A preparation method of inonotus obliquus fermentation product and its product and application

Abstract

The present invention relates to a preparation method of a fermentation product of Inonotus birchii, and its product and application, comprising the following steps: inoculating Inonotus birchii into a culture medium for cultivation, sterilizing, filtering, inoculating yeast and lactic acid bacteria into the filtrate, fermenting, sterilizing, filtering, and obtaining the fermentation product of Inonotus birchii. The preparation method of the present invention using staged fermentation can effectively increase the content of active ingredients such as polysaccharides and polyphenols in the fermentation product, and the probiotics will perform secondary treatment on the metabolites of Inonotus birchii during the fermentation process, which greatly increases the value of the effective ingredients, and the product is mild and non-irritating, and has good effects in calming the skin, soothing anti-allergy, anti-inflammatory, anti-oxidation, anti-aging, etc., and can be used as a raw material in cosmetics or pharmaceutical products.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and specifically relates to a preparation method of a fermentation product of Inonotus obliquus and a product and application thereof. Background Art

[0002] Inonotus obliquus is a large edible and medicinal fungus of the subphylum Basidiomycetes. It mainly grows in cold countries and regions. It grows slowly under natural conditions and exists in the form of sclerotia. As a precious medicinal fungus, Inonotus obliquus has attracted much attention due to its unique biological activity and pharmacological effects. Its fermentation products contain a large number of active substances, such as polysaccharides, triterpenes, polyphenols, etc., which have excellent antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, and immunomodulatory activities. It has broad application prospects and economic value in the fields of cosmetics and medicine. However, the active ingredients of Inonotus obliquus are low in natural state, and the stability of active products cannot be guaranteed. Therefore, the research on active substances has increased greatly, and it also limits the development and application of related products.

[0003] Traditionally, the active ingredients of Inonotus birchii are mainly obtained through direct extraction or simple processing, but this method often has problems such as low content of effective substances and poor activity retention. The traditional method of extracting these effective substances from Inonotus birchii has many limitations. First, the method of direct drying and grinding and extraction is often inefficient, and a large amount of effective substances are still retained in the cell wall and difficult to release completely. Secondly, although the high-temperature extraction process can accelerate the dissolution of substances, it will also destroy the stability of many heat-sensitive active ingredients, resulting in reduced efficacy. Furthermore, the traditional fermentation process is not precise enough in the selection of strains, culture medium formulation and fermentation conditions, and it is difficult to maximize the promotion of the biosynthesis of effective substances.

[0004] Therefore, developing a preparation method that can efficiently and stably improve the content and activity of effective substances in Inonotus obliquus is of great significance for promoting its research and application. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a fermentation product of Inonotus obliquus and its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a fermentation product of Inonotus obliquus, the preparation method comprising the following steps:

[0008] (1) Inonotus obliquus is inoculated into a culture medium, cultured, sterilized, and filtered;

[0009] (2) Yeast and lactic acid bacteria are introduced into the filtrate, and the filtrate is fermented, sterilized, and filtered to obtain the inonotus birch fermentation product.

[0010] The preparation method of the fermented product of Inonotus birchii involved in the present invention creatively adopts a staged fermentation method. On the one hand, the content of active ingredients such as polysaccharides, triterpenes and polyphenols in the fermented product of Inonotus birchii can be effectively increased. At the same time, the probiotics will perform secondary treatment on the metabolites of Inonotus birchii during the fermentation process, which greatly increases the value of the effective ingredients. On the other hand, the preparation method can increase the utilization rate of the basic components of the culture medium by the bacteria, while limiting the growth of miscellaneous bacteria and improving the safety of the product. The fermented product of Inonotus birchii prepared by the method of the present invention is mild and non-irritating, and has good effects in calming the skin, soothing anti-allergy, anti-inflammatory, anti-oxidation, anti-aging, etc., and can be used as a raw material in cosmetics or pharmaceutical products.

[0011] Preferably, the birch mushroom in step (1) is inoculated in the form of seed liquid, and the inoculation amount is 4-6% (for example, 4%, 4.5%, 5%, 5.5%, 6%, etc.), and the inoculation amount is the ratio of the volume of the transplanted seed liquid to the volume of the culture liquid after inoculation. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0012] Preferably, the culturing in step (1) is carried out under aerobic conditions of 3-7% (e.g., 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, etc.), and other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0013] The culture temperature is 25-35°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0014] The stirring speed of the culture is 100-200rpm, for example, 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0015] The culture time is 120-168h, for example 120h, 126h, 132h, 138h, 144h, 150h, 156h, 162h, 168h, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0016] Preferably, the sterilization in step (1) is performed by pasteurization.

