly washing with distilled water to remove the waste liquid, photograph the wells of the test group using a stereomicroscope and record the entire set of photographs.

[0228] Add 300 μL of acetic acid to each well to fully dissolve the cells at the bottom, then transfer to a 96-well plate and measure the absorbance at 570 nm.

[0229] 4. Experimental Results

[0230] Depend on Figure 4 , Figure 5 It can be seen that 200 mg / kg copper-enriched yeast extract solutions with concentrations ranging from 0.0244% to 3.12500% can all increase the HaCaT proliferation rate, and 200 mg / kg copper-enriched yeast extract solutions with a concentration of 3.125% can reduce the HaCaT adhesion amount from 257.1% to 172.3%.

[0231] Therefore, copper-rich yeast extract can significantly reduce HaCaT adhesion and has significant repair effects.

[0232] Example 4: Human Efficacy Test

[0233] Test methods

[0234] 1.1 Experimental Principles

[0235] Double-blind, randomized, self-controlled before-and-after.

[0236] 1.2 Subject Screening

[0237] Number of participants: The experiment was divided into two groups, with 40 participants in each group;

[0238] Selection criteria:

[0239] (1) Age 30-60 years old, gender not limited;

[0240] (2) Facial skin is loose and lacks elasticity;

[0241] (3) The skin around the eyes has obvious crow's feet and under-eye wrinkles;

[0242] 2. Sample preparation

[0243] The copper-enriched yeast extract was the copper-enriched yeast extract prepared in Example 3 (a copper-enriched yeast extract solution with a copper content of 200 mg / kg was prepared from the copper-enriched yeast extract powder). The test sample formulations for the experimental group and the control group are shown in the table below:

[0244] Table 7. Sample Formulation

[0245]

[0246] 3. Efficacy testing process

[0247] No products may be used on the test site on the day of the test. Before the test, subjects must cleanse their faces, pat dry with lint-free tissues, and sit quietly for 30 minutes in the human efficacy evaluation room (temperature: 20~22℃, humidity: 40~60%). During this time, they must not drink water or beverages. Subjects should remain relaxed, with their faces exposed, and avoid touching them.

[0248] After cleansing, apply an appropriate amount of product evenly to the face until fully absorbed. Use once in the morning and once in the evening for 56 consecutive days. During the trial, subjects are prohibited from using repairing, soothing, firming, or anti-wrinkle preparations; subjects are prohibited from ingesting, injecting, or otherwise taking preparations that may affect the repairing, soothing, firming, or anti-wrinkle test results; subjects should primarily engage in indoor activities and avoid prolonged exposure to outdoor sunlight.

[0249] 4. Instrument measurement parameters and related equipment

[0250] Table 8. Instrument Measurement Parameters and Related Equipment

[0251]

[0252] Human trials using copper-enriched yeast extract showed that after 56 days of product use, the percentage of skin redness, skin F4 value, skin TWEL value, percentage of crow's feet, and percentage of under-eye wrinkles decreased to 30.5%, 45.9%, 53.1%, 43.2%, and 50.8%, respectively, while the skin stratum corneum moisture content increased to 134.6%. This demonstrates that copper-enriched yeast extract has significant soothing, repairing, moisturizing, firming, and anti-wrinkle effects.

[0253] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing copper-enriched yeast extract, characterized in that, Includes the following steps: Step 1: Take copper-rich yeast and heat-shock it; Step 2: Enzymatic hydrolysis with 200~2000 U / mL of protease; the protease includes acidic protease, neutral protease, alkaline protease and / or papain; Step 3: Solid-liquid separation and decolorization to obtain a decolorized solution; The temperature of the heat shock treatment in step 1 is 80~95℃; the time of the heat shock treatment is 5~50s; The copper-rich yeast is a yeast strain with accession number CCTCC NO:M 2017781; The heat shock treatment described in step 1 also includes the step of preparing a 5-30 wt% solution of copper-rich yeast; The pH value of the enzymatic hydrolysis in step 2 is 4.0~8.5; the temperature of the enzymatic hydrolysis is 45~65℃; The enzymatic hydrolysis time described in step 2 is 10-20 hours; The protease described in step 2 is selected from any of the following: alkaline protease and neutral protease; neutral protease, acidic protease and papain; or acidic protease and neutral protease.

2. The preparation method according to claim 1, characterized in that, Step 3 is followed by drying the decolorized solution to obtain copper-rich yeast extract powder.

3. The preparation method according to claim 2, characterized in that, The copper-rich yeast extract includes the decolorized solution and / or the copper-rich yeast extract powder.

4. The preparation method according to claim 1, characterized in that, The copper-rich yeast mentioned in step 1 includes any one or a combination of at least two of the following: copper-rich yeast fermentation broth obtained by fermentation using copper-rich yeast strains, copper-rich yeast milk, copper-rich yeast sludge, and active or inactive copper-rich dry yeast obtained by drying.

5. The preparation method according to claim 1, characterized in that, The decolorization described in step 3 includes decolorization using any one or a combination of at least two of the following methods: gelation, macroporous resin, activated carbon, or membrane separation.

6. The preparation method according to claim 2, characterized in that, The drying process includes concentrating the decolorized solution to 10-30 wt% dry matter and then spray drying it.

7. A copper-rich yeast extract prepared by any one of claims 1 to 6.

8. The copper-rich yeast extract as described in claim 7, characterized in that, The copper-rich yeast extract includes the decolorized solution and / or the copper-rich yeast extract powder; The copper content of the copper-rich yeast extract powder is 3000~30000 mg / kg.

9. The copper-rich yeast extract as described in claim 8, characterized in that, The protein content of the copper-rich yeast extract powder is 40-65 wt%.

10. The copper-rich yeast extract as described in claim 8, characterized in that, The solubility of the copper-rich yeast extract powder in water ranges from 30% to 60%.

11. The use of the copper-rich yeast extract as described in any one of claims 7 to 10 in the preparation of cosmetics with anti-UV, soothing, repairing, moisturizing, firming or anti-wrinkle properties.

12. The application as described in claim 11, characterized in that, The application includes any of the following: (I) Reduce the proportion of red area on the skin; (II) Reduce skin F4 value; (III) Reduce skin TWEL value; (IV) Reduce the proportion of the area covered by crow's feet; (V) Reduce the proportion of under-eye wrinkles; (VI) Increase the moisture content of the stratum corneum of the skin.

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