Method for directly preparing high-viscosity fermented emulsion through biological fermentation and product

Abstract

The invention provides a method for directly preparing high-viscosity fermented emulsion through biological fermentation. The method comprises the following steps: step 1, preparing schizophyllumcommuneh mycelia through liquid fermentation culture; step 2, preparing a substrate from the schizophyllumcommuneh mycelium and water in proportion, sterilizing, inoculating a schizophyllumcommuneh seed solution, performing fermentation culture, centrifuging, taking supernate, and sterilizing to obtain fermentation liquor. The invention also provides the high-viscosity fermented emulsion which is prepared by the method. According to the high-viscosity fermented emulsion prepared by directly biologically fermenting the mycelium, the viscosity of the fermented liquid is improved, the skin feeling is improved, and the high-viscosity fermented emulsion has good soothing, anti-wrinkle and moisturizing properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to a method and product for directly preparing high-viscosity fermented emulsion by biological fermentation. Background Art

[0002] Usually, emulsions are formulated, and the water phase and the oil phase are emulsified by the action of emulsifiers to finally obtain water-in-oil type (O / W type) or oil-in-water type (W / O type). Emulsions have a high water content, good fluidity, low viscosity, and are difficult to maintain stability. They are prone to stratification during storage. In the formulation design and preparation of emulsions, in order to make the density of the dispersed phase and the dispersion medium as close as possible to obtain a stable emulsion system, thickeners such as water-soluble colloid raw materials or water-soluble polymer compounds are often added to the formula. The selection of emulsifiers and emulsification equipment has high requirements.

[0003] The present invention directly uses a biological fermentation method, takes oil directly as a pre-fermentation substrate, and obtains high-viscosity fermented milk by direct separation after fermentation through the fermentation of microorganisms. The method of the present invention does not require the addition of an emulsifier, and there is no formula design and emulsification process in the later stage. It is purely biologically manufactured, and the obtained fermented milk is white to light yellow in color, moisturizing the skin, easy to absorb, and natural. Summary of the invention

[0004] A brief overview of the disclosure is given below in order to provide a basic understanding of certain aspects of the disclosure. It should be understood that this overview is not an exhaustive overview of the disclosure. It is not intended to identify key or important parts of the disclosure, nor is it intended to limit the scope of the disclosure. Its purpose is simply to give certain concepts in a simplified form as a prelude to a more detailed description discussed later.

[0005] In order to solve the above technical problems, the technical solution provided by the present disclosure is:

[0006] In a first aspect, the present disclosure provides a method for directly preparing a high-viscosity fermented emulsion by biological fermentation, comprising:

[0007] Step 1, preparing white ginseng mycelium by liquid fermentation culture;

[0008] Step 2: prepare the white ginseng fungus mycelium and water in proportion to form a substrate, sterilize, inoculate white ginseng fungus seed liquid, perform fermentation culture, centrifuge and sterilize the supernatant to obtain the fermentation liquid.

[0009] In the above preparation method, as a preferred embodiment, the white ginseng fungus is Schizophyllum commune YSC1, and its preservation number is CGMCC No.17788.

[0010] In the above preparation method, as a preferred embodiment, in step 1, the preparation method of the white ginseng fungus mycelium is: add 2% potatoes and 2% sucrose to water and then perform homogenization, then inoculate white ginseng fungus seed liquid for fermentation culture, and finally separate and obtain the white ginseng fungus mycelium. In a specific example, for example, 6g potatoes and 6g sucrose can be added to 300mL of water, then homogenized, then inoculated with white ginseng fungus seed liquid for fermentation culture, and finally separate and obtain the white ginseng fungus mycelium.

[0011] In the above preparation method, as a preferred embodiment, in step one and / or step two, the volume proportion of the white ginseng mycelium in the white ginseng seed liquid is 75-90% (such as 78%, 80%, 82%, 85%, 88%, etc.) of the entire liquid culture medium, preferably 80-90%; the inoculation ratio, that is, the volume ratio of the seed liquid to the substrate is (0.1-5):30 (such as 0.5:30, 1:30, 2:30, 3:30, 4:30, etc.), preferably 1:30. More preferably, the volume of the mycelium of the white ginseng fungus in the white ginseng fungus seed liquid accounts for 85-90% of the entire liquid culture medium. The preparation method of the white ginseng fungus seed liquid comprises the following steps: (1) using a sterilized puncher to take out 2-3 solid fungus species with a diameter of about 0.5 cm from the white ginseng fungus solid fungus species grown on a PDA plate, together with the culture medium, and inoculating them into 300 mL of potato glucose water culture medium, and culturing them at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 1, wherein the bacterial balls of the white ginseng fungus seed liquid 1 are of different sizes; (2) after homogenizing the white ginseng fungus seed liquid 1, 10 mL of the white ginseng fungus seed liquid is taken as the seed liquid and inoculated into 300 mL of potato glucose water culture medium, and culturing them at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 2, wherein the volume of the mycelium of the white ginseng fungus accounts for about 85-90% of the entire liquid culture medium.

[0012] In the above preparation method, as a preferred embodiment, in step 2, the mass ratio of the white ginseng mycelium in the substrate to water is 1:5 to 1:15 (such as 1:8, 1:10, 1:12, 1:14, etc.); preferably 1:5-1:10.

[0013] In the above preparation method, as a preferred embodiment, step 2 further includes sucrose, and the amount of sucrose added is 0-2% (such as 0.1%, 0.2%, 0.5%, 1%, 1.5%, etc.); preferably 0.5%.

[0014] In the above preparation method, as a preferred embodiment, in step 2, mung bean starch is also included, and the amount of mung bean starch added is 0-3% (for example, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, etc.), preferably 0.5%; further, macadamia seed oil is also included, and the amount of macadamia seed oil added is 0-3% (for example, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, etc.), preferably 1-1.5%.

[0015] In the above preparation method, as a preferred embodiment, after the liquid fermentation in step one is completed (without separation), it is directly homogenized and then used as a substrate for step two treatment; more preferably, in step two, the substrate also includes oil, and the oil is selected from at least one of white meadowfoam seed oil, soybean oil, linseed oil, and corn germ oil, and the added amount of the oil is 0.5-3% (such as 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, etc.), preferably 1%.

[0016] In the above preparation method, as a preferred embodiment, the fermentation culture temperature is 24-30°C (such as 25°C, 26°C, 27°C, 28°C, 29°C, etc.), and the time is 48-96h (such as 52h, 60h, 72h, 84h, 88h, 92h, etc.). Furthermore, the fermentation culture is carried out in a shaker, and the rotation speed in the shaker is 150rpm-200rpm (such as 160rpm, 170rpm, 180rpm, 190rpm, 195rpm, etc.).

[0017] In a second aspect, the present disclosure provides a high-viscosity fermented emulsion product prepared by the above preparation method.