[0017] Preferably, the sterilization comprises the following steps: heating to 72-75°C (for example, 72°C, 72.5°C, 73°C, 73.5°C, 74°C, 74.5°C, 75°C, etc.) for 15-30s (15s, 16s, 17s, 18s, 19s, 20s, 22s, 24s, 26s, 28s, 30s, etc.), and then cooling to 20-25°C (for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc.). Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0018] Preferably, in step (2), the yeast and lactic acid bacteria are inoculated in the form of seed liquid, and the total inoculation amount is 2-4% (for example, 2%, 2.5%, 3%, 3.5%, 4%, etc.), and the inoculation amount is the ratio of the volume of the transplanted seed liquid to the volume of the culture liquid after inoculation. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0019] Preferably, the ratio of the live counts of yeast and lactic acid bacteria is (2-4):1, for example 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc. Other specific points within this numerical range can be selected and will not be described in detail here.

[0020] The present invention creatively discovered that when the ratio of the number of live yeasts to that of lactic acid bacteria is (2-4):1, the content of active ingredients in the fermentation product can be further increased, thereby obtaining better antioxidant, soothing, anti-allergic and anti-inflammatory effects.

[0021] Preferably, the yeast comprises Saccharomyces cerevisiae and / or Brettanomyces cerevisiae.

[0022] Preferably, the yeast is Saccharomyces cerevisiae.

[0023] Preferably, the lactic acid bacteria include any one of Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium longum or Lactococcus lactis, or a combination of at least two thereof.

[0024] Preferably, the lactic acid bacteria is a combination of Lactobacillus plantarum and Lactococcus lactis.

[0025] The present invention creatively discovered that when the lactic acid bacteria select a combination of Lactobacillus plantarum and Lactococcus lactis, compared with other lactic acid bacteria, the content of active ingredients in the fermentation product can be further increased, thereby obtaining better antioxidant, soothing, anti-allergic and anti-inflammatory effects.

[0026] Saccharomyces cerevisiae, Lactobacillus plantarum and Lactococcus lactis can play a synergistic role in increasing the content of active ingredients in the fermentation products of Inonotus birchii and improving the antioxidant, soothing, anti-allergic and anti-inflammatory effects of the fermentation products.

[0027] Preferably, the ratio of the viable counts of Lactobacillus plantarum and Lactococcus lactis is (1-3):(1-3), wherein (1-3) can be selected as 1, 1.5, 2, 2.5, 3, etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0028] Preferably, the brewer's yeast is selected from the brewer's yeast Saccharomyces cerevisiae YK-S001 strain with a preservation number of CGMCC No. 28276.

[0029] The plant lactobacillus is selected from the plant lactobacillus Lactiplantibacillus plantarum YK-L002 strain with a preservation number of CGMCC No. 29289.

[0030] The lactococcus lactis was selected from the lactococcus lactis subspecies YK-L006, which was purchased from Yokomeigu (Shandong) Biotechnology Co., Ltd.

[0031] The present invention also creatively discovered that when specific strains of brewer's yeast, Lactobacillus plantarum and Lactococcus lactis are selected, the content of active ingredients in the fermentation product of Inonotus obliquus, and the antioxidant, soothing and anti-inflammatory effects of the fermentation product can be further improved.

[0032] Preferably, the fermentation in step (2) is microaerobic fermentation.

[0033] Preferably, the fermentation in step (2) is carried out under aerobic conditions of 3-7% (e.g., 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, etc.), and other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0034] Preferably, the fermentation temperature is 30-35°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0035] Preferably, the fermentation time is 12-36h, for example 12h, 14h, 16h, 18h, 20h, 24h, 28h, 30h, 36h, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0036] Preferably, the pH measured after fermentation is 5.0-5.5, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, and other specific points within this numerical range can be selected, which will not be described here one by one.

[0037] Preferably, ultrasonic treatment is performed after sterilization and before filtration.

[0038] Preferably, the ultrasonic treatment comprises 70-80°C (e.g., 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, etc.) for 5-15min (e.g., 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.), and other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0039] Preferably, the sterilization in step (2) comprises heating to 80-85°C (for example, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, etc.) and sterilizing for 15-25min (for example, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, etc.). Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0040] Preferably, the filtering in step (2) includes filtering using a 0.1-3 μm (e.g., 0.1 μm, 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, etc.) filter. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0041] Preferably, the culture medium in step (1) comprises glucose, yeast powder, peptone, birch juice concentrate, pear juice, brown rice extract, royal jelly, black tea, potassium dihydrogen phosphate, magnesium sulfate, vitamin B1 and vitamin B3.

[0042] The present invention creatively finds that adding pear juice, brown rice extract, royal jelly and black tea to the culture medium plays a synergistic role in increasing the content of active substances in the fermentation product of Inonotus obliquus and improving the antioxidant, soothing and anti-inflammatory effects of the fermentation product.

[0043] Preferably, the culture medium in step (1) comprises 30-40 g / L of glucose, 3-7 g / L of yeast powder, 3-7 g / L of peptone, 1-3 g / L of birch juice concentrate, 1-3 g / L of pear juice, 1-3 g / L of brown rice extract, 0.1-2 g / L of royal jelly, 0.1-1 g / L of black tea, 0.1-2 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 0.01-0.1 g / L of vitamin B1 and 0.01-0.1 g / L of vitamin B3.

[0044] Among them, 30-40g / L can choose 30g / L, 32g / L, 34g / L, 36g / L, 38g / L, 40g / L, etc.

[0045] 3-7g / L can choose 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, etc.

[0046] 1-3g / L can choose 1g / L, 1.5g / L, 2g / L, 2.5g / L, 3g / L, etc.

[0047] 0.1-2g / L can choose 0.1g / L, 1g / L, 1.2g / L, 1.4g / L, 1.6g / L, 1.8g / L, 2g / L, etc.

[0048] 0.1-1g / L can choose 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc.

[0049] 0.01-0.1g / L can choose 0.01g / L, 0.02g / L, 0.04g / L, 0.06g / L, 0.08g / L, 0.1g / L, etc.

[0050] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0051] In a second aspect, the present invention provides a fermentation product of Inonotus obliquus obtained according to the preparation method described in the first aspect.

[0052] In a third aspect, the present invention provides a use of the fermentation product of Inonotus obliquus according to the second aspect in the preparation of a product having any one or at least two of the following effects;

[0053] 1) Anti-oxidation; 2) Anti-inflammatory; 3) Soothing and anti-allergic.

[0054] The product includes a cosmetic or a pharmaceutical product.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The preparation method of the inonotus birch probiotic fermentation product involved in the present invention creatively adopts a staged fermentation method. On the one hand, the content of active ingredients such as polysaccharides, triterpenes and polyphenols in the inonotus birch fermentation product can be effectively increased. At the same time, the probiotics will perform secondary treatment on the inonotus birch metabolites during the fermentation process, which greatly improves the value of the effective ingredients. On the other hand, the preparation method can improve the utilization rate of the basic components of the culture medium by the bacteria, while limiting the growth of miscellaneous bacteria and improving the safety of the product. The inonotus birch fermentation product prepared by the method of the present invention is mild and non-irritating, and has good effects in calming the skin, soothing anti-allergy, anti-inflammatory, anti-oxidation, anti-aging, etc., and can be used as a raw material in cosmetics or pharmaceutical products.

[0057] The YK-S001 strain involved in the present invention is classified and named as Saccharomyces cerevisiae, and the depository unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit time is August 28, 2023, the deposit number is CGMCC No. 28276, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0058] The YK-L002 strain involved in the present invention is classified and named as Lactiplantibacillus plantarum, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation time is December 11, 2023, the preservation number is CGMCC No. 29289, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0059] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0060] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0061] The yeast powder and peptone involved in the following examples were purchased from Shandong Weimei Biotechnology Co., Ltd.

[0062] Birch juice concentrate was purchased from Heleba Import and Export Trading Company;

[0063] Pear juice was purchased from Dachuan Food Industry Co., Ltd.;

[0064] Royal jelly was purchased from Baihua Bee Industry Technology Development Co., Ltd.;

[0065] For dark tea, choose Anhua dark tea;

[0066] The preparation method of the brown rice extract is as follows: the brown rice is crushed and passed through an 80-mesh sieve, mixed with water according to a solid-liquid ratio of 1:20 g / mL, heated at 80° C. for extraction for 2 hours, filtered, concentrated, and dried to obtain the brown rice extract.

[0067] The YK-S001 strain involved in the following embodiment is classified and named as Saccharomyces cerevisiae, and the depository unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit time is August 28, 2023, the deposit number is CGMCC No. 28276, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] The YK-L002 strain involved in the following embodiment is classified and named Lactiplantibacillus plantarum, and the depository is the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit time is December 11, 2023, the deposit number is CGMCC No. 29289, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0069] The YK-L006 strain involved in the following examples is Lactococcus lactis subspecies YK-L006, which was purchased from Yokomeigu (Shandong) Biotechnology Co., Ltd.

[0070] The following examples involve the separation and purification of Inonotus obliquus by the following method:

[0071] Isolation medium: birch bark powder 15 g / L, agar 20 g / L, sterilize at 121°C for 30 min, add 0.1 g of ampicillin before use.

[0072] PDA medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L, sterilized at 121°C for 30 min.

[0073] (1) Select fresh Inonotus obliquus, remove surface impurities, disinfect the surface with 75% alcohol three times, separate it using the tissue separation method, cut a 0.7 cm small piece under sterile conditions, and place it in the middle of the separation culture medium plate;

[0074] (2) Cultivate at 28°C for 6 days. After mycelium grows out, select the mycelium at the edge, take a small piece of 0.5×0.5 cm, and place it in PDA culture medium;

[0075] (3) Grow on PDA medium for 10 days and repeat the above operation twice to obtain purified Inonotus obliquus strains;

[0076] (4) The purified strain was inoculated on PDA slant medium for growth. After the mycelium covered the slant medium, it was placed in a refrigerator and stored at 4°C.