[0018] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0019] 1. This application directly increases the viscosity of the fermentation liquid and improves the skin feel by using the method of biological fermentation of mycelium;

[0020] 2. This application directly uses the biological fermentation method to use oil as the pre-fermentation substrate, and through the fermentation of microorganisms, directly separates after the fermentation to obtain a high-viscosity fermented emulsion. The method of this application does not require the addition of emulsifiers, and there is no formula design and emulsification process in the later stage. It is purely biologically manufactured. The color of the obtained fermented milk is white to light yellow, and it feels moisturizing, easy to absorb, and natural;

[0021] 3. The high-viscosity fermented emulsion prepared in this application has good soothing, anti-wrinkle and moisturizing properties;

[0022] 4. The preparation method of the present application preferably adopts an in-situ fermentation process, that is, the mycelium acquisition stage is used as the first step of fermentation. The mycelium is no longer collected separately, but the mycelium and fermentation liquid of the first step of fermentation are directly processed together, and the second step of fermentation is directly carried out. It is easier to achieve large-scale production, omitting the mycelium separation and preservation steps, and saving production energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are photos of white ginseng fungus seed solution (YSC-1 seed solution, 290 seed solution);

[0024] Figure 2 The photos are of the fermentation supernatant samples obtained in Examples 1-5, Comparative Examples 1, 2.1, 2.2, 3, and Examples 6-8;

[0025] Figure 3 (a) and (b) show the DPPH radical scavenging ability curves of the fermentation supernatant freeze-dried powder obtained in Example 6 and Comparative Example 2.1, respectively;

[0026] Figure 4 This is a sensory comparison chart of the fermentation broth obtained in Example 6 and Comparative Example 2.1;

[0027] Figure 5 This is a sensory evaluation comparison chart of the fermentation broth obtained in Example 6 and Comparative Example 2.1 and a commercially available facial mask (natural fermentation facial mask);

[0028] Figure 6 This is a sensory evaluation comparison chart of the emulsions made from the fermented broths obtained in Examples 7 and 8;

[0029] Figure 7 This is a sensory comparison chart of the emulsion made from the fermented broth obtained in Examples 7 and 8 and a commercially available emulsion;

[0030] Figure 8 The anti-wrinkle performance test results of the facial mask liquids prepared in Example 6 and Comparative Example 2.1 and the emulsions prepared in Example 7 and Example 8 are shown;

[0031] Fig. 9 The moisturizing performance test results of the facial mask liquids prepared in Example 6 and Comparative Example 2.1 and the emulsions prepared in Example 7 and Example 8 are shown;

[0032] Fig.10 The upper and lower figures respectively show the zebrafish inflammatory factor gene expression inhibition test results of the sample prepared in Example 6 (concentration 5 g / L) and the Cordyceps cicadae CH2347 fermentation broth sample (concentration 5 g / L);

[0033] Fig.11The upper and lower figures respectively show the test results of the zebrafish neutrophil inhibition rate of the sample (concentration 5 g / L) prepared in Example 6 and the typical diagram of soothing and anti-inflammatory effects (the red dotted line area is the quantitative counting area, and the red arrows are neutrophils);

[0034] Fig.12 The upper and lower figures respectively show the zebrafish neutrophil inhibition rate test results of the Cordyceps cicadae CH2347 fermentation broth sample (concentration 5g / L) and the soothing and anti-inflammatory typical diagram (the red dotted line area is the quantitative counting area, and the red arrow is the neutrophil);

[0035] Fig.13 The sample photos are respectively prepared for Examples 9-14 and Comparative Examples 6-8. DETAILED DESCRIPTION

[0036] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without violating the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention belongs to the scope of the present invention.

[0037] The technical solution of the present disclosure will be described below in conjunction with exemplary embodiments. The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0038] The white ginseng fungus (290, i.e., YSC-1) used in the examples / comparative examples is specifically Schizophyllum commune YSC1, whose preservation number is CGMCC No.17788. The strain has been recorded in CN202111516706.X and CN202111483413.6.

[0039] The preparation method of the white ginseng fungus seed liquid (also known as 290 seed liquid, YSC-1 seed liquid) used in the embodiment / comparative example comprises the following steps: (1) the solid strain of white ginseng fungus YSC-1 (with culture medium, 2 to 3 solid strains with a diameter of about 0.5 cm are taken out by a sterilized puncher) grown on a PDA plate is inoculated into 300 mL of potato glucose water culture medium, and cultured at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 1, at which time the bacterial balls in the white ginseng fungus seed liquid 1 are of different sizes; (2) the white ginseng fungus seed liquid 1 obtained in step (1) is homogenized by a sterile glass homogenizer, and 10 mL is taken as the seed liquid and inoculated into 300 mL of potato glucose water culture medium, and cultured at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 2, at which time the YSC-1 bacterial balls are suspended in the fermentation liquid, see Figure 1The volume of the mycelium of white ginseng mushroom accounts for about 85-90% of the entire liquid culture medium. Unless otherwise specified, this is the white ginseng mushroom seed liquid used in the preparation of subsequent experimental samples.

[0040] The white meadowfoam seed oil used in the examples / comparative examples was purchased from Qingdao Bio-Uti Chemical Technology Co., Ltd., macadamia seed oil was purchased from Heying International Trade (Shanghai) Co., Ltd., linseed oil, soybean oil, and corn germ oil were all purchased from JD.com; the potato glucose water culture medium used was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. (Product No.: HB0233-4). The potatoes used in the examples / comparative examples were all fresh potatoes.

[0041] The viscosity was measured using a DV2T viscometer, Brookfield Ametek, with the parameters of rotor No. 2 at 60 rpm. The solid content was measured using a LG18T solid solution concentration meter, Guangzhou Mingrui Electronic Technology Co., Ltd.

[0042] 1. Examples 1-5 and Comparative Examples 1.1-3

[0043] In Examples 1 and 5, only the mycelium of white ginseng fungus was used as a substrate for fermentation; while Examples 2-4 used the mycelium of white ginseng fungus plus certain supplementary ingredients as a substrate for fermentation. In Comparative Example 1.1, only the mycelium of white ginseng fungus was used as a substrate for fermentation, but the seed liquid used was different from that in Example 1; the mycelium used as a fermentation substrate in Comparative Examples 1.2 and 1.3 was not freshly prepared mycelium; in Comparative Examples 2.1 and 2.2, different proportions of potato homogenate were used as a substrate for conventional liquid fermentation with white ginseng fungus; Comparative Example 3 used a 1% rice substrate formula for conventional liquid fermentation with white ginseng fungus.

[0044] Example 1

[0045] (1) 300 mL of potato glucose water culture medium was inoculated with 10 mL of white ginseng fungus seed solution (also referred to as 290 seed solution or YSC-1 seed solution in this application) at 28° C. and 180 rpm. After 3 days of culture, mycelium was collected by centrifugation.

[0046] (2) According to the ratio of 1:10 (mass ratio), fresh mycelium and water were homogenized and packaged, 300 mL per bottle, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, cultured at 28°C, 180 rpm, for 3 days, and centrifuged to obtain the fermentation supernatant (i.e., fermentation liquid). The viscosity of the supernatant was measured to be 302 Pa·s, the solid content (abbreviated as "solid content") was 1.30%, and the pH was 6.31.

[0047] Example 2

[0048] (1) Mycelium preparation was the same as in Example 1.

[0049] (2) Fresh mycelium and water were homogenized in a ratio of 1:10 (mass ratio), and 0.5% sucrose (1.5 g) was added to each bottle (300 mL). After sterilization at 121°C for 15 min, 10 mL of YSC-1 seed liquid was inoculated and cultured at 28°C and 180 rpm for 3 days. The fermentation supernatant was obtained by centrifugation and the viscosity, solid content and pH were measured.