[0077] The inonotus obliquus, yeast and lactic acid bacteria involved in the following examples are inoculated in the form of seed liquid, and the seed liquid preparation method is as follows:

[0078] (1) Inonotus obliquus seed solution:

[0079] Seed culture medium: prepare the culture medium with 10g / L malt extract, 4g / L yeast powder and 10g / L glucose, and sterilize at 115℃ for 20min.

[0080] When the culture medium temperature drops below 30°C, the purified mycelium of Inonotus birchii is scraped and inoculated into the seed culture medium, and cultured at 28°C for 3 days.

[0081] (2) Yeast seed liquid:

[0082] Seed culture medium: Cut 200g fresh potatoes into pieces and cook until soft, then filter out the potatoes. Add water to the remaining liquid to make up to 1L, add 20g glucose as the culture medium, and sterilize at 115℃ for 20min.

[0083] When the culture medium temperature drops below 30°C, inoculate the yeast into the seed culture medium and culture at 30°C for 24 hours.

[0084] (3) Lactic acid bacteria seed liquid:

[0085] Seed culture medium: prepare MRS medium (peptone 10 g / L, beef extract 10 g / L, glucose 15 g / L, lactose 15 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​salt 0.5 g / L), sterilize at 115 ℃ for 20 min.

[0086] When the culture medium temperature drops below 30°C, inoculate the lactic acid bacteria into the seed culture medium and culture at 37°C for 24 hours.

[0087] Example 1

[0088] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus:

[0089] (1) Accurately weigh 35 g of glucose, 5 g of yeast powder, 5 g of peptone, 2 g of birch juice concentrate, 2 g of pear juice, 2 g of brown rice extract, 1 g of royal jelly, 0.5 g of black tea, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.05 g of vitamin B1, and 0.05 g of vitamin B3. Add deionized water to make up to 1 L, sterilize at 115°C for 30 min, and use as a liquid culture medium for Inonotus obliquus.

[0090] (2) Inonotus obliquus culture: Inoculate the seed liquid of Inonotus obliquus into the liquid culture medium of Inonotus obliquus under sterile conditions, avoid introducing other strains or adding additional chemical reagents during the inoculation process, the inoculation amount is 5%, 5% aerobic conditions, 30℃, 150rpm shaking culture for 6 days, continuous culture transfer for two generations. Use pasteurization method, quickly heat to 74℃, hold for 22s, then immediately cool to 23℃, and store for later use;

[0091] (3) Filter the sterilized liquid obtained in step (2), inoculate the sterilized filtrate with Saccharomyces cerevisiae YK-S001 seed liquid, Lactobacillus plantarum YK-L002 seed liquid and Lactococcus lactis subspecies YK-L006 seed liquid at a ratio of 3:0.5:0.5 of viable bacteria count, with a total inoculation amount of 3%. Ferment at 32°C for 24 h under 5% aerobic conditions, heat to 83°C for sterilization for 20 min, and ultrasonically treat at 75°C for 10 min, then filter using a 1 μm filter to obtain a fermentation product of Inonotus obliquus.

[0092] The fermentation product of Inonotus obliquus is a brown-yellow transparent liquid, has a pH of 5.1, and a conductivity of 410 μS / cm.

[0093] Example 2

[0094] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus:

[0095] (1) Accurately weigh 33 g glucose, 7 g yeast powder, 3 g peptone, 3 g birch juice concentrate, 1 g pear juice, 3 g brown rice extract, 0.5 g royal jelly, 1 g black tea, 0.5 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 0.03 g vitamin B1, and 0.07 g vitamin B3, dilute to 1 L with deionized water, and sterilize at 115°C for 30 min. This is used as a liquid culture medium for Inonotus obliquus.

[0096] (2) Inonotus obliquus culture: Inoculate the seed liquid of Inonotus obliquus into the culture medium under sterile conditions, avoid introducing other strains or adding additional chemical reagents during the inoculation process, the inoculation amount is 4%, 4% aerobic conditions, 28°C, 130rpm shaking culture for 5 days, continuous culture transfer for two generations. Use pasteurization method, quickly heat to 75°C for 15 seconds, then immediately cool to 20°C and store for later use;

[0097] (3) The sterilized liquid obtained in the filtering step (2) is inoculated with the seed liquid of Saccharomyces cerevisiae YK-S001, the seed liquid of Lactobacillus plantarum YK-L002 and the seed liquid of Lactococcus lactis subspecies YK-L006 in a ratio of 2:0.4:0.6 of viable bacteria count in the sterilized filtrate, with a total inoculation amount of 2%. The mixture is fermented at 33°C for 22 h under 4% aerobic conditions, heated to 80°C for sterilization for 23 min, and ultrasonically treated at 80°C for 5 min, and then filtered using a 1 μm filter to obtain a fermentation product of Inonotus obliquus.

[0098] Example 3

[0099] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus:

[0100] (1) Accurately weigh 37 g glucose, 3 g yeast powder, 7 g peptone, 1 g birch juice concentrate, 3 g pear juice, 1 g brown rice extract, 1.5 g royal jelly, 0.3 g black tea, 1.5 g potassium dihydrogen phosphate, 0.3 g magnesium sulfate, 0.07 g vitamin B1, and 0.03 g vitamin B3, dilute to 1 L with deionized water, and sterilize at 115°C for 30 min. This is used as a liquid culture medium for Inonotus obliquus.

[0101] (2) Inonotus obliquus culture: Inoculate the seed liquid of Inonotus obliquus into the culture medium under sterile conditions to avoid the introduction of other strains or the addition of additional chemical reagents during the inoculation process. The inoculation amount is 6%, and the culture is carried out under aerobic conditions at 6%, 32°C, and 170rpm shaking on a shaker for 7 days. The culture is continuously transferred for two generations. Use the pasteurization method, quickly heat to 72°C for 30 seconds, then immediately cool to 25°C and store for later use;

[0102] (3) Filter the sterilized liquid obtained in step (2), inoculate the sterilized filtrate with Saccharomyces cerevisiae YK-S001 seed liquid, Lactobacillus plantarum YK-L002 seed liquid and Lactococcus lactis subspecies YK-L006 seed liquid with a live bacterial count ratio of 4:0.6:0.4, with a total inoculation amount of 4%, and perform microaerobic fermentation at 6% aerobic conditions at 31°C for 26 h, heat to 85°C for sterilization for 17 min, and ultrasonically treat at 70°C for 15 min, then filter using a 1 μm filter to obtain a fermentation product of Inonotus obliquus.

[0103] Example 4

[0104] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), while keeping the total viable count unchanged, the seed solution of Saccharomyces cerevisiae YK-S001 and the seed solution of Lactobacillus plantarum YK-L002 are inoculated at a ratio of 3:0.5, and other conditions remain unchanged.

[0105] Example 5

[0106] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), while keeping the total viable count unchanged, the seed solution of Saccharomyces cerevisiae YK-S001 and the seed solution of Lactococcus lactis subspecies YK-L006 are inoculated at a ratio of 3:0.5, and other conditions remain unchanged.

[0107] Example 6

[0108] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), while keeping the total viable bacterial count unchanged, Lactobacillus plantarum YK-L002 seed solution and Lactococcus lactis subspecies YK-L006 seed solution are inoculated at a ratio of 1:1, and other conditions remain unchanged.

[0109] Example 7

[0110] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), the viable bacteria count of Lactobacillus plantarum YK-L002 seed solution is replaced by Bifidobacterium animalis subsp. lactis CICC 21712 seed solution, and other conditions remain unchanged.

[0111] Example 8

[0112] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), the viable bacteria count of the Saccharomyces cerevisiae YK-S001 seed solution is replaced by the Saccharomyces cerevisiae CICC 1001 seed solution, and other conditions remain unchanged.

[0113] Example 9

[0114] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), the viable bacterial count of Lactobacillus plantarum YK-L002 seed solution is replaced by Lactobacillus plantarum ATCC 8014 seed solution, and other conditions remain unchanged.

[0115] Example 10

[0116] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that in step (3), the viable bacterial count of the seed solution of Lactococcus lactis subspecies YK-L006 is replaced by the seed solution of Lactococcus lactis subspecies CICC 21052, and other conditions remain unchanged.

[0117] Embodiment 11

[0118] The present embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from the embodiment 1 only in that in step (3), while the total viable bacterial count remains unchanged, the seed liquid of Saccharomyces cerevisiae YK-S001, the seed liquid of Lactobacillus plantarum YK-L002 and the seed liquid of Lactococcus lactis subspecies YK-L006 with a viable bacterial count ratio of 1:0.5:0.5 are inoculated into the sterilized filtrate, and other conditions remain unchanged.

[0119] Example 12

[0120] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that pear juice is not added in step (1), and its mass is distributed to brown rice extract, royal jelly and black tea according to the mass ratio of Embodiment 1, while other conditions remain unchanged.

[0121] Example 13

[0122] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that brown rice extract is not added in step (1), and its mass is distributed to pear juice, royal jelly and dark tea according to the mass ratio of Embodiment 1, while other conditions remain unchanged.

[0123] Embodiment 14

[0124] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that royal jelly is not added in step (1), and its mass is distributed to pear juice, brown rice extract and black tea according to the mass ratio of Embodiment 1, while other conditions remain unchanged.

[0125] Embodiment 15

[0126] This embodiment provides a method for preparing a fermentation product of Inonotus obliquus, which differs from Embodiment 1 only in that no dark tea is added in step (1), and its mass is distributed to pear juice, brown rice extract and royal jelly according to the mass ratio of Embodiment 1, while other conditions remain unchanged.