[0050] Example 3

[0051] (1) Mycelium preparation was the same as in Example 1.

[0052] (2) Fresh mycelium and water were homogenized in a ratio of 1:10 (mass ratio), and 0.5% mung bean starch (1.5 g) was added to each bottle (300 mL). After sterilization at 121°C for 15 min, 10 mL of YSC-1 seed liquid was inoculated and cultured at 28°C and 180 rpm for 3 days. The fermentation supernatant was obtained by centrifugation and the viscosity, solid content and pH were measured.

[0053] Example 4

[0054] (1) Mycelium preparation was the same as in Example 1.

[0055] (2) Fresh mycelium and water were homogenized in a ratio of 1:10 (mass ratio), and 0.5% mung bean starch (1.5 g) and 1.34% macadamia seed oil (4 g) were added to each bottle (300 mL). After sterilization at 121°C for 15 min, 10 mL of YSC-1 seed liquid was inoculated and cultured at 28°C and 180 rpm for 3 days. The fermentation supernatant was obtained by centrifugation and the viscosity, solid content and pH were measured.

[0056] According to the data, adding nutrients, such as sucrose, will help increase viscosity, and adding oil will also help increase viscosity.

[0057] Example 5

[0058] (1) Mycelium preparation was the same as in Example 1.

[0059] (2) Fresh mycelium and water were homogenized in a ratio of 1:5 (mass ratio), and 0.5% sucrose (1.5 g) was added to each bottle (300 mL). After sterilization at 121°C for 15 min, 10 mL of YSC-1 seed liquid was inoculated and cultured at 28°C and 180 rpm for 3 days. The fermentation supernatant was obtained by centrifugation and the viscosity, solid content and pH were measured.

[0060] Example 5.1

[0061] (1) Mycelium preparation was the same as in Example 1.

[0062] (2) Fresh mycelium and water were homogenized in a ratio of 1:5 (mass ratio), 300 mL per bottle, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed solution, cultured at 28°C and 180 rpm for 3 days, and the fermentation supernatant was obtained by centrifugation. The viscosity, solid content and pH were measured.

[0063] Example 5.2

[0064] (1) Mycelium preparation was the same as in Example 1.

[0065] (2) Fresh mycelium and water were homogenized in a ratio of 1:5 (mass ratio), and 0.5% sucrose (1.5 g) was added to each bottle (300 mL). After sterilization at 121°C for 15 min, 10 mL of YSC-1 seed liquid was inoculated and cultured at 28°C and 180 rpm for 3 days. The fermentation supernatant was obtained by centrifugation and the viscosity, solid content and pH were measured.

[0066] Comparative Example 1.1

[0067] The only difference between this comparative example and Example 1.1 is that the seed liquid inoculated in steps (1) and (2) is white ginseng fungus seed liquid 1 instead of white ginseng fungus seed liquid 2, and the viscosity of the fermentation liquid finally obtained is 9 Pa·s.

[0068] Comparative Example 1.2

[0069] The steps and process conditions of this comparative example are the same as those of Example 5.1, except that the mycelium obtained in step (1) is fermented for the second time after being stored for 25 days, and the viscosity of the fermentation liquid finally obtained is 650 Pa·s.

[0070] Comparative Example 1.3

[0071] The steps and process conditions of this comparative example are the same as those of Example 5.1, except that the mycelium obtained in step (1) is fermented for the second time after being stored for 25 days. The viscosity of the fermentation liquid obtained finally is 1125 Pa·s.

[0072] Comparative Example 2.1

[0073] 5% potato homogenate was used as a substrate for liquid fermentation of white ginseng fungus (5% potato and water homogenate was divided into triangular flasks, 300 mL per bottle, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, and cultured at 28°C and 180 rpm for 3 days). The viscosity of the obtained fermentation liquid was 224 Pa·s.

[0074] Comparative Example 2.2

[0075] 1% potato homogenate was used as a substrate for liquid fermentation of white ginseng fungus (1% potato and water homogenate was divided into triangular flasks, 300 mL per bottle, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, and cultured at 28°C and 180 rpm for 3 days). The viscosity of the obtained fermentation liquid was 58.5 Pa·s.

[0076] Comparative Example 3

[0077] 1% rice was used as a substrate for liquid fermentation of white ginseng fungus (rice was added to a triangular flask at a ratio of 1%, 300 mL of water was added to each flask, sterilized at 121°C for 15 min, 10 mL of YSC-1 seed solution was inoculated, and cultured at 28°C and 180 rpm for 3 days). The resulting fermentation liquid had a viscosity of 170 Pa·s and had an unpleasant odor.

[0078] See the fermentation supernatant obtained in the above examples 1-5 and comparative examples 1-3 for photos. Figure 2 As can be seen from the figure, the comparative examples and the examples are all light yellow to yellow, transparent or turbid fermentation broths. Comparative examples 1 to 3 are more transparent and clear; examples 1 to 5 are yellowish, more turbid, and thicker.

[0079] The fermentation supernatants obtained in Examples 1-5 and Comparative Examples 1-3 were tested, and the results are shown in Table 1 below.

[0080] Table 1 Process conditions and physicochemical properties of fermentation broth of Examples 1-5.2 and Comparative Examples 1.1-3

[0081]

[0082] It can be seen that the viscosity of the fermentation liquid obtained by secondary fermentation with mycelium as the substrate increases. Further supplementation of nutrients, such as sucrose, in the secondary fermentation will further contribute to the increase of the viscosity of the fermentation liquid, and the addition of oil will also contribute to the increase of the viscosity of the fermentation liquid; while the comparative example 2.1-3 uses conventional white ginseng liquid fermentation, and the resulting fermentation liquid has a lower viscosity. At the same time, the viscosity of the fermentation liquid is different due to the different proportion of mycelium in the substrate.

[0083] The skin feel of the fermented liquid obtained in Example 3 and Comparative Example 2.1 was compared, and the results are shown in Table 2 below:

[0084] Table 2 Skin feel test score table of the fermented liquid obtained in Example 3 and Comparative Example 2.1

[0085]

[0086] It can be seen that the mycelium fermented liquid is smoother than the potato homogenate fermented liquid; the potato homogenate fermented liquid is lighter and thinner than the mycelium fermented liquid. Both are not tight or muddy after use.

[0087] The inventors summarize the shortcomings of the above-mentioned secondary fermentation process as follows:

[0088] (1) According to Examples 5.1-5.2 and Comparative Examples 1.2-1.3, it can be seen that the length of mycelium storage time affects the viscosity of the final fermentation supernatant. The reason is that the mycelium will precipitate water as the storage time increases, resulting in unstable substrate components after homogenization according to the proportion, thereby affecting the effect of secondary fermentation.

[0089] (2) After the fermentation supernatant with mung bean starch added was sterilized and applied to the face, it had a faint unpleasant odor. The white ginseng fungus fermentation liquid of rice in Comparative Example 3 also had an unpleasant odor.

[0090] (3) The above-mentioned secondary fermentation process is relatively difficult to achieve in large-scale production; the separation and preservation of mycelium are difficult; and the energy consumption is relatively high.