[0127] Comparative Example 1

[0128] This comparative example provides a method for preparing a fermentation product of Inonotus obliquus:

[0129] (1) Grinding the Inonotus obliquus, passing through a 40-mesh sieve, mixing the sieved Inonotus obliquus powder with water in a mass ratio of 1:9, heating and extracting at 80° C. for 2 h, cooling and filtering to obtain an Inonotus obliquus extract;

[0130] (2) Accurately weigh 35 g of glucose, 5 g of yeast powder, 5 g of peptone, 2 g of birch juice concentrate, 2 g of pear juice, 2 g of brown rice extract, 1 g of royal jelly, 0.5 g of black tea, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.05 g of vitamin B1, 0.05 g of vitamin B3, and 5 g of Inonotus obliquus extract, dilute to 1 L with deionized water, sterilize at 115°C for 30 min, and use as liquid culture medium for later use;

[0131] (3) In the liquid culture medium prepared in step (2), the seed liquid of Saccharomyces cerevisiae YK-S001, the seed liquid of Lactobacillus plantarum YK-L002 and the seed liquid of Lactococcus lactis subspecies YK-L006 were inoculated with a live bacterial count ratio of 3:0.5:0.5, respectively, with a total inoculation amount of 3%. Under 5% aerobic conditions, the mixture was fermented at 32°C for 24 h, heated to 83°C for sterilization for 20 min, and ultrasonically treated at 75°C for 10 min, and then filtered using a 1 μm filter to obtain a fermentation product of Inonotus birchii.

[0132] Comparative Example 2

[0133] This comparative example provides a method for preparing a fermentation product of Inonotus obliquus:

[0134] (1) Accurately weigh 35 g of glucose, 5 g of yeast powder, 5 g of peptone, 2 g of birch juice concentrate, 2 g of pear juice, 2 g of brown rice extract, 1 g of royal jelly, 0.5 g of black tea, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.05 g of vitamin B1, and 0.05 g of vitamin B3. Add deionized water to make up to 1 L, sterilize at 115°C for 30 min, and use as liquid culture medium for later use;

[0135] (2) Under sterile conditions, 5% inoculum of Inonotus obliquus, 3% inoculum of Saccharomyces cerevisiae YK-S001, Lactobacillus plantarum YK-L002 and Lactococcus lactis subsp. YK-L006 were inoculated into liquid culture medium (the ratio of the number of live bacteria of YK-S001, YK-L002 and YK-L006 was 3:0.5:0.5), and the culture medium was fermented at 30°C for 7 days under 5% aerobic conditions, and then the temperature was raised to 83°C for sterilization for 20 min, and then ultrasonically treated at 75°C for 10 min, and then filtered through a 1 μm filter to obtain the fermentation product of Inonotus obliquus.

[0136] Test Example 1

[0137] In this test example, the solid content, polyphenol content, and polysaccharide content of the fermentation products obtained in Examples 1-15 and Comparative Examples 1-2 were measured. The results are shown in Table 1.

[0138] (1) Solid content detection: Weigh 2 g (m0) of the product and dry it at 95 °C for 120 min. Take it out and put it in a dryer. After cooling to 25 °C, weigh it as m1. Then calculate the solid content according to the formula: solid content = (m1 / m0) × 100%.

[0139] (2) Detection of polyphenol content: 1.5 mL of the product was measured and placed in a 10 mL test tube with a scale and stopper. 1 mL of Folin phenol reagent was added and shaken. 2 mL of 7.5% sodium carbonate solution was added and shaken. The volume was made up to 10 mL and shaken. The mixture was placed in a 75°C water bath for 15 min. The mixture was taken out and cooled to 25°C. The absorbance was measured at a wavelength of 760 nm. The results were measured three times for each group, and the average value was taken to calculate the polyphenol content.

[0140] (3) Polysaccharide content detection

[0141] The method in NY / T 1676-2008 “Determination of crude polysaccharide content in edible fungi” was used to prepare a standard curve with glucose as the standard substance, and the content of polysaccharides in the extracted product was determined.

[0142] Table 1

[0143]

[0144] It can be seen from the data in Table 1 that the preparation method of the fermentation product of Inonotus obliquus involved in the present invention can effectively increase the solid content, polyphenol content and polysaccharide content of the fermentation product of Inonotus obliquus.

[0145] By comparing the results of Example 1 and Examples 4-6, it can be seen that Saccharomyces cerevisiae, Lactobacillus plantarum and Lactococcus lactis achieve a synergistic effect in increasing the content of active substances in the fermentation product of Inonotus obliquus.

[0146] By comparing the results of Example 1 and Example 7, it can be seen that when specific lactic acid bacteria are selected, the content of active substances in the fermentation product of Inonotus obliquus is further improved.

[0147] By comparing the results of Example 1 and Examples 8-10, it can be seen that when specific strains of Saccharomyces cerevisiae, Lactobacillus plantarum and Lactococcus lactis are selected, the content of active substances in the fermentation product of Inonotus obliquus is further improved.

[0148] By comparing the results of Example 1 and Example 11, it can be seen that the use of a specific ratio of yeast and lactic acid bacteria can further increase the content of active substances in the fermentation product of Inonotus obliquus.