[0091] To this end, the inventor continued to explore and obtained another improved implementation method:

[0092] In the in-situ fermentation process, the mycelium acquisition stage is used as the first step of fermentation. The mycelium obtained is no longer collected separately, but the mycelium and fermentation liquid obtained in the first step of fermentation are directly processed together and the second step of fermentation is directly performed. See the following examples for details.

[0093] 2. Examples 6-8 and Comparative Examples 4-5

[0094] Example 6 adopts the above-mentioned in-situ fermentation process for secondary fermentation, while Comparative Examples 4 and 5 do not adopt the in-situ fermentation process. Examples 7-8 supplement certain ingredients as the second-step fermentation substrate during the second fermentation process.

[0095] Example 6

[0096] Take 18g fresh potatoes and 18g sucrose and add them to 900mL water for homogenization (potatoes account for 2% and sucrose accounts for 2%), take 300mL homogenate into a conical flask, sterilize at 121℃ for 15min, inoculate 10mL YSC-1 seed liquid, culture at 28℃, 180rpm, and culture for 2 days, pour out and homogenize the whole, pour the homogenate back into the conical flask for sterilization and secondary inoculation of 10mL YSC-1 seed liquid, culture at 28℃, 180rpm, and culture for 3 days. Centrifuge, take the fermentation supernatant, and sterilize. The viscosity of the supernatant was measured to be 912.5Pa·s, the solid content was 3.1%, and the pH was 4.98.

[0097] Comparative Example 4

[0098] Take 18g fresh potatoes and 18g sucrose and add them to 900mL water for homogenization (potatoes account for 2% and sucrose accounts for 2%), take 300mL and put it into a conical flask, sterilize at 121℃ for 15min, inoculate 10mL YSC-1 seed liquid, culture at 28℃, 180rpm, after 2 days, no homogenization operation, sterilize directly, inoculate 10mL YSC-1 seed liquid for the second time, culture at 28℃, 180rpm, and culture for 3 days. Centrifuge, take the fermentation supernatant, and sterilize. The viscosity of the supernatant was measured to be 366.5Pa·s, the solid content was 3.2%, and the pH was 5.13.

[0099] Comparative Example 5

[0100] 18g of fresh potatoes and 18g of sucrose were added to 900mL of water for homogenization (potatoes accounted for 2% and sucrose accounted for 2%), 300mL was put into a conical flask, sterilized at 121℃ for 15min, inoculated with 10mL of YSC-1 seed liquid, cultured at 28℃, 180rpm, and the supernatant was sterilized after centrifugation. The viscosity of the supernatant was 104.5Pa·s, the solid content was 3.2%, and the pH was 5.07.

[0101] By comparing Example 6, Comparative Example 4 and Comparative Example 5, it can be seen that the second fermentation step is helpful to improve the viscosity of the final fermentation liquid (the viscosity of Comparative Example 5 is 104.5 Pa·s, the viscosity of Comparative Example 4 after the second fermentation is 366 Pa·s, and the viscosity of Example 6 reaches 912.5 Pa·s after the homogenization step and the second fermentation. The homogenization operation before the second fermentation is indispensable and an important step to increase the viscosity of the final fermentation system. The direct sterilization has no greater destructive effect on the mycelium than the homogenization operation, resulting in the viscosity of the fermentation liquid of Comparative Example 4 being much lower than that of Example 6.

[0102] Example 7

[0103] Take 18g of fresh potatoes and 18g of sucrose and add them to 900mL of water for homogenization (potatoes account for 2% and sucrose accounts for 2%), take 300mL and put it into a triangular flask, sterilize at 121℃ for 15min, inoculate 10mL of YSC-1 seed liquid, culture at 28℃, 180rpm, and after 2 days, pour out and homogenize the whole, and pour the homogenate back into the triangular flask. Add 1.34% of white pondweed seed oil (4g / 300mL), sterilize and inoculate 10mL of YSC-1 seed liquid for the second time, culture at 28℃, 180rpm, and culture for 3 days. Centrifuge, take the fermentation supernatant, and sterilize. The viscosity of the supernatant was measured to be 1363Pa·s, the solid content was 2.9%, and the pH was 4.97.

[0104] Example 8

[0105] Take 18g fresh potatoes and 18g sucrose and add them to 900mL water for homogenization (potatoes account for 2% and sucrose accounts for 2%), take 300mL and put it into a triangular flask, sterilize at 121℃ for 15min, inoculate 10mL YSC-1 seed liquid, 28℃, 180rpm, culture for 2 days, pour out and homogenize the whole, and pour the homogenate back into the triangular flask. Add 3% white pondweed seed oil (15g / 300mL), sterilize and inoculate 10mL YSC-1 seed liquid for the second time, 28℃, 180rpm, and culture for 3 days. Centrifuge, take the fermentation supernatant, and sterilize. Determine the viscosity of the supernatant to be 1245Pa·s, the solid content to be 2.9%, and the pH to be 6.

[0106] See the photos of the fermentation supernatant prepared in Example 6-8. Figure 2 It can be seen that the samples of Examples 6 to 8 are all yellow to dark yellow turbid fermentation broths, among which the samples of Examples 7 and 8 are more turbid and thicker.

[0107] Efficacy studies

[0108] 1.DPPH free radical scavenging ability test

[0109] The fermentation supernatants obtained in Example 6 and Comparative Example 2.1 were freeze-dried to obtain freeze-dried powders, which were named in-situ fermentation samples and ordinary fermentation samples, respectively, and the following efficacy studies were conducted on the two samples.

[0110] The experimental method refers to the literature (Wu Di, Liu Pingping, Li Meng, Wang Changtao, Zhao Dan, Zhang Jiachan. Determination of the in vitro antioxidant and anti-aging effects of Pueraria lobata water extract and Pueraria lobata fermentation broth [J]. Food Industry Science and Technology, 2019, 40 (12): 285-290 + 294.), specifically: prepare a 4 mg / mL DPPH solution with anhydrous ethanol, add 2 mL of samples of different concentrations to 2 mL DPPH solution, mix well, let stand for 30 minutes, centrifuge at 3000r / min for 10 minutes, take the supernatant and measure the absorbance at 517nm. The ability of the test substance to remove DPPH can be expressed by the clearance rate. The greater the clearance rate, the stronger the clearance ability. The formula is: clearance rate I = [I-(A i -A j ) / A0]×100%; where: A i -2mL DPPH·solution+2mL sample; A j -2mL DPPH·solution solvent + 2mL sample; A0-2mL DPPH·solution + 2mL sample solvent.

[0111] Figure 3 (a) and (b) in the figure show the DPPH free radical scavenging ability curves of the in situ fermentation sample and the common fermentation sample, respectively. Figure 3It can be seen that the DPPH radical scavenging ability of the in situ fermentation samples increases with the increase of concentration, and the free radical scavenging rate is close to 90% at 4000 mg / mL; the DPPH radical scavenging ability of the ordinary fermentation samples also increases with the increase of concentration, but the scavenging rate is below 35%.