[0149] By comparing the results of Example 1 and Examples 12-15, it can be seen that the addition of pear juice, brown rice extract, royal jelly and dark tea to the culture medium plays a synergistic role, which is beneficial to the growth of Inonotus obliquus and the metabolic accumulation of nutrients, thereby further increasing the content of active substances in the fermentation product of Inonotus obliquus.

[0150] By comparing the results of Example 1 and Comparative Examples 1-2, it can be seen that the staged fermentation method involved in the present invention can effectively increase the content of active substances in the fermentation product of Inonotus obliquus compared with other fermentation methods such as co-fermentation. During the co-fermentation process, the growth rate of yeast and lactic acid bacteria will be faster than that of Inonotus obliquus, which will inhibit the growth of Inonotus obliquus.

[0151] Test Example 2

[0152] This test example tests the antioxidant and anti-aging properties of the fermentation products obtained in Examples 1-15 and Comparative Examples 1-2:

[0153] DPPH scavenging experiment

[0154] According to the “Cosmetics-Free Radical (DPPH) Scavenging Test Method (T / SHRH006-2018)”, the DPPH free radical scavenging rate (%) of the products was tested respectively.

[0155] Experimental method: A 96-well plate was used, with three replicate wells set up in each group, and the system was 200 μL. Sample group: freeze-dried the products of each group, and took an appropriate amount of freeze-dried powder of each group of products and dissolved it in 100 μL of distilled water, so that the final concentration of the sample to be tested in the system was 10%, and 100 μL of 0.1 mM DPPH solution was added to the reaction system; control group: 100 μL of distilled water was taken, and then 100 μL of 0.1 mM DPPH solution was added. After the reaction system was constructed, it was shaken in the dark for 10 minutes, and the absorbance at 520 nm was tested using an ELISA reader.

[0156] Calculation method of clearance rate: Clearance rate (%) = [(A0-A x ) / A0]×100%, A0 is the absorbance of the control group, A x is the absorbance of the sample group. The DPPH free radical scavenging test results are shown in Table 2.

[0157] Table 2

[0158]

[0159] It can be seen from the data in Table 2 that the fermented product of Inonotus obliquus obtained by the specific process of the present invention has significant antioxidant and anti-aging properties. The staged fermentation method can effectively improve the DPPH clearance rate of the fermented product of Inonotus obliquus. At the same time, the composition of the culture medium and the selection of specific strains will also affect the DPPH clearance rate of the fermented product of Inonotus obliquus.

[0160] Test Example 3

[0161] This test example tests the soothing and anti-allergic performance of the fermented products obtained in Examples 1-15 and Comparative Examples 1-2:

[0162] Hyaluronidase activity inhibition experiment:

[0163] (3.1) Solution preparation:

[0164] Hyaluronidase solution: Accurately weigh 0.01 g of hyaluronidase and add 4 mL of acetate buffer solution to a final concentration of 1250 μg mL -1 .

[0165] 0.5mg·mL -1 Sodium hyaluronate solution: Accurately weigh 0.005 g of sodium hyaluronate, add it to 10 mL of acetate buffer solution, and set aside after it is fully dissolved.

[0166] Acetate buffer solution (pH=5.6): Take 1155μL of glacial acetic acid and dilute it to 100mL in a volumetric flask. Mix well and take 4.8mL as solution A; take 2.72g of sodium acetate crystals, dissolve and dilute to 100mL, mix well and take 45.2mL as solution B; mix solutions A and B, dilute to 100mL with deionized water and mix well. Accurately measure its pH value and adjust the pH value to 5.6 with solution A or B.

[0167] Ehrlich reagent: Accurately weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol for later use.

[0168] Acetylacetone solution: Dissolve 3.5 mL of acetylacetone in 50 mL of 1.0 mol·L -1 The acetylacetone solution must be prepared before use.

[0169] Test sample solution: freeze-dry each group of products, take 1 g of the freeze-dried product, dissolve it in 10 mL of deionized water to make a 10% solution for later use.

[0170] (3.2) Experimental process:

[0171] Add 0.1 mL of 0.25 mmol·L -1 CaCl2 solution and 0.5 mL hyaluronidase solution were incubated at 37°C for 20 min; 0.5 mL of the test sample solution was added and incubated at 37°C for 20 min; 0.5 mL of sodium hyaluronate solution was added and incubated at 37°C for 30 min; then the mixture was allowed to stand at room temperature for 5 min and 0.4 mol·L was added. -1 0.1 mL of NaOH solution and 0.5 mL of acetylacetone solution were heated in a boiling water bath for 15 min and then immediately placed in an ice bath for 5 min. 1.0 mL of Ehrlich reagent was then added and diluted with 3.0 mL of anhydrous ethanol. After standing for 20 min, the color was developed and the absorbance was measured. The inhibition rate was calculated by substituting it into the following formula.

[0172] Hyaluronidase inhibition rate (%): {[(AB)-(CD)] / (AB)}×100%, where: A is the absorbance value of the control solution (using acetate buffer solution instead of the sample solution); B is the absorbance value of the blank control solution (using acetate buffer solution instead of the sample solution and enzyme solution); C is the absorbance value of the sample solution; D is the absorbance value of the sample blank solution (using acetate buffer solution instead of the enzyme solution). The test results are shown in Table 3.