[0112] 2. Sensory evaluation (Testing company: Haotu Technology (Shanghai) Co., Ltd., HT-SE-2023-133-R-V1 Water Product Sensory Evaluation Test Report)

[0113] 2.1 Mask Liquid Evaluation

[0114] The two samples are: Example 6 (the in-situ fermentation material produced by the factory, that is, the fermentation supernatant directly prepared by the process of Example 6, sample label: SMF-factory) and Comparative Example 2.1 (the fermentation supernatant prepared by directly fermenting 5% potato homogenate with 290, sample label: original process).

[0115] (1) Comparison between two samples, see Figure 4 In terms of product appearance, the texture of the sample "SMF-Factory" is thicker and the brushing is longer; the sample "original process" is more transparent; when using: the sample "SMF-Factory" is easier to apply, the application is smoother, and the product has a stronger oily feel; after use: the skin of the sample "SMF-Factory" is brighter / the skin feels more oily (immediately), and the skin feels more watery (2 minutes); Overall, the two water products have great differences in sensory attributes: the sample "SMF-Factory" has a thicker texture and longer brushing, and it is easier to apply when used, the application is smoother, and the product feels more oily. After use, the skin is brighter / the skin feels more oily (immediately), and the skin feels more watery (2 minutes); the sample "original process" is more transparent.

[0116] (2) The above two samples were compared with a commercially available fermented facial mask (natural fermented facial mask), see Figure 5. In terms of product appearance, the sample "SMF-Factory" is more transparent, the thickest, and has the longest brushing; the sample "Original Craft" is the most transparent, the thinnest, and has longer brushing; the sample "Natural Fermentation Mask" is the most opaque, thicker, and has the shortest brushing. When using: the sample "SMF-Factory" is easier to apply, the smoothest to apply, the thicker to apply, the skin feels cooler, and the product feels more oily; the sample "Original Craft" is easier to apply, the application is smoother, and the product feels the weakest oil; the sample "Natural Fermentation Mask" is the least easy to apply, the application is the least smooth, and the product feels more oily. After use: the skin of the sample "SMF-Factory" feels more hydrated (2 minutes); the skin of the sample "Natural Fermentation Mask" is brighter (2 minutes). In general, the sensory properties of the three water products are quite different: the sample "SMF-Factory" (product in Example 6) is more transparent, the thickest, the longest drawing, easier to apply when using, the smoothest application, thicker application, a stronger cooling feeling on the skin, a stronger oily feeling of the product, and the skin feels more watery after use (2 minutes); the sample "Original Process" (product in Comparative Example 2.1) is the most transparent, the thinnest, the longer drawing, easier to apply when using, a smoother application, and the weakest oily feeling of the product; the sample "Natural Original Fermentation Mask" (commercially available product) is the most opaque, thicker, the shortest drawing, the least easy to apply when using, the least smooth application, the product feels more oily, and the skin feels brighter after use (2 minutes).

[0117] 2.2 Emulsion evaluation

[0118] The two samples are: the fermentation supernatant sample prepared in Example 7 (sample label: post-emulsification sample 1) and the fermentation supernatant sample prepared in Example 8 (sample label: post-emulsification sample 2).

[0119] (1) Comparison between two samples, see Figure 6 In terms of product appearance, post-emulsification sample 1 is more transparent; when used, there is no difference between the two water products; after use, the skin is brighter after using the post-emulsification sample 2 (immediately); Overall, the two water products have little difference in sensory attributes, except that: post-emulsification sample 1 is more transparent; the skin is brighter after using the post-emulsification sample 2 (immediately).

[0120] (2) The above two samples were compared with a commercially available lotion (sample label: essence lotion), see Figure 7. In terms of product appearance, the essence milk is the most opaque. When used, the essence milk is the most difficult to spread and the least smooth to apply. After use, the skin of post-emulsified sample 1 is smoother (2 minutes); the skin of the essence milk is the least smooth / the skin feels more hydrated (1 minute). Overall, there is no difference in sensory attributes between post-emulsified sample 1 and post-emulsified sample 2, and the sensory attributes of post-emulsified sample 1, post-emulsified sample 2 and the essence milk are quite different: compared with the essence milk, the skin of post-emulsified sample 1 is smoother after use (2 minutes); the essence milk is the most opaque, the most difficult to spread, the least smooth to apply, and the skin is the least smooth / the skin feels more hydrated after use (1 minute).

[0121] 3. HSF cell COL-I assay (anti-wrinkle performance test)

[0122] Test principle: By measuring the COL-I content of human skin fibroblasts, we can compare whether the sample stimulation is improved compared with the blank control group without sample treatment. The COL-I is determined by ELISA kit. The kit was purchased from Cloud-Clone, batch number SEA571Hu, named Type I collagen (COL1) detection kit (enzyme-linked immunosorbent assay).

[0123] The experiment included a blank control group (Control, no sample but cells), a sample group (Samples, samples and cells), and a positive control group (Positive control, TGF-β as a positive control, 100 ng / mL). Well-growing human skin fibroblasts (HSFs) were selected, and the number of cells was controlled to 1.5×10 6 pc·mL -1 , 2 mL per well were plated in a 6-well plate, and then incubated overnight at 37°C, 5% CO2 incubator, and treated with samples for 72 hours. The cell supernatant was collected and the COL-I (collagen I, the higher the value, the better) content of the supernatant was detected. The indicator was the ratio of the COL-I content of each treatment group to the COL-I content of the control group.

[0124] The samples in this experiment are: Example 6 (factory in-situ fermentation material, i.e. fermentation supernatant directly obtained by the process of Example 6, sample label: SMF), Comparative Example 2.1 (fermentation supernatant obtained by direct 290 fermentation of 5% potato homogenate, sample label: original process), Example 7 (sample label: post-emulsification sample 1), Example 8 (sample label: post-emulsification sample 2). All liquid samples have a loading concentration of 5% (i.e., a solution sample with a concentration of 5% prepared by adding water to the fermentation liquid), and TGF-β is used as a positive control, 100 ng / mL.

[0125] Results Figure 8, it can be seen that: compared with the Control group, TGF-β significantly promotes the generation of COL-I; samples "original process" and sample "SMF" also significantly increase the generation of COL-I, and there is no significant difference compared with the positive control TGF-β; post-emulsification sample 1 and post-emulsification sample 2 also have the effect of significantly increasing the generation of COL-I, but compared with the positive control, the significance is lower. Therefore, in terms of promoting the generation of COL-I, sample "SMF" and sample "original process" have better effects.

[0126] 4. HaCaT cell AQP3 assay (moisturizing performance test)

[0127] AQP3 is a water channel protein 3, which is related to the transport of water molecules. An increase in the value indicates that it helps water enter cells and is related to moisturizing. The determination of AQP3 uses an ELISA kit. The kit was purchased from Cloud-Clone, batch number SEA581Hu, and the name is: Aquaporin 3 (AQP3) Detection Kit (Enzyme-Linked Immunosorbent Assay).

[0128] The experiment set up a blank control group (Control, no sample but cells), a sample group (Samples, samples and cells), and a positive control group (Positive control, Ciglitazone, Cig, 5μM). Select well-growing HaCaT cells and control the cell number to 1.5×10 6 pc·mL -1 , 2 mL per well were plated in a 6-well plate, and then incubated at 37°C, 5% CO2 overnight. Samples were added for 48 hours, cells were collected, and adherent cells were lysed with lysis buffer. After centrifugation, the supernatant was stored in a -20°C refrigerator for detection of intracellular AQP3 content. The index was the ratio of AQP3 content in each treatment group to AQP3 content in the control group.