[0173] Table 3

[0174]

[0175] It can be seen from the data in Table 3 that the fermented product of Inonotus obliquus obtained by the specific process of the present invention has significant soothing and anti-allergic properties. The staged fermentation method can effectively improve the hyaluronidase activity inhibition rate of the fermented product of Inonotus obliquus. At the same time, the composition of the culture medium and the selection of specific strains will also affect the hyaluronidase activity inhibition rate of the fermented product of Inonotus obliquus.

[0176] Test Example 4

[0177] This test example tests the soothing and anti-inflammatory properties of the fermented products obtained in Examples 1-15 and Comparative Examples 1-2:

[0178] RAW264.7 macrophages were used as the research object, and the cell inflammation model was established by stimulating the cells with lipopolysaccharide (LPS) (bacterial endotoxin). Macrophages (1.0×10 4 The cells were plated in 12-well plates (100 μg / well) and incubated in an incubator at 37°C and 5% CO2 for 24 hours. Then, 4 μL of 1% (v / v) dilution obtained by diluting the fermentation product (lyophilized form) with water was added, and LPS was added to the dilution to a final concentration of 1 μg / mL 2 hours later as the sample group. A control group 1 (no dilution of the fermentation product was added, only the dilution of LPS was added) and a control group 2 (no dilution of LPS was added, only the dilution of the fermentation product was added) were set up. The supernatant was collected and centrifuged, and the release level of the pro-inflammatory inflammatory factor TNF-α in RAW264.7 macrophages was detected using an ELISA kit.

[0179] The TNF-α inhibition rate was calculated according to the following formula:

[0180] TNF-α inhibition rate (%) = (inflammatory factor concentration in control group 1 - inflammatory factor concentration in sample group) / (inflammatory factor concentration in control group 1 - inflammatory factor concentration in control group 2) × 100%.

[0181] The higher the TNF-α inhibition rate, the better the anti-inflammatory effect of the sample. The test results of TNF-α inhibition are shown in Table 4.

[0182] Table 4

[0183]

[0184] It can be seen from the data in Table 4 that the fermented product of Inonotus obliquus obtained by the specific process of the present invention has significant soothing and anti-inflammatory properties. The staged fermentation method can effectively improve the TNF-α inhibition rate of the fermented product of Inonotus obliquus. At the same time, the composition of the culture medium and the selection of specific strains will also affect the TNF-α inhibition rate of the fermented product of Inonotus obliquus.

[0185] Test Example 5

[0186] The skin irritation and eye irritation of the fermentation products obtained in Examples 1-3 were measured according to the 2015 edition of the "Technical Specification for Safety of Cosmetics". The results are shown in Table 5.

[0187] Table 5

[0188]

[0189] The results show that the fermentation product prepared by the present invention has no skin irritation and eye irritation and has good safety.

[0190] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0191] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0192] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a fermentation product of Inonotus obliquus, characterized in that: The preparation method comprises the following steps: (1) Inoculate the seed liquid of Inonotus obliquus into the culture medium at an inoculum amount of 4-6%, culture it under 3-7% aerobic conditions for 120-168 hours at a culture temperature of 25-35°C, sterilize it, and filter it; (2) inoculating yeast seed liquid and lactic acid bacteria seed liquid into the filtrate with a total inoculation amount of 2-4%, fermenting under 3-7% aerobic conditions for 12-36 hours at a fermentation temperature of 30-35° C., sterilizing, ultrasonically treating, and filtering to obtain the inonotus birch fermentation product; Wherein, the culture medium in step (1) comprises 30-40 g / L of glucose, 3-7 g / L of yeast powder, 3-7 g / L of peptone, 1-3 g / L of birch juice concentrate, 1-3 g / L of pear juice, 1-3 g / L of brown rice extract, 0.1-2 g / L of royal jelly, 0.1-1 g / L of black tea, 0.1-2 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 0.01-0.1 g / L of vitamin B1 and 0.01-0.1 g / L of vitamin B3; The ratio of the number of live yeast and lactic acid bacteria in step (2) is (2-4):1; The yeast includes Saccharomyces cerevisiae and / or Brettanomyces cerevisiae; The lactic acid bacteria include any one of Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium longum or Lactococcus lactis, or a combination of at least two thereof.

2. The method for preparing the Inonotus obliquus fermentation product according to claim 1, characterized in that: The sterilization in step (1) is performed by pasteurization; The sterilization comprises the following steps: heating to 72-75°C for 15-30 seconds, and then cooling to 20-25°C.

3. The method for preparing the Inonotus obliquus fermentation product according to claim 1, characterized in that: The lactic acid bacteria is a combination of Lactobacillus plantarum and Lactococcus lactis; The ratio of the viable counts of Lactobacillus plantarum and Lactococcus lactis is (1-3):(1-3).

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