[0129] The samples in this experiment are: Example 6 (the in-situ fermentation material from the factory, i.e., the fermentation supernatant, sample label: SMF), Comparative Example 2.1 (the fermentation supernatant obtained by direct 290 fermentation of 5% potato homogenate, sample label: original process), Example 7 (sample label: post-emulsification sample 1), Example 8 (sample label: post-emulsification sample 2). All liquid samples have a loading concentration of 5%, and Ciglitazone (Cig, 5 μM) is used as a positive control.

[0130] Results Fig. 9, it can be seen that: compared with the Control group, Cig significantly promotes the generation of AQP3; samples "original process" and sample "SMF" also significantly increase the generation of AQP3, and compared with the positive control Cig, sample "SMF" has no significant difference; post-emulsification sample 1 and post-emulsification sample 2 also have the effect of significantly increasing the generation of AQP3, and there is no significant difference with the positive control. Therefore, in terms of promoting the generation of AQP3, sample "SMF", post-emulsification sample 1 and post-emulsification sample 2 have better effects.

[0131] 5. Soothing effect (zebrafish inflammatory factor gene expression inhibition test)

[0132] The response of zebrafish skin to inflammatory stimulation is similar to that of human skin. After being subjected to inflammatory stimulation, the skin can induce the expression of large amounts of related inflammatory factor genes. A zebrafish inflammation induction model was constructed using copper sulfate. After treatment with the test substance, the relative expression levels of inflammatory-related factor genes (IL-1β, IL-6, NF-κB and TNFα) were detected to evaluate whether the cosmetics had soothing and anti-inflammatory effects.

[0133] Zebrafish embryos on the third day of development were selected as experimental organisms. The main reagents included copper sulfate (modeling drug), dexamethasone (positive drug), MS-222, calcium chloride, magnesium sulfate, sodium bicarbonate, potassium chloride, etc.; the main consumables included 24-well plates, centrifuge tubes, disposable Pasteur pipettes, etc. The upstream primer of the internal reference gene (β-actin) was 5'-GCTGACAGGATGCAGAAGGA-3', and the downstream primer was 5'-TAGAAGCATTTGCGGTGGA-3'; the upstream primer of IL-1β was 5'-CTCAGCCTGTGTGTTTGGGA-3', and the downstream primer was 5'-GGGACATTTGACGGACTCG-3'; the upstream primer of TNF-α was 5'-GCTGGATCTTCAAAGTCGGGTGTA-3', and the downstream primer was 5'-TGTGAGTCTCAGCACACTTCCATC-3'; the upstream primer of IL-6 was 5'-ACGACATCAAACACAGCACC-3', and the downstream primer was 5'-TCGATCATCACGCTGGAGAA-3'; the upstream primer of NF-κB was 5'-ACAAGACGCAAGGAGCCCAG-3', and the downstream primer was 5'-AACTGTCTCTTGCACAAAGGGCTCA-3'.

[0134] The instruments and equipment mainly include: fluorescence quantitative PCR instrument (Applied Biosystems QuantStudio 5), low-temperature refrigerated centrifuge, ultra-micro spectrophotometer (Thermo Scientific Nanodrop 2000), analytical balance (1 / 10,000), pipette, refrigerator, clean bench, zebrafish breeding system, constant temperature incubator, etc.

[0135] The specific experimental procedures are as follows: a blank control group, a model group (1 μM), a positive drug group (0.02 g / L) and a sample group (5.0 g / L, prepared by adding water to the fermentation liquid) were set up. Each experimental group was set up with 3 replicates, and each replicate had 10 zebrafish embryos, which were placed in a 24-well plate. 2 mL of the working solution corresponding to each concentration group was added to each well; the 24-well plate to be tested was placed in a constant temperature incubator at 28.5 ± 0.5 ° C, and samples were taken after incubation for 24 hours. Total RNA was extracted, reverse transcribed and amplified by fluorescence quantitative PCR from each group of zebrafish. The β-actin gene was used as the internal reference gene, and 2 -△△Ct Methods The relative expression levels of inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) in each group were calculated, and the gene expression inhibition rate of the sample group relative to the model group was calculated.

[0136] The gene expression inhibition rate was calculated using the following formula: inhibition rate (%) = (MT) / M × 100%; where T is the relative gene expression level in the sample group; M is the relative gene expression level in the model group; a T test was performed on the relative gene expression levels in the sample group and the model group, and p < 0.05 indicated significant differences.

[0137] The test results are as follows: Fig.10 The relative expression of zebrafish inflammatory factor genes in each group is shown. It can be seen that compared with the model group, the sample at a concentration of 5.0g / L inhibited the expression of zebrafish inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) by 53.97%, 39.54%, 52.12% and 37.86%, respectively, with significant differences (p<0.05) (see Fig.10 The positive drug group inhibited the expression of zebrafish inflammatory factor genes by 26.99%, 35.06%, 20.91% and 30.35% respectively. That is, under the experimental conditions, the sample "SMF-2" (the second batch of materials obtained by the method of Example 6) can significantly inhibit the expression of zebrafish inflammatory factor genes at a concentration of 5.0g / L (p<0.05), and has a soothing effect.

[0138] For comparison, the inventors studied the soothing effect of the fermentation broth sample of Pseudomonas cicadae (i.e. Cordyceps cicadae CH2347, deposit number CGMCC No.40399, deposit date April 10, 2023, classification name: Cordyceps cicadae CH2347, deposit unit name is China National Microbiological Culture Collection Center, referred to as CGMCC, address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code 100101). The details are as follows: the culture medium formula is 2% potato + 2% sucrose. First, 300mL of a mixed solution containing 2% sucrose is prepared, and then 6g of fresh peeled potatoes are added to a homogenizer, and the 300mL solution prepared above is added for homogenization, and then all are put into a 500mL Erlenmeyer flask, sterilized, inoculated with 20mL of Paecilomyces cicadae seed solution, and fermented at 28℃180rpm for 3 days, then the homogenate is taken out, and all are sterilized again (121℃20min), and inoculated with 20mL of Paecilomyces cicadae seed solution, and fermented at 28℃180rpm for 3 days, and the supernatant (fermentation liquid) is centrifuged and sterilized (121℃20min), and 1% of a preservative (the preservative system is 1:1 of fenvalerate and hexanediol) is added to obtain sample No. 300. Sample No. 300 was tested for inhibition of zebrafish inflammatory factor gene expression according to the above method. Compared with the model group, the sample at a concentration of 5.0 g / L inhibited the expression of zebrafish inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) by 31.85%, 61.22%, 37.90% and 22.05%, respectively, with significant differences (p<0.05) ( Fig.10 The positive drug group inhibited the expression of zebrafish inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) by 26.99%, 35.06%, 20.91% and 30.35%, respectively. It can be seen that sample "300" can significantly inhibit the expression of zebrafish inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) at ​​a concentration of 5.0g / L (p<0.05), and also has a soothing effect, but its inhibition rate is significantly lower than that of the sample of this application.

[0139] 6. Soothing effect (zebrafish neutrophil inhibition rate test)

[0140] The response of zebrafish skin to inflammatory stimulation is similar to that of human skin. After the skin is subjected to inflammatory stimulation, it can induce neutrophils to gather on the skin surface. A zebrafish inflammation induction model was constructed using copper sulfate. After treatment with the test substance, the number of neutrophils on the skin surface was measured to evaluate whether the sample had a soothing effect.

[0141] Zebrafish embryos on the third day of development were selected as experimental organisms. The main reagents included copper sulfate (modeling drug), dexamethasone (positive drug), MS-222, Sudan black, calcium chloride, magnesium sulfate, sodium bicarbonate, potassium chloride, etc.; the main consumables included 24-well plates, centrifuge tubes, disposable Pasteur pipettes, etc. The instruments and equipment mainly included stereo microscopes, zebrafish breeding systems, analytical balances (one ten-thousandth), pipettes, refrigerators, clean benches, constant temperature incubators, etc.

[0142] The specific experimental procedures are as follows: a blank control group, a model group (1 μM), a positive drug group (0.02 g / L) and a sample group (5.0 g / L) were set up. Each experimental group was set up with 3 replicates, and 10 zebrafish embryos were placed in a 24-well plate in each replicate. 2 mL of the working solution corresponding to each concentration group was added to each well; the 24-well plate to be tested was placed in a constant temperature incubator at 28.5 ± 0.5 ° C, and samples were taken after incubation for 4 hours. After the incubation, no less than 12 zebrafish embryos were randomly selected from each group and fixed with 4% paraformaldehyde overnight. The juveniles were washed with PBS 4 times, 10 min / time. Sudan black solution was added for staining, incubated in the dark for 40 minutes, washed with 70% ethanol under light-proof conditions until the body color was transparent, washed with PBS 3 times, and the fish were uniformly adjusted to the lateral position and photographed using a stereo microscope. The number of neutrophils on the skin surface of a specific area of ​​each fish was counted.

[0143] The neutrophil aggregation inhibition rate was calculated using the following formula: inhibition rate (%) = (MT) / M × 100%; wherein: T is the average number of neutrophils in a specific area of ​​the test group fish embryos; M is the average number of neutrophils in a specific area of ​​the model group fish embryos; a two-tailed T test was performed on the neutrophils in the specific area of ​​the test group fish embryos and the neutrophils in the specific area of ​​the model group fish embryos, and p < 0.05 was considered to be significantly different.

[0144] The test results are as follows: For the analysis of the mean values ​​of neutrophils in specific regions of zebrafish embryos in each group, see Fig.11 's picture above. Fig.11 The figure below is a typical picture of soothing and anti-inflammatory effects (the red dotted line is the quantitative counting area, and the red arrow is the neutrophil). It can be seen that compared with the model group, the sample at a concentration of 5.0g / L has an inhibition rate of 88.85% on neutrophil aggregation in specific areas of zebrafish, which is significantly different (p<0.05). Under the conditions of this experiment, the sample "SMF-2" (the second batch of materials obtained by reproducing according to the method of Example 6) can significantly inhibit the aggregation of neutrophils in specific areas of zebrafish (p<0.05), and has a soothing effect.

[0145] Similarly, the above sample 300 was tested for zebrafish neutrophil inhibition rate according to the above method, and the results are shown in Fig.12The upper and lower figures show that compared with the model group, the inhibition rate of sample 300 on neutrophil aggregation in specific areas of zebrafish at a concentration of 5.0g / L was 75.09%, which was significantly different (p<0.05). Under the conditions of this experiment, sample 300 can significantly inhibit the aggregation of neutrophils in specific areas of zebrafish (p<0.05), and has a soothing effect, but its inhibition rate is also significantly lower than that of the samples in this application.

[0146] III. Examples 9-14 and Comparative Examples 6-8

[0147] Examples 9-14 and Comparative Examples 6-8 are another batch of experiments. In Examples 9-14, freshly prepared 290 mycelium was used as the main substrate for fermentation, and the preparation method of 290 mycelium was the same as that of Example 1.

[0148] Example 9

[0149] Weigh the freshly prepared 290 mycelium, add water at a ratio of 1:10 and homogenize, then divide into Erlenmeyer flasks, 300 mL per bottle, sterilize at 121°C for 30 min, inoculate with 290 seed solution, inoculation amount of 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant.

[0150] Example 10

[0151] Weigh the freshly prepared 290 mycelium, add water at a ratio of 1:10 and homogenize into triangular bottles, 300 mL per bottle. Add sucrose at a ratio of 0.5%, sterilize at 121°C for 30 min, inoculate with 290 seed liquid with an inoculum size of 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant.

[0152] Embodiment 11

[0153] After weighing the freshly prepared 290 mycelium, add water at a ratio of 1:10 to homogenize, and dispense into triangular bottles, 300 mL per bottle, add sucrose at a ratio of 0.5%, sterilize at 121°C for 30 min, inoculate 290 seed liquid, inoculation amount 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant. The process of this embodiment is the same as that of embodiment 10, except that the experimental batches are different.

[0154] Example 12

[0155] Weigh the freshly prepared 290 mycelium, add water at a ratio of 1:10 and homogenize, and divide into triangular bottles, 300 mL per bottle, add mung bean starch at a ratio of 0.5%, sterilize at 121°C for 30 min, inoculate with 290 seed liquid, with an inoculum size of 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant.

[0156] Example 13

[0157] After weighing the freshly prepared 290 mycelium, add water at a ratio of 1:10 and homogenize, and divide into triangular bottles, 300 mL per bottle, add mung bean starch at a ratio of 0.5%, sterilize at 121°C for 30 min, inoculate 290 seed liquid, inoculation amount 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant. The process of this embodiment is the same as that of Example 12, except that the experimental batches are different.

[0158] Embodiment 14

[0159] Weigh the freshly prepared 290 mycelium, add water at a ratio of 1:10 and homogenize, then divide into Erlenmeyer flasks, 300 mL per bottle, add 4 g macadamia oil, sterilize at 121°C for 30 min, inoculate with 290 seed liquid, inoculation amount of 10 mL, ferment at 28°C, 180 rpm for 3 days, and centrifuge to obtain the supernatant.

[0160] Comparative Example 6

[0161] Take fresh potatoes at a mass ratio of 1%, add water to homogenize, put 300g into each triangular bottle, sterilize at 121℃ for 30min, inoculate 290 seed liquid, inoculation amount 10mL / bottle, ferment at 28℃, 180rpm for 3 days, and centrifuge to obtain the supernatant.

[0162] Comparative Example 7

[0163] Take fresh potatoes at a mass ratio of 5%, add water to homogenize, put 300g into each triangular bottle, sterilize at 121℃ for 30min, inoculate 290 seed liquid, inoculation amount 10mL / bottle, ferment at 28℃, 180rpm for 3 days, and centrifuge to obtain the supernatant.

[0164] Comparative Example 8

[0165] Each bottle contains 300g of material. According to the mass ratio of 1%, rice flour is added with water and sterilized at 121℃ for 30min. 290g of seed liquid is inoculated with an inoculation amount of 10mL / bottle. Ferment at 28℃ and 180rpm for 3 days and the supernatant is obtained by centrifugation.

[0166] The viscosity and total sugar test results of the samples prepared in Examples 9-14 and Comparative Examples 6-8 are shown in Table 3 below. Fig.13 .

[0167] Table 3 Viscosity and total sugar test results of samples obtained from Examples 9-14 and Comparative Examples 6-8

[0168]

[0169] From Table 3, it can be seen that the total sugar content of samples with high viscosity is not high, and the polypeptide content is low in the samples tested; Fig.13 It can be seen that the comparative examples and example samples are all light yellow to yellow, transparent or turbid fermentation broths; comparative examples 6 to 8 are lighter and more transparent; examples 9 to 14 are yellowish, more turbid, and thicker.

[0170] Comparative Example 9

[0171] First, potato glucose water culture medium was used, sterilized and inoculated with birch inonotus seed liquid (laboratory number 415, seed liquid preparation method was the same as white ginseng seed liquid), the inoculation amount (volume ratio of seed liquid to culture medium) was 1:30, 28°C, 180rpm, and after 3 days of culture, the mycelium was collected by centrifugation. Then, the freshly prepared 415 mycelium was weighed, added with water at a ratio of 1:10, homogenized, and divided into triangular bottles, 300mL per bottle, sterilized at 121°C for 30min, inoculated with 415 seed liquid, inoculation amount 10mL / bottle, fermented at 28°C, 180rpm for 3 days, and centrifuged to obtain the supernatant.

[0172] Comparative Example 10

[0173] First, potato glucose water culture medium was used, sterilized and inoculated with the seed solution of the fungus Inonotus birchii (laboratory number 415, the seed solution was prepared in the same way as the seed solution of the white ginseng fungus), the inoculation amount was 1:30, 28°C, 180rpm, and after culturing for 3 days, the mycelium was collected by centrifugation. Then, the freshly prepared 415 mycelium was weighed, and water was added at a ratio of 1:10 and the homogenate was divided into triangular bottles, 300mL per bottle, sucrose was added at a ratio of 0.5%, and sterilized at 121°C for 30min, inoculated with 415 seed solution, the inoculation amount was 10mL, and fermented at 28°C, 180rpm for 3 days, and the supernatant was taken by centrifugation.

[0174] Comparative Example 11

[0175] First, a potato glucose water culture medium was used, and after sterilization, the seed solution of the fungus Inonotus obliquus (laboratory number 415, the seed solution preparation method was the same as the seed solution of the white ginseng fungus) was inoculated at an inoculation amount of 1:30, 28°C, 180rpm, and after culturing for 3 days, the mycelium was collected by centrifugation; then, the freshly prepared 415 mycelium was weighed, water was added at a ratio of 1:10, and the mixture was homogenized and packaged in triangular bottles, 300mL per bottle, and mung bean starch was added at a ratio of 0.5%, sterilized at 121°C for 30min, inoculated with 415 seed solution, the inoculation amount was 10mL, and fermented at 28°C, 180rpm for 3 days, and the supernatant was obtained by centrifugation.

[0176] The viscosity, total sugar, and polypeptide of the samples obtained in Comparative Examples 9-11 (containing preservatives) were tested. The results are shown in Table 4.

[0177] Table 4 Process and performance test results of Examples 9-14 and Comparative Examples 9-11

[0178]

[0179] As can be seen from Table 4, the viscosity of the fermentation liquid of the mycelium of Inonotus obliquus 415 is much lower than that of the fermentation liquid of the mycelium of the present application, and the total sugar content is lower than that of the fermentation liquid of the mycelium of the present application, while the polypeptide content is higher than that of the fermentation liquid of the mycelium of the present application.

[0180] Finally, it should be noted that, if any, in this disclosure, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises", or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or device. In the absence of further restrictions, the elements defined by the statement "comprises a ..." do not exclude the presence of other identical elements in the process, method, article, or device including the elements.

[0181] Although the present disclosure has been disclosed above through the description of the specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed by the present disclosure.

Claims

1. A method for directly preparing a high-viscosity fermented emulsion by biological fermentation, characterized in that: include: Step 1, preparing white ginseng mycelium by liquid fermentation culture; Step 2: The white ginseng mycelium and water are mixed in proportion to form a substrate, sterilized, inoculated with white ginseng seed liquid, fermented and cultured, and the supernatant is sterilized by centrifugation to obtain a fermentation liquid.

2. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to claim 1, characterized in that: The white ginseng fungus is Schizophyllum commune YSC1, and its preservation number is CGMCC No.17788.

3. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to claim 1 or 2, characterized in that: In step 1, the preparation method of the white ginseng mycelium includes: adding 2% potato and 2% sucrose to water and then performing homogenization, then inoculating white ginseng seed liquid for fermentation and culture, and finally separating and obtaining the white ginseng mycelium.

4. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 3, characterized in that: The volume of the mycelium of the white ginseng fungus in the white ginseng fungus seed liquid accounts for 75-90% of the entire liquid culture medium, and the volume ratio of the seed liquid to the substrate is (0.1-5):30, preferably 1:30; Preferably, the volume of the mycelium of the white ginseng fungus in the white ginseng fungus seed liquid accounts for 85-90% of the entire liquid culture medium. The preparation method of the white ginseng fungus seed liquid comprises the following steps: (1) taking 2-3 solid fungus species with a diameter of 0.5 cm from the solid fungus species grown on the PDA plate together with the culture medium using a sterilized puncher, inoculating them into 300 mL of potato glucose water culture medium, and culturing them at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 1; (2) homogenizing the white ginseng fungus seed liquid 1, taking 10 mL as the seed liquid and inoculating it into 300 mL of potato glucose water culture medium, and culturing them at 28° C. and 180 rpm for 3 days to obtain white ginseng fungus seed liquid 2.

5. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 4, characterized in that: In step 2, the mass ratio of the white ginseng mycelium to water in the substrate is 1:5 to 1:15, preferably 1:5 to 1:

10.

6. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 5, characterized in that: In step 2, the substrate further includes sucrose, and the amount of sucrose added is 0-2%, preferably 0.5%; or; In step 2, the substrate further includes mung bean starch, and the amount of mung bean starch added is 0-3%, preferably 0.5%; further, the substrate further includes macadamia seed oil, and the amount of macadamia seed oil added is 0-3%, preferably 1-1.5%.

7. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 6, characterized in that: After the liquid fermentation in step 1 is completed, it is directly homogenized and then used as a substrate for step 2 treatment; Preferably, in step 2, the substrate further comprises oil, and the oil is selected from at least one of meadowfoam seed oil, soybean oil, linseed oil and corn germ oil, and the added amount of the oil is 0.5-3%, preferably 1%.

8. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 7, characterized in that: The fermentation culture temperature is 24-30°C and the fermentation time is 48-96h.

9. The method for directly preparing high-viscosity fermented emulsion by biological fermentation according to any one of claims 1 to 8, characterized in that: The fermentation culture is carried out in a shaker with a rotation speed of 150 rpm-200 rpm.

10. A high viscosity fermented emulsion product, characterized in that: The method is prepared according to any one of claims 1 to 9.

